A synthetic antibiotic class with a deeply-optimized design for overcoming bacterial resistance

A synthetic antibiotic class with a deeply-optimized design for overcoming bacterial resistance | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article A synthetic antibiotic class with a deeply-optimized design for overcoming bacterial resistance Xu Wang, Jin Feng, Le Zheng, Wanqing Ma, Defeng Weng, Depeng Peng, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3667988/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract The lack of new drugs that are effective against antibiotic-resistant bacteria has caused increasing concern in global public health. As antibiotic resistance continues to escalate worldwide, the development of new antibiotics that can effectively treat bacterial infections is crucial. Based on this study, we report the development of a hybrid antimicrobial drug that is rationally designed through drug structural hybridization-based structure-guided design and component-based synthesis. The optimal modified compound, F8, was identified, which demonstrated excellent in vitro and in vivo anti-resistant bacterial activity and effectively mitigated the development of resistance. F8 exhibits significant bactericidal activity against bacteria resistant to antibiotics such as methicillin, polymyxin B, florfenicol, doxycycline, ampicillin and sulfamethoxazole. In the mouse model of drug-resistant bacterial bacteremia, F8 was found to increase survival and significantly reduce bacterial load in infected mice. Multi-omics analysis (transcriptomics, proteomics, and metabolomics) have indicated that ornithine carbamoyl transferase (arcB) is a novel antimicrobial target of F8. Further molecular docking, Isothermal Titration Calorimetry (ITC), and Differential Scanning Fluorimetry (DSF) studies verified arcB as a novel and effective target for F8. Finally, mechanistic studies suggest that F8 competitively binds to arcB, disrupting the bacterial cell membrane and inducing a certain degree of oxidative damage. The findings of this study highlight F8 as a promising candidate drug for the development of novel antibiotic formulations to combat antibiotic-resistant bacteria-associated infections. Biological sciences/Microbiology/Antimicrobials/Antimicrobial resistance Biological sciences/Drug discovery/Drug delivery antibiotic resistance structural hybridization arcB bacteria drug target Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Introduction The rapid increase of drug-resistant pathogens, particularly the emergence of superbugs, poses a significant threat to public health 1 – 3 . The discovery and clinical application of antibiotics undoubtedly mark a milestone in human and modern medical history. They have driven advancements in various medical practices and are indispensable life-saving weapons 4 – 7 ; however, the rise and spread of bacteria that have developed resistance to most or all existing antibiotics have raised concerns about an impending global infectious disease crisis 1 , 8 – 11 . In the United States alone, there are over 2.8 million antibiotic-resistant infections each year, resulting in more than 35,000 deaths 12 . In Europe, antibiotic resistance leads to about 33,000 deaths per year, with estimated hospital costs exceeding €900 million 12 . Any plan to address this issue relies on the discovery of new antibiotics that are effective against modern bacterial pathogens, making the development of antibiotics with unique mechanisms crucial 1 , 13 , 14 . The development of antibiotics is continuously challenging nowadays, leading to the rarity of new antibiotics being introduced clinically. Therefore, there is an urgent need to identify new targets and drugs to fill the gaps in antibiotic discovery and development, and to combat bacterial infections. ArcB is a catabolic ornithine carbamoyl transferase involved in the degradation of arginine, and is crucial for bacterial arginine biosynthesis and catabolism metabolism. It also has a key role in gene regulation, cell growth, biofilm formation, and biological mechanisms 15 – 20 . Arginine metabolism is tightly regulated in bacteria, as it is vital for cell growth and biofilm formation 15 . The expression of arcB is known to be affected by treatment with antibiotics such as gentamicin and polymyxin B, indicating that cell membrane stress affects arcB activity 21 , 22 . In addition, arcB is related to nitrogen metabolism, which is an essential nutrient for synthetic metabolism and is crucial for the synthesis of amino acids. Cells must balance the activity of synthetic metabolic pathways with the overlapping pathways used for energy production through chemical interactions 23 . It is worth noting that arcB not only participates in the regulation of cellular processes, but is also involved in cell death regulation due to the modulation of reactive oxygen species levels. Importantly, mutants of arcB may affect bacterial growth 24 , therefore, arcB appears to be a promising target for antibacterial drug development. In recent years, hybrid antimicrobial agents have garnered significant attention as a novel strategy for combating bacterial resistance. They utilize the principle of structural hybridization, which is a rational drug design method where new chemical entities are formed by combining two or more drugs or pharmacophores from different bioactive compounds into a single entity 25 – 27 . The resulting compounds have multiple potential advantages, such as broadening the spectrum of antimicrobial activity, enhancing activity against resistant strains, and reducing the emergence of bacterial resistance. Compared to combination therapy, drug structural hybridization better addresses issues of differential bioavailability, pharmacokinetics, and metabolism, resulting in improved therapeutic safety and avoidance of drug-drug interactions. Currently, several hybrid antimicrobial molecules have been synthesized and tested, with some entering clinical trial stages 28 – 31 , showing promise as prospects for development. Given the clear advantages of structural hybrids, the concept of structural hybridization is attractive; however, the complexity of molecular structures, challenging chemical synthesis, and the rigorous work required to establish modes of action and benefits of structural hybrids compared to conventional drugs can make the method daunting. Cell permeability is also a key problem, as antibacterials with high molecular weight will not pass through non-selective protein channels, limiting their cell uptake to receptor-mediated endocytosis or passive diffusion 25 . In fact, permeability barriers due to high molecular weight are the main reason for the limited activity of most antibiotics against bacteria 32 – 34 . Despite the challenges, the structural hybridization strategy remains a viable approach to expand our existing antimicrobial drug library. To find compounds with potent antimicrobial activity and a reduced propensity for resistance, this study employed the approach of drug structure hybridization, leading to the discovery of the optimized modified compound, F8. We found that F8 exhibited excellent in vitro and in vivo anti-resistant bacterial activity, and through multi-omics analysis (transcriptomics, proteomics, and metabolomics), we inferred that the antibacterial target of F8 may be the key enzyme of arcB in the arginine degradation pathway. This study evaluated the therapeutic effects of F8 both in vitro and in vivo . F8 demonstrates significant antibacterial activity against both gram-positive and gram-negative bacteria, with a minimum inhibitory concentration (MIC) range of 2 to 8 µM, which is notably superior to the positive control florfenicol (FLO) (4×MIC, and 2×MIC). In a mouse model of FLO-resistant S. aureus bacteraemia, the bacterial burden in tissues was significantly reduced (Log 10 CFU/mL as low as 2 ~ 3), and the survival rate of the mice within 72 h is as high as 50% (all deaths within 24 h were in the control group). Furthermore, the antibacterial mechanism of F8 targeting arcB was systematically investigated. Overall, our research findings suggest that F8 competitively binds to arcB, thereby activating multiple pathways to exert its antimicrobial effects. The results of this study provides a foundation and strategy for the development of novel antimicrobial drugs, and reaffirmed the ability of chemical synthesis to supplement our antibiotic arsenal, providing broad-spectrum drugs that can overcome increasingly severe drug resistance mechanisms. Results Design and synthesis of broad-spectrum antibacterial small molecule compounds based on structural hybridization strategy In order to find compounds with effective antibacterial activity and that are less likely to develop resistance, we endeavoured to design and synthesize broad-spectrum antibacterial hybrid small-molecule compounds using a drug structure hybridization strategy and computer assisted drug design (CADD) 35 . Guided by the hybrid strategy, we chose FLO, a compound with simple structure, low molecular weight, and easy synthesis, as our main skeleton structure. With peptidyl transferase centre (PTC) of the bacterial 50S ribosome subunit as the target, the key area of FLO, we applied CADD and developed a modular synthesis route. By forming an ester bond to connect these components, we produced a variety of antibacterial candidate drugs. Therefore, we studied a wide range of structural changes and evaluated their antibacterial activities. First, we conducted molecular docking studies using the crystal structure of the PTC region of the 50S subunit from the protein data bank (PDB: 6c4h) 36 . We then modelled the binding pocket of the PTC region of the 50S subunit (method) and established an atomic property field (Fig. 1 a). The structure-activity relationship of FLO has shown that its main scaffold occupies the conserved region of the binding pocket of the 50S subunit's PTC region, therefore, we have chosen to keep this region static and instead structurally modified the β-hydroxy position. The hydroxyl group provides a considerable advantage for the synthesis of small molecules. Even for the novel compounds generated, it must conform to a mechanism that can be easily synthesized, otherwise, it would have minimal significant broad influences 37 . Next, we selected various simple small molecule structures, including tricyclic, tetracyclic, pentacyclic, and hexacyclic structures, to hybridize with the skeleton structure (ChemDraw software), generating over a hundred novel hybrid structures for automated molecular docking. According to the scoring of SYBYL-X, some of the lead compounds exhibited improved binding power, reduced intermolecular collision force, and tighter binding with the receptor protein when compared to the skeleton structure (Fig. 1 b, c). Based on docking simulations, ligands with smaller fragments have lower crash values (a crash value close to 0 is beneficial), suggesting that structures with smaller spatial conformation within the binding pocket can lower the level of internal self-collisions that the ligand might experience, thereby enhancing the stability of binding. Among these studies defined within relatively narrow parameters, we comprehensively selected 11 compounds with the best scores (Fig. 1 c), total scores and polarity all higher than the total scores and polarity of the scaffold compounds (score = 5.1467), and with good binding specificity and affinity. These were then visually compared and synthesized against the PTC region of the 50S subunit. The structure was confirmed by LCMS-IT-TOF, 1 H-NMR, and 13 C-NMR measurements (Fig. 1 d, Supplementary Table 1, and Supporting information). We identified a hybrid structure 8 (F8, 3-(1-Piperidinyl) propanoic acid) as the optimal modification, which further enhanced the antibacterial effects against pathogens such as E. coli , S. aureus , S. typhi , P. multocida , and H. parasuis , and imparted measurable activity against the particularly challenging P. aeruginosa . The antibacterial activity test results are shown in Tables 1 and 2 . As shown in Table 1 , hybrid structures 1, 2, 6, 7, 8, and 14 exhibit good antibacterial effects. When hybrid small molecule fragments consist of tetracyclic and pentacyclic structures, including thiazole and cyclobutane classes (hybrid structures 1, 2, and 7), their antibacterial activity is weaker compared to the skeleton structure. When the hybrid small molecule fragments feature hexacyclic structures (hybrid structure 14), the antibacterial activity is similar to the skeleton structure. Further using hexacyclic structures, we found that quinoline and morpholine structures (hybrid structures 11 and 13) virtually lose their antibacterial activities. We found that in general, short chain small fragments are more active than their homologues. Furthermore, it was found that the piperidine structure (hybrid structure 8) is sensitive to antibacterial activity and antibacterial spectra, and its effect is superior to that of the skeleton structure. Docking simulation shows that F8 can extend to a deeper position at the end of the PTC region of the 50S subunit (Fig. 1 b). We observed changes in the binding amino acid sites, with a greater number of hydrogen bonds between F8 and the target involved in ligand-protein binding than the skeleton structure, which may directly lead to an increase in binding affinity. Moreover, F8 binds near U2585 (key site), which may compete with the substrate of the a-site (one of the core functional regions of the ribosome), interrupting the binding of the a-site with tRNA and preventing the formation of peptide bonds, thereby playing a crucial role. The software also scored F8 (score = 6.1738) higher than the scaffold structure (score = 5.1467) (Fig. 1 c), providing support for our design strategy. Table 1 MIC of broad-spectrum antibacterial small molecule compounds based on structural hybridization strategy. Organism MIC (µM) F F1 F2 F3 F4 F6 F7 F8 F11 F13 F14 F15 E. coli 8 16 32 > 128 > 128 16 16 8 > 128 > 128 16 > 128 S. typhi 4 16 32 > 128 > 128 8 16 2 > 128 > 128 8 > 128 P. aeruginosa > 128 > 128 > 128 > 128 > 128 128 > 128 128 > 128 > 128 > 128 > 128 S. aureus 16 32 64 > 128 > 128 16 32 8 > 128 > 128 16 > 128 B. subtilis 4 16 16 > 128 128 4 4 2 > 128 > 128 4 > 128 E. faecalis 16 16 32 > 128 128 8 8 4 > 128 > 128 8 > 128 P. multocida 8 16 8 > 128 64 4 4 2 > 128 > 128 2 > 128 A. pleuropneumoniae 8 32 32 > 128 128 4 16 4 > 128 > 128 8 > 128 S. suis 16 64 64 > 128 > 128 16 16 8 > 128 > 128 32 > 128 H. parasuis 32 64 64 > 128 > 128 16 32 8 > 128 > 128 16 > 128 Table 2 MBC of broad-spectrum antibacterial small molecule compounds based on structural hybridization strategy. Organism MBC (µM) F F1 F2 F3 F4 F6 F7 F8 F11 F13 F14 F15 E. coli 16 32 64 > 128 > 128 16 32 8 > 128 > 128 32 > 128 S. typhi 8 16 64 > 128 > 128 8 32 4 > 128 > 128 16 > 128 P. aeruginosa > 128 > 128 > 128 > 128 > 128 128 > 128 128 > 128 > 128 > 128 > 128 S. aureus 16 32 64 > 128 > 128 16 32 8 > 128 > 128 32 > 128 B. subtilis 8 16 32 > 128 128 4 4 4 > 128 > 128 8 > 128 E. faecalis 16 32 64 > 128 128 8 16 4 > 128 > 128 16 > 128 P. multocida 8 16 8 > 128 128 4 8 4 > 128 > 128 2 > 128 A. pleuropneumoniae 16 32 32 > 128 128 4 16 4 > 128 > 128 8 > 128 S. suis 32 128 128 > 128 > 128 16 32 8 > 128 > 128 64 > 128 H. parasuis 32 64 64 > 128 > 128 16 32 8 > 128 > 128 16 > 128 F8 is an excellent candidate antibiotic The chemical structure of F8 is shown in Fig. 1 d. To evaluate its in vitro antibacterial activity, we determined the MIC against various bacteria, including both gram-negative and gram-positive bacteria. F8 exhibited significant antibacterial activity against both gram-positive and gram-negative bacteria, with MIC values ranging from 2 to 8 µM, superior to the positive control FLO (4×MIC and 2×MIC). Measurable activity was noted against the particularly challenging P. aeruginosa , with an MIC of 128 µM (Table 1 ). F8 exhibits significant bactericidal activity against methicillin-resistant S. aureus (MRSA), polymyxin B-resistant E. hormaechei , FLO-resistant S. suis , FLO-resistant H. parasuis , doxycycline-resistant S. typhi , ampicillin-resistant S. typhi and sulfamethoxazole-resistant S. typhi , among other drug-resistant bacteria (Table 3 ). Additionally, we used a representative gram-positive bacterium, S. aureus , and a gram-negative bacterium, E. coli , as models to investigate the antibacterial activity and mechanism of action of F8. Table 3 MIC of F8 against antibiotic-resistant strains. Organism MIC (µM) Methicillin-resistant S. aureus B1-1 8 Polymyxin B-resistant E. hormaechei wb 4 16 Florfenicol-resistant S. suis 1136 8 Florfenicol-resistant S. suis 1194 4 Florfenicol-resistant S. suis 1197 8 Florfenicol-resistant S. suis 1655 8 Florfenicol-resistant S. suis 1658 4 Florfenicol-resistant S. suis 1669 16 Florfenicol-resistant H. parasuis 1565 8 Florfenicol-resistant H. parasuis 1614 4 Florfenicol-resistant H. parasuis 1651 8 Ampicillin-resistant S. typhi BYG 9 4 Sulfamethoxazole-resistant S. typhi BYG 21 4 Doxycycline-resistant S. typhi BYG 11 4 Doxycycline-resistant S. typhi BYG 25 8 Doxycycline-resistant S. typhi BYG 31 8 To confirm its biological activity, the agar diffusion method was employed to test the antibacterial activity of F8 against S. aureus and E. coli . We observed that F8 consistently inhibited the growth of the pathogens, displaying distinct inhibition zones (Fig. 2 a). The growth inhibition effect of F8 on S. aureus and E. coli was further assessed (Fig. 2 b, c), and growth curves demonstrated that F8 effectively suppressed S. aureus and E. coli at 4×MIC, 2×MIC, and MIC, notably diminishing the growth of S. aureus at half the MIC concentration. Additionally, we monitored the bacterial viability of S. aureus and E. coli exposed to different concentrations of F8 at various time points (Fig. 2 d, e and Supplementary Fig. 1a, b), and found that F8 at 4×MIC, 2×MIC, and MIC could kill the bacteria within 8 hours and eliminate the majority of bacteria for an extended period. Based on the minimum bactericidal concentration (MBC) and MIC values (Table 1 , Table 2 and Table 3 ), we confirmed F8 as an effective candidate antibacterial agent. To further evaluate the in vivo therapeutic potential of F8, we used a murine sepsis model induced by the intraperitoneal injection of S. aureus to test the therapeutic effect of F8 on bacterial infection. Bacterial colony counts revealed that F8 at 60 mg/kg could effectively reduce bacterial load in the liver, spleen, kidneys, and blood (Fig. 2 f-i). The S. aureus infection group displayed extensive tubular necrosis with an indistinct structure in kidney tissues (cortical and medullary) (Fig. 2 j). Inflammatory cell infiltration centred on granulocytes within the tubules, along with necrosis and conglomeration of inflammatory cells, significant tubular dilation, and focal haemorrhages were also notable in the infected group. Hepatic cells demonstrated vacuolar degeneration and inflammatory cell infiltration, with visible bleeding within vessels. The boundary between the red pulp and white pulp in the spleen was indistinct, with an increase in the number of neutrophils and macrophages within the red pulp. However, in the F8 treatment group, nearly no significant pathological changes were observed. To further characterize the potential of F8 as an antimicrobial agent, we also investigated the effect of F8 on organ damage. Results showed that no notable abnormalities were observed in the liver, kidneys, and intestines in the F8-treated group at 1500 mg/kg, with no significant pathological changes in the spleen, kidneys, and bone marrow (Fig. 3 a-c, j and Supplementary Fig. 2); however, the positive control FLO group exhibited minor vacuolar degeneration and inflammatory cell infiltration in hepatic cells, with mild bleeding visible inside vessels. Kidney structure was abnormal with indistinct boundaries, a disappearance of tubular lumens, and glomerular atrophic degeneration. Extensive fibrosis with slight necrotic degeneration and shedding was observed in the intestinal tissues. Thymic medullary lymphocytes showed relatively loose arrangements with serum exudation, the number of lymphocytes was lower than in the control group, and minor inflammatory cell infiltration was observed within the medulla. The boundary between the red pulp and white pulp in the spleen was indistinct, with increased numbers of neutrophils and macrophages in the red pulp (Fig. 3 j). The ELISA results showed no significant differences in the levels of IL-6, IL-2, and Hsp70 in the blood between the F8 treatment group and the control group (Fig. 3 d-f). As a highly conserved stress protein, Hsp70 significantly increases in expression level when exposed to harmful stimuli or stress factors, playing a crucial role in immune regulation. Compared with the control group, the concentration of Hsp70 in the thymus and spleen of the F8 group showed no significant difference, but significantly increased in the bone marrow (Fig. 3 g-i). In addition, no significant differences in the number of white blood cells (WBCs), neutrophils (Neu), lymphocytes (Lym), red blood cells (RBCs), haemoglobin (HGB), and platelets (PLT) were observed between the F8-treated group and the control group, indicating that F8 has low toxicity to mice (Fig. 3 k-p). In TUNEL staining analysis, the immune organs (thymus, spleen, and bone marrow) treated with F8 did not show intense fluorescence compared with the positive control FLO, indicating fewer instances of cell death (Fig. 3 q). Additionally, F8 exhibited excellent tolerance in a 14-day study of acute toxicity in mice. At a maximum dose of 5000 mg/kg, no abnormal clinical signs were observed (Supplementary Fig. 3a, b). The cytotoxicity of F8 to mammalian cells was also low (Supplementary Fig. 4a-c). Furthermore, we found that F8 has an extremely low haemolysis rate on sheep red blood cells, showing negligible haemolysis even at 256 µM (Supplementary Fig. 5a, b). In conclusion, these results suggest that F8 possesses good pharmacological efficacy in vivo ; however, further studies should be carried out to investigate its pharmacokinetic characters and impacts on a wider range of clinical infections. F8 overcomes resistance and is effective in vivo Bacterial resistance is also an important indicator of the potential clinical application of antimicrobial drugs. To evaluate the development of F8 resistance, we conducted an in vitro study, continuously exposing S. aureus and E. coli to sub-inhibitory concentrations of F8 for 30 consecutive days. Compared with the positive control FLO, the trend of bacteria developing resistance to F8 was lower (Fig. 4 a, b). Subsequently, the inhibitory effect of F8 on induced FLO-resistant S. aureus and FLO-resistant E. coli was also tested, and it was found that F8 significantly enhanced the inhibitory effect on these FLO-resistant bacteria, which was significantly superior to the positive control FLO (Fig. 4 c). These results suggest that F8 holds greater advantages in avoiding bacterial resistance. To further evaluate the in vivo antibacterial action of F8, a mouse sepsis model with FLO-resistant S. aureus was established. Excitingly, the survival curve showed that the survival rate of all mice infected with drug-resistant bacteria was as high as 50% within 72 h after treatment with 60 mg/kg of F8, while under the same conditions, the survival rate of the positive control FLO decreased to 0 within 48 h (Fig. 4 i). This survival analysis showed that the F8 treatment group was able to significantly inhibit drug-resistant bacteria in vivo and had a significant survival advantage. Subsequent evaluation of bacterial load measurements was consistent with the survival rate analysis. After 24 h of F8 treatment, the bacterial load in the liver, spleen, kidneys, and blood showed significant reduction (Fig. 4 d-g), and furthermore, F8 exhibited a considerable inhibitory effect on the invasion of FLO-resistant S. aureus . Moreover, histopathological examinations revealed noticeable tissue necrosis and damage in the FLO-resistant S. aureus group, while F8 treatment significantly alleviated the necrosis and damage in the liver, kidney, and spleen tissues caused by bacterial infection (Fig. 4 h). Overall, our results suggest that F8 effectively alleviates the severity of FLO-resistant S. aureus infection. Discovery of arcB as the antibacterial target of F8 from multi-omics analysis To uncover the potential targets of F8 in its anti- S. aureus activity, we analysed the bacterial changes post-F8 treatment using transcriptomics, proteomics, and metabolomics (Fig. 5 a-d). From transcriptomic analysis, a total of 1,212 differentially-expressed genes (DEGs) (598 up-regulated and 614 down-regulated) showed significant expression patterns before and after F8 treatment (FC 2, p < 0.05) (Fig. 6 a). Kyoto encyclopedia of genes and genomes (KEGG) and gene ontology (GO) analysis revealed that pathways for cellular components (cytoplasmic, membrane, and plastid parts), molecular function (amino acid biosynthesis process, small molecule metabolic process, and oxidoreductase activity), and energy metabolism (arginine biosynthesis, nitrogen metabolism, histidine metabolism, glycolysis, and alanine, aspartate and glutamate metabolism) were down-regulated, whereas activities of RNA processing, RNA metabolic processes, and cellular macromolecule biosynthetic processes were up-regulated (Fig. 6 b-f). In conclusion, these up-regulated DEGs are largely associated with the metabolic disorder and degradation of secondary metabolites, whereas processes like amino acid biosynthesis, nitrogen source metabolism, and glycolysis are significantly suppressed. Proteomic analysis revealed that after F8 treatment, there were 457 differentially-expressed proteins (DEPs) (224 up-regulated and 233 down-regulated), with significantly down-regulated genes mainly concentrated in the pathways of histidine metabolism, nitrogen metabolism, arginine biosynthesis, and alanine, aspartate, and glutamate metabolism (Fig. 7 ). In the metabolomics data, there were 326 different metabolites between the control group and F8 group (Fig. 8 ). KEGG pathway analysis revealed a significant down-regulation in tricarboxylic acid (TCA) cycle and cofactor biosynthesis. Furthermore, after F8 treatment, molecules associated with purine and pyrimidine metabolism, as well as ornithine, pyruvate, aspartate, glutamate, and arginine metabolism were significantly downregulated. Subsequently, multi-omics pathway analysis was utilized to further scrutinize the data across the three omics (Fig. 5 ). In the integrated transcriptomics-proteomics analysis, a strong correlation was observed in nitrogen metabolism, amino acid synthesis, and the stress response. Similarly, in the integrated transcriptomics-metabolomics analysis, amino acid synthesis, nitrogen metabolism, and energy metabolism were significantly downregulated. After conducting KEGG enrichment analysis of significantly down-regulated proteins and metabolites, we noticed a significant down-regulation in arginine metabolism, alanine, aspartate, and glutamate metabolism. Finally, multi-omics analysis suggested that F8 down-regulates the biosynthesis of nitrogen metabolism, arginine biosynthesis, and alanine, aspartate, and glutamate metabolism. Arginine metabolism in bacteria is tightly regulated, and arginine is crucial for cell growth and biomembrane formation 15 , thus, we infer that F8 exerts its antibacterial effects through multifaceted pathways including participation in the arginine degradation metabolic pathway, bacterial cell membranes, and energy metabolism. Based on the multi-omics analysis, we speculate that the antibacterial activity of F8 is associated with multiple pathways, including the arginine degradation metabolic pathway, the bacterial cell membrane, and energy metabolism. Among these, downregulation of nitrogen metabolism and arginine biosynthesis was observed in all three omics datasets, hence, we hypothesize that an important antibacterial target of F8 against S. aureus could potentially involve the arginine degradation metabolic pathway. Within the arginine degradation metabolic pathway, arginine deiminase (arcA), arcB, and carbamate kinase (arcC) are categorized as an arginine deiminase pathway (ADI) 38 . The RT-PCR results are consistent with the transcriptomics data (Fig. 9 c, Supplementary Fig. 6a-i and Supplementary Table 2). Notably, since arcB is involved in arginine degradation and is also indispensable for arginine biosynthesis, studies have shown that arcB levels might limit arginine biosynthesis of S. gordonii 15 . Expression of arcB is also known to be affected following treatment with antibiotics, including gentamicin and polymyxin B 21 , 22 . Therefore, we preliminarily infer arcB as a possible target of F8. ArcB binding by F8 Given that F8 significantly impacts the expression of arcB in multi-omics analysis, we posit that F8 exerts its antibacterial activity by targeting the arcB protein and affecting its function. To further detail the binding mode of F8 and arcB, we searched for the crystal structure of the arcB protein (PDB: 2ksd) in the PDB and performed molecular docking using the SYBYL-X software. We found that F8 is capable of binding with the arcB protein and occupying the binding pocket (Fig. 9 a, b and Supplementary Fig. 7), and that the cyclic structure of F8 nestles into the cavity of the receptor protein, enhancing the tightness of the binding. As shown in Fig. 9 a, F8 binds directly to arcB through hydrogen bonding interactions with LEU-31 of arcB. To further validate that F8 can directly bind to arcB, we expressed the arcB protein and then examined their interactions (Fig. 9 d and Supplementary Fig. 8a). Isothermal titration calorimetry (ITC) measurements further confirmed the predicted interaction between F8 and the active pocket of arcB. The results showed that F8 directly and specifically binds to the arcB protein (Kd = 1.081 ± e − 5 M) (Fig. 9 e, f and Supplementary Fig. 8b, c). Subsequently, differential scanning fluorimetry (DSF) was employed to assess protein thermal stability induced by compound binding to evaluate the interaction. A thermal shift (ΔT m change) was observed when the arcB protein was incubated with F8 compared to the blank control (Fig. 9 g), providing evidence for the binding of F8 to arcB protein. These results collectively demonstrate that F8 can directly bind to arcB with high affinity in vitro . Bactericidal mechanism of F8 Given the wide-ranging regulatory ability of arcB, which is crucial for cell growth and biofilm formation, we hypothesize that F8 may exert its antibacterial effect through bacterial cell membrane damage. To this end, we first examined the bacterial extracellular contents, showing an increasing trend in the concentration of DNA, RNA, and protein at various time intervals after F8 treatment at different concentrations (4×MIC, 2×MIC, and MIC) (Fig. 10 a, b and Supplementary Fig. 9). The reason for this observation may be that F8 can alter bacterial cell membrane permeability, resulting in an increased efflux of these three substances. Alternatively, F8 might have a destructive effect on the bacterial cell membrane structure, resulting in a direct efflux of bacterial contents. Regardless of the reason, the reduction in bacterial DNA, RNA, and protein content can disrupt its normal physiological activities, leading to bacteriostatic or bactericidal effects. Further examination of bacterial cytoplasmic membrane permeability showed that F8 induced increased membrane permeability in S. aureus (Fig. 10 c, d and Supplementary Fig. 10). This demonstrates that F8 can alter bacterial cell membrane permeability or disrupt cell membrane structure, subsequently exerting bacteriostatic or bactericidal effects. Consistent with this, changes in membrane rigidity disrupted the bacterial homeostasis, leading to fundamental metabolic disarray, including the dissipation of proton motive force (PMF) 39 . Thus, we evaluated the ΔpH, a key component of PMF, in S. aureus using the fluorescent probe BCECF-AM 40 . The experiment was conducted by treating the bacteria with 4×MIC of F8. Within 1 h after the medication was added, the ΔpH significantly dissipated (Fig. 10 e, f and Supplementary Fig. 11), which is consistent with the bactericidal effect. This may indicate that changes in bacterial membrane rigidity disrupt the bacterial steady state, causing fundamental metabolic disarray and leading to the dissipation of proton motive force. On the other hand, disruption of membrane homeostasis often contributes to the accumulation of reactive oxygen species (ROS) 41 . Moreover, previous studies have shown that the deletion of arcB in E. coli leads to an increased sensitivity to hydrogen peroxide under aerobic conditions 42 . Our investigations revealed that F8 increased intracellular ROS content (Fig. 10 g, h and Supplementary Fig. 12), which may correspondingly exacerbate membrane damage and further disrupt bacterial homeostasis. Endogenous ROS plays a crucial role in bactericidal activity 43 . F8 is expected to stimulate the production of ROS in various ways, while also inhibiting the activity of arcB, leading to a decrease in antioxidant capacity. This dual approach may lead to more severe oxidative damage, thereby achieving better bactericidal effects and potentially avoiding the resistance effect caused by a single factor. Interestingly, we observed that ATP levels increased in a dose-dependent manner following F8 treatment (Fig. 10 i, j and Supplementary Fig. 13). Such results are in line with previous research, suggesting that bactericidal antibiotics are associated with accelerated respiration 44 . These findings provide convincing phenotypic support for the notion that F8 disrupts bacterial cell membranes and induces a certain degree of oxidative damage. Discussion Antimicrobial drug resistance has evolved into a global health crisis. Antibiotics have been indispensable in the battle against bacteria, saving millions of lives 45 – 48 ; however, the overuse of antibiotics has led to a rapid increase in the number and types of resistant bacteria, particularly the emergence of superbugs, making it crucial to develop new and more effective strategies to treat bacterial infections 5 , 49 , 50 . Nowadays, the development of antibiotics is constantly challenged, resulting in few new antibiotics being introduced into clinical practice 14 . Despite considerable efforts, almost no new antibiotics have been approved for clinical use in recent years, therefore, there is an urgent need for feasible approaches to increase the number of therapeutic options. Based on the structural hybridization method, we determined that F8 is the optimal modifying compound. It further enhanced its antibacterial effect against pathogenic bacteria such as E. coli , S. aureus , S. typhi , P. multocida , and H. parasuis , and imparted measurable activity against the particularly challenging P. aeruginosa . The most notorious drug-resistant bacterium, MRSA, exhibits resistance to a multitude of antibiotics and is spreading at an alarming rate worldwide. Currently, there is a significant lack of antibiotics capable of combating MRSA 51 . Our research findings indicate that F8 has a strong inhibitory effect on MRSA, with a MIC value of 8 µM, consistent with the results observed in the standard strains. Polymyxins, particularly polymyxin B, have become the last line of therapy against multi-drug resistant gram-negative bacteria 52 . However, the rapid development of resistance has led to the emergence of more and more polymyxin B resistant bacteria. The MIC value of F8 against polymyxin B-resistant E. hormaechei is 16 µM, which is a promising lead compound for resisting polymyxin B resistance. Sulfamethoxazole is reported to be one of the most widely used sulfonamide antibiotics in the world; however, the increasingly drug resistance greatly limits its utility 53 . The MIC value of F8 against sulfamethoxazole-resistant S. typhi only 4 µM. In addition, F8 also showed excellent bactericidal effects on FLO-resistant S. suis , FLO-resistant H. parasuis and doxycycline-resistant S. typhi . Recently, many structurally-hybrid antibiotics have entered trials, but only a few have reportedly entered clinical trials 54 – 56 . Structural hybridization has obvious advantages, but the complexity of molecular structure, difficult-to-handle chemical synthesis, and the rigorous work required to establish the mode of action and benefits of structural hybridization compared to conventional drugs make structural hybridization methods daunting 25 , 57 , 58 . In this study, we first chose structural hybridization to perform at the β-hydroxy position of the bioactive scaffold. The hydroxyl group offers significant advantages for the fusion of small molecules, enabling the generation of novel structures with straightforward synthetic routes and conforming to easily-synthesizable mechanisms. The increase in molecular weight is a major limitation of the structural hybridization approach. In fact, the permeability barriers caused by a high molecular weight are the primary reasons for the limited antibacterial activity exhibited by most antibiotics 25 . Given the bacterial cell wall and membrane structure, antibacterial compounds with a high molecular weight (> 600 g/mol) do not pass through non-selective protein channels, restricting uptake by bacteria to receptor-mediated endocytosis or passive diffusion 25 , 59 . In this study, we have selected the simple, low-molecular-weight, and easily-hybridisable FLO as the primary bioactive scaffold. Targeting the key bacterial ribosome 50S subunit PTC region of FLO, we employed a computer-aided drug design to develop a modular synthetic route, and investigated a broad range of structural variations. We discovered that, in general, short fragments have higher activity compared to their homologs. Smaller spatial conformations within the binding pocket reduce the level of inherent self-collision incidences the ligand may experience, leading to increased stability in binding. This also aligns with the lower molecular weight required for cell permeability. Although the conceptualization of an ideal blueprint may seem trivial, the discovery of F8 undoubtedly reaffirms that structural hybridization and chemical synthesis remain powerful means to supplement our arsenal of antibiotics. F8 slows down the development of drug resistance and exhibits significant in vitro and in vivo antibacterial effects on resistant bacteria, which means that compared to traditional antibiotics, F8 has a novel mechanism of action or exerts its antibacterial effect through the combination of several mechanisms of action. Although the structural hybridization strategy is crucial and effective, the resulting compounds are ultimately influenced by the same type of resistance mechanism, which evolves and spreads in response to the resistance mechanism of previously-used compounds. In contrast, F8, due to its novel mechanism of action, bypasses existing drug resistance mechanisms and exhibits excellent in vitro and in vivo activity against drug resistance. Multi-omics analysis indicates that F8 affects multiple metabolic routes, including nitrogen metabolism, arginine biosynthesis, and alanine, aspartic acid, and glutamate biosynthesis. Arginine metabolism is strictly regulated in bacteria and arginine is essential for cell growth and biofilm formation 15 . In the arginine catabolism pathway, arcA, arcB, and arcC are classified as the arginine deiminase pathway 38 . Research indicates that in S. aureus , the activation of the arginine deiminase pathway confers resistance to vancomycin 60 , which can explain why S. aureus showed a lower resistance to F8 in the process of continuous passage. Meanwhile, in ITC and DSF assays, we discovered and validated the target protein arcB of F8. This finding is consistent with our multi-omics analysis. As ArcB is involved in arginine degradation metabolism biosynthesis 17 – 20 , the report by Jakubovics et al. 15 suggests that arcB levels may limit the biosynthesis of arginine in S. gordonii . Our research results also indicate that F8 can alter bacterial cell membrane permeability or disrupt the cell membrane structure, thus exerting an antibacterial or bactericidal effect. The expression of arcB is also known to be affected after treatment with antibiotics, including gentamicin and polymyxin B, suggesting that cell membrane stress influences ArcB activity 21 , 22 . In addition, our research indicates that F8 increases the content of intracellular ROS, which may correspondingly exacerbate membrane damage and further disrupt bacterial homeostasis. Cells are sensitive to ROS, which is composed of oxygen and its derivatives (peroxides, superoxides, and hydroxyl radicals), as they can cause damage to bacterial DNA, proteins, and lipids 61 . Therefore, bacteria have evolved methods to clear ROS, reducing them to less harmful byproducts 62 . Several studies have determined that arcA/arcB play a role in this ROS resistance, and in fact, the deletion of arcA or arcB has been shown to lead to increased sensitivity to hydrogen peroxide under aerobic conditions in E. coli 42 . It has been demonstrated that the arcA/arcB can regulate the synthesis and transport of proteins and amino acids, thereby affecting the adaptability of E. coli under ROS stress 42 , thus, it is hypothesized that bactericidal antibiotics lead to metabolic instability and toxic ROS formation as part of their lethal action 41 , 44 , 63 – 65 . Considering the potential significant role of arcB in antimicrobial activities, further extensive work is required focusing on the study of arcB, including target knockout and key amino acid residue mutations, among other approaches. The role of F8 in arcB (a simple and immutable target) has expanded our understanding of antibiotic development to avoid drug resistance, and has guided the rational design of compounds with long-term clinical lifespan. In summary, our research findings suggest that F8 is a broad-spectrum antibacterial small molecule compound, exhibiting potent in vivo and in vitro antibacterial activity against both standard and drug-resistant strains, with a low propensity for the development of resistance. This mechanism of antibacterial action is thought to be based on competitive binding with arcB, thereby activating multiple pathways. Together, our results suggest that F8 may be a promising candidate drug for developing new antibiotic formulations to combat antibiotic-resistant bacteria-associated infections. Methods Strains, media, and growth conditions E. coli (ATCC 25922), S. aureus (ATCC 29213), S. typhi (CICC 110420), P. aeruginosa (ATCC 9027), B. subtilis (CMCC 63501), E. faecalis (ATCC 29212), P. multocida (ATCC 43137), A. pleuropneumoniae (ATCC 27088), S. suis (CVCC 606), H. parasuis (ATCC 19417), methicillin-resistant S. aureus (B1-1), polymyxin B-resistant E. hormaechei (wb 4), FLO-resistant S. suis (1136, 1194, 1197, 1655, 1658, 1669), FLO-resistant H. parasuis (1565, 1614, 1651), ampicillin-resistant S. typhi (BYG 9), sulfamethoxazole-resistant S. typhi (BYG 21) and doxycycline-resistant S. typhi (BYG 11, BYG 25, BYG 31) were used in this study. Trypticase soy broth (TSB), Mueller-Hinton broth (MHB), Muller-Hinton agar (MHA), LB, and LB nutrient agar were bought from Haibo Biotechnology (Qingdao, China). Cells were grown at 37°C on a rotating shaker at 300 rpm in flasks, or at 900 rpm in plate shakers. Molecular docking studies Preparation of the binding site: The X-Ray crystal structure of the 50S subunit PTC during translation termination (the PTC region) with the PDB code 6c4h was used for the virtual screening. The binding pockets were confirmed using SYBYL-X Suite with default parameters. Hydrogen atoms were added to the structure, and considerations were made regarding correct orientation of Asn and Gln sidechains, ligands, and protein charges. All waters were removed except for the iron/sulphur complex. A diverse subset of the compound library (i.e., SPECS with 1,084,348 small-molecule compounds and a Topscience compound library with 16,594 natural products) was prepared to generate three-dimensional configurations by Surflex for searching SYBYL-X with all options set as default. Docking projects were conducted using Surflex-Dock GeomX in the SYBYL-X Suite. Scoring calculations were calculated by CScore in the SYBYL-X Suite. Design and synthesis of broad-spectrum antibacterial small molecule compounds The synthesis work was performed as follows: N,N-dimethylformamide (DMF, 20 mL) was added to a round-bottom flask, followed by the addition of 3-(1-Piperidinyl)propanoic acid (CAS: 26371-07-3, 2 g) until complete dissolution. Subsequently, 4-dimethylaminopyridine (DMAP, 1.5 g), 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride (EDC, 3 g), and FLO (2,2-Dichloro-N-[(1R,2S)-3-fluoro-1-hydroxy-1-(4-methylsulfonylphenyl)propan-2-yl]acetamide) (3 g) (Longxiang, China) were sequentially added. The reaction mixture was stirred at 25°C for 24 h. The output was monitored by thin-layer chromatography (TLC) (hexyl hydride/ethyl acetate [hexane/EtOAc] = 1/1, retention factor [Rf] = 0.25) and visualized by a 5% vanillin sulfuric acid/ethanol solution. The product was purified by column chromatography on a silica gel (hexane/EtOAc = 2/1). Other derivatives, such as F1, F2, F3, F4, F6, F7, F11, F13, F14, and F15, were synthesized using the same procedure. Broad-spectrum antibacterial small molecule compounds were characterized by various techniques, including MS, 1 H-NMR, and 13 C-NMR (Fig. 1 d, Supplementary Table 1 and Supporting information). In vitro susceptibility The MICs of antibiotics were determined by the broth microdilution method following the Clinical and Laboratory Standards Institute (CLSI) guidelines. Briefly, single bacterial colonies were cultured in MHB at 37°C at 220 rpm for 8–12 h. Subsequently, the potential lead candidates or other antibiotics were diluted two-fold in MHB and mixed with an equal volume of bacterial suspensions in MHB containing approximately 1 × 10 6 CFU/mL in a clear UV-sterilized 96‐well microtiter plate. The plate was placed in the incubator for 18 h at 37°C, then the MIC values were read. MIC values were defined as the lowest concentrations of antibiotics with no visible growth of bacteria. Growth curves of bacteria A single colony of E. coli ATCC 25922 or S. aureus ATCC 29213 was selected and inoculated into MHB for cultivation. The cultures were incubated at 37°C with shaking at 200 rpm for overnight growth. The overnight culture was standardized to a 0.5 McFarland turbidity standard. It was then diluted at 1:100 in MHB, and adjusted to approximately 1 × 10 6 CFU/mL. Different concentrations of FLO or F8 were added to a 96-well microplate and mixed with an equal volume of bacterial dilution, then sealed with a plate lid. The plate was incubated at 37°C, and the wavelength of 600 nm was measured every 1 h using an Infinite M200 Microplate reader (Tecan). Time-kill curves A single colony of E. coli ATCC 25922 or S. aureus ATCC 29213 was selected and inoculated into MHB for cultivation. The cultures were incubated at 37°C with shaking at 200 rpm for overnight growth. The overnight cultured bacteria were diluted at 1:100 in MHB and adjusted to approximately 1 × 10 6 CFU/mL. Different concentrations of FLO and F8 (4 × MIC, 2 × MIC, MIC, 1/2 × MIC) were added to the diluted bacterial suspension. The cultures were then incubated at 37°C with shaking at 200 rpm. After incubation for 30 min, 1, 2, 4, 8, and 24 h, 100 µL aliquots were taken, serially diluted 10-fold, then inoculated on MHA plates (with the inclusion of three parallel plates), and the colony forming units (CFU) were calculated after incubation for 24 h at 37°C. Membrane integrity assay Overnight cultures of the bacterial fluid were centrifuged and resuspended in 0.01 mol/L PBS (pH 7.4), and the bacterial suspensions were adjusted to an OD600 nm of approximately 0.5. Subsequently, FLO or F8 was added at a final concentration of 4 × MIC, and the bacterial suspensions were incubated at 37°C for 2 h without light. The suspensions were then incubated with propidium iodide (PI, Thermo Scientific, P1304MP) at a final concentration of 10 nmol/L for 20 min before the fluorescence values were quantified using an Infinite M200 Microplate reader (Tecan) at an excitation wavelength of 535 nm and an emission wavelength of 615 nm. Membrane permeability measurement Overnight cultures of the bacterial fluid were centrifuged and resuspended in 0.01 mol/L PBS (pH 7.4), and the bacterial suspensions were adjusted to approximately 1 × 10 6 CFU/mL. The diluted bacterial cultures were supplemented with different concentrations of FLO and F8 (4 × MIC, 2 × MIC, MIC) and incubated at 37°C with shaking at 150 rpm. After incubation for 2, 4, and 8 h, the bacterial suspensions were collected, and the supernatant was obtained by centrifugation at 12,000 rpm for 2 min. The DNA, RNA, and protein contents were measured using a Nano Drop microspectrophotometer. ΔpH measurement Overnight cultures of the bacterial fluid were centrifuged and resuspended in 0.01 mol/L PBS (pH 7.4), and the bacterial suspensions were adjusted to an OD600 nm of approximately 0.5. Subsequently, 10 µL of FLO or F8 (final concentration of 4 × MIC) was added, followed by the addition of a pH-sensitive fluorescent probe, BCECF-AM, at a final concentration of 10 µmol/L. The mixture was then incubated at 37°C and fluorescence measurements were taken every 5 min using the Infinite M200 Microplate reader (Tecan) with an excitation wavelength of 488 nm and an emission wavelength of 535 nm. ATP determination Extracellular and intracellular ATP levels were determined using the Enhanced ATP Assay Kit (Beyotime, Cat. No. S29213). Overnight cultures of the bacterial fluid were centrifuged and resuspended in 0.01 mol/L PBS (pH 7.4), and the bacterial suspensions were adjusted to an OD600 nm of approximately 0.5. After treatment with 4 × MIC of FLO or F8 for 2 h, the bacterial cultures were centrifuged at 12,000 rpm for 5 min at 4°C, and the supernatant was collected to determine extracellular ATP levels. Meanwhile, the bacterial precipitate was lysed with lysozyme, centrifuged, and the supernatant was prepared to determine the intracellular ATP levels. The assay solution was added to a 96-well plate and incubated for another 5 min at room temperature. The supernatants were added to the well and mixed quickly before recording in the model of luminescence using the Infinite M200 Microplate reader (Tecan). ROS measurement Overnight cultures of the bacterial fluid were centrifuged and resuspended in 0.01 mol/L PBS (pH 7.4), and the bacterial suspensions were adjusted to an OD600 nm of approximately 0.5. The fluorescent probe, 2’,7’-dichlorofluorescein diacetate (DCFH-DA) (10 µmol/L), was used to detect ROS accumulation in the bacterial fluids after FLO or F8 treatment, following the manufacturer's instructions (Beyotime, Cat. No. S0033). Briefly, DCFH-DA was added to the bacterial suspension and incubated at 37°C for 20 min. After washing three times with 0.01 mol/L PBS, 190 µL of the bacterial suspension was added to a 96-well microplate and mixed with 10 µL of FLO or F8 (final concentration of 4 × MIC). After incubation for 2 h, the fluorescent values were measured using the Infinite M200 Microplate reader (Tecan) with an excitation wavelength of 488 nm and an emission wavelength of 525 nm. Transcriptomics Untreated and FLO- or F8-treated S. aureus ATCC 29213 were used for transcriptomics studies. FLO or F8 (final concentration of 4 × MIC) was added to the bacterial suspension and incubated for 2 h. The bacterial cultures were then washed three times with 0.01 mol/L PBS (pH 7.4) and centrifuged at 8,000 rpm for 10 min at 4°C to collect the bacterial precipitates. RNA-Seq was performed using the Illumina platform at Shanghai Majorbio Bio-pharm Technology Co., Ltd. (Shanghai, China). Total RNA was extracted from the samples, and the concentration and purity of the extracted RNA were determined using Nanodrop 2000. The integrity of the RNA was assessed by agarose gel electrophoresis, and the RNA integrity was further evaluated using the Agilent 2100 Bioanalyzer. Genes with adjusted p < 0.05, identified by DESeq, were considered differentially expressed. According to the KEGG analysis, differentially-significant genes were assigned to different functional groups. The data was uploaded to the Majorbio cloud platform ( https://cloud.majorbio.com ) after the database search for data analysis. Metabolomics Untreated and FLO- or F8-treated S. aureus ATCC 29213 were used for metabolomics studies. FLO or F8 (final concentration of 4 × MIC) was added to the bacterial suspension and incubated for 2 h. The bacterial cultures were then washed three times with 0.01 mol/L PBS (pH 7.4) and centrifuged at 8,000 rpm for 10 min at 4°C to collect the bacterial precipitates. 50 mg bacterial precipitates were accurately weighed, and the metabolites were extracted using a 400 µL methanol:water (4:1, v/v) solution with 0.02 mg/mL L-2-chlorophenylalanin as an internal standard. The mixture was allowed to settle at -10°C, and was treated using the high throughput tissue crusher, Wonbio-96c (Shanghai Wanbo Biotechnology Co., LTD), at 50 Hz for 6 min, followed by sonication at 40 kHz for 30 min at 5°C. The samples were placed at -20°C for 30 min to precipitate the proteins. After centrifugation at 13,000 g at 4°C for 15 min, the supernatant was carefully transferred to sample vials for LC-MS/MS analysis. The instrument platform for this LC-MS analysis was UHPLC-Q Exactive HF-X system of Thermo Fisher Scientific. The data was uploaded to the Majorbio cloud platform ( https://cloud.majorbio.com ) after the database search for data analysis. Proteomics Untreated and FLO- or F8-treated S. aureus ATCC 29213 were used for proteomics studies. FLO or F8 (final concentration of 4 × MIC) was added to the bacterial suspension and incubated for 2 h. The bacterial cultures were then washed three times with 0.01 mol/L PBS (pH 7.4) and centrifuged at 8,000 rpm for 10 min at 4°C to collect the bacterial precipitates. The bacterial precipitates were removed and put on ice. An appropriate amount of protein lysate was then added, (8 M urea, 1% SDS) and the precipitates were sonicated for 2 min at a low temperature, then split for 30 min. After centrifugation at 12,000g at 4°C for 30min, the concentration of protein supernatant was determined through the Bicinchoninic acid (BCA) method using the BCA Protein Assay Kit (Pierce, Thermo, USA). Protein samples at 100 µg, TEAB (Triethylammonium bicarbonate buffer), and TCEP (tris (2-carboxyethyl) phosphine) was combined for reaction for 60 min at 37°C. IAM (Iodoacetamide) was added to the final concentration at 40 mM, and reacted for 40 min at room temperature under dark conditions. A certain percentage (acetone: sample v/v = 6:1) of pre-cooled acetone was added to each sample, then allowed to settle for 4 h at -20°C. After centrifugation for 20 min at 10,000 g, the sediment was collected, and 100 µL of 100mM TEAB solution was added. Finally, the mixture was digested with Trypsin overnight at 37°C, and added at a 1:50 trypsin-to-protein mass ratio. Trypsin-digested peptides were analysed using online nano flow liquid chromatography tandem mass spectrometry performed on an EASY-nLC 1200 system (Thermo, USA) connected to a Q Exactive HF-X quadrupole orbitrap mass spectrometer (Thermo, USA) through a nanoelectrospray ion source. The data was uploaded to the Majorbio cloud platform ( https://cloud.majorbio.com ) after the database search for data analysis. Protein expression and purification Plasmids pET28a-AcrA and pET28a-AcrB were constructed after codon optimization in E. coli . The plasmid was amplified in DH5α-competent cells, then transformed into BL21 (DE3)- competent cells. After overnight incubation, single colonies were picked and transferred to LB medium. The cultures were shaken at 37°C for 4–6 h until the OD600 reached approximately 0.8, before induction of fusion protein expression by 0.1 mM isopropyl thio-β-d-galactoside (IPTG) at 37°C for 5 h. Then, the cells were harvested by centrifugation and purified. Briefly, the cells were sonicated in PBS, the homogenate was centrifuged (12,000 g, 10 min, 4°C), and the pellet was collected. The supernatant was passed through the Ni-NTA column at a flow rate of 1.0 mL/min by a peristaltic pump at 4°C overnight, then passed onto the binding buffer (50 mM sodium phosphate buffer pH 8, 500 mM NaCl and 10 mM imidazole). The bacterial proteins were then removed with elution buffer A, which contained 30 mM imidazole (50 mM sodium phosphate buffer pH 8, 500 mM NaCl, and 30 mM imidazole) in 20 mL. Next, the column was eluted with elution buffer B and 200 mM imidazole (50 mM sodium phosphate buffer pH 8, 500 mM NaCl, and 200 mM imidazole) for 20 mL to obtain the target protein. The crude product was washed with PBS via a 10 kDa millipore centrifugal ultrafiltration tube to remove imidazole. The fractions of pure product, total protein, supernatant, and precipitate were then analysed by SDS-PAGE and WB using an anti-His tag antibody (AE003, ABclonal, China). Real-time quantitative polymerase chain reaction (RT-qPCR) Cellular total RNA was extracted using the RNA Isolater Total RNA Extraction Reagent (Vazyme, Cat. No. RC112-01), following the manufacturer's instructions. Complementary DNA (cDNA) was synthesized using HiScript II Q Select RT SuperMix for qPCR with gDNA wiper (Vazyme, Cat. No. R233-01) in a total volume of 10 µL. RT-qPCR was performed using 2X Universal SYBR Green Rapid qPCR Mix (Abclonal, Cat. No. RK21203) with 100 ng of cDNA and 5 nM primer pairs at a time. The results were monitored using the CFX96 Real-Time PCR Assay System (Bio-Rad, USA). Supplementary Table 2 lists all primers used in the quantitative PCR. Isothermal titration calorimetry (ITC) To assess the interaction between the ligand and the protein, ITC experiments were conducted using MicroCal ITC at 25°C. All titrations were performed at 25°C while stirring at 300 rpm in PBS. A control experiment of titrant into buffer was performed to account for the heat of dilution. All titrations were repeated at least three times with similar results. For ligand-protein titrations, an approximate protein concentration of 50 µM was used. The concentration of the ligand is approximately ten times higher than that of the protein. All ITC experiments were carried out and analysed using Launch NanoAnalyze Software. Differential scanning fluorimetry (DSF) For the DSF experiments, 20 µL samples were prepared in duplicate using 100 µM of protein and a compound concentration of 50 µM. The samples were heated from 20 to 95°C with increments of 1°C/min before incubation for 20 min, and fluorescence was measured at each step in nanoDSF (Nano Temper Prometheus NT.48, Germany). The change in melting temperature (T m ) values was calculated and recorded by the instrument. Data analysis and image generation were performed using PR. ChemControl Software. Haemolytic activity Sterile defibrinated sheep haemocytes were washed three times with PBS, then 100 uL of 8% haemocytes was added to 100 uL of FLO or F8 at different concentrations (0, 1, 2, 4, 8, 16, 32, 64, 128, 256, and 512 µM). Meanwhile, 0.2% Triton X-100 and PBS were used as positive and negative controls, respectively. The solution was placed in a 96-well microtiter plate and was incubated at 37°C for 1 h before centrifugation at 3,000 g for 10 min. Then, 100 µL of the supernatant was taken, and its absorbance was determined at 576 nm by an Infinity M200 Microplate reader (Tecan). Cytotoxicity The cytotoxicity of FLO or F8 on pk15, Vero, and L-02 cells was estimated using the CCK-8 Cell Proliferation and Cytotoxicity Assay Kit (CCK-8, Solarbio, Cat. No. CA1210). Cells were seeded, counted, and evenly distributed into 96-well microtiter plates at a cell density of 10 5 cells per well, and incubated for 1 day at 37°C and 5% CO 2 to promote cell growth. Subsequently, cells were treated with different concentrations of FLO or F8, with six replicates for each condition. After incubating at 37°C and 5% CO 2 for 24 h, 10µL of CCK-8 solution was added to each well, then further incubated for 2 h. The absorbance was then measured at 450 nm using an Infinite M200 Microplate reader (Tecan). Resistance-development studies A single colony of E. coli ATCC 25922 or S. aureus ATCC 29213 was picked and inoculated into MHB for overnight cultivation. Overnight cultures were inoculated in fresh MHB containing 1/2, 1, 2, and 4 × MIC of FLO or F8. The bacterial cultures were then incubated at 37°C for 24 h at 200 rpm with continuous shaking. Subsequently, the MIC of bacteria from the second-highest concentrations with visible growth (OD600 nm ≥ 0.3) was determined by broth microdilution in fresh MHB media containing different concentrations of FLO or F8. The cultures were serially passaged for 30 days. Animal studies All experimental procedures were conducted in accordance with animal welfare guidelines, and were previously approved by the Animal Welfare and Ethics Committee of the Huazhong Agricultural University Wuhan, China (approval permit numbers: 202311010007 and 202311010008). All specific pathogen-free (SPF) Kunming mice (6–7 weeks old, weighing approximately 25 g) and SPF C57BL/6 mice (6–7 weeks old, weighing 18 ± 2 g) were purchased from Hubei Provincial Laboratory Animal Center (Wuhan, China). Animals were housed under standard humidity (50 ± 10%), temperature (25 ± 2°C), and light-dark cycle (12 h each) conditions with free access to food and water. To study the toxicity of FLO and F8, SPF Kunming mice were randomly divided into three groups and weighed once a day. Each group was given a dose of 1,500 mg/kg of body weight (bw) of FLO or F8 for seven consecutive days, and their health conditions were observed daily. The control mice were given the same volume of solvent. These doses did not cause any deaths and were considered safe in this experiment. The mice were euthanized and dissected seven days later, and the blood, spleen, kidneys, thymus, liver, small intestine, and femur were removed and collected for further analysis. For acute toxicity assays of FLO and F8, survival and mortality status were recorded after a single administration of 5,000 mg/kg bw, followed by 14 days of observation of the surviving mice once a day. Mouse systemic infection study To evaluate the anti-infective effects of FLO and F8, a mouse systemic infection model was established. Briefly, the experiments were performed using SPF C57BL/6 mice (6–7 weeks old, weighing 18 ± 2 g), and the mice were infected by intraperitoneal injection with S. aureus ATCC 29213 at a dose of 5 × 10 6 CFU, or a pre-induced FLO-resistant S. aureus suspension (n = 6). One hour after the infection, mice were administered a dose of 60 mg/kg bw of either FLO or F8. The control mice were given an equivalent volume of the solvent. The health status of the mice was observed, and once an infected mouse died, the blood, liver, spleen, and kidneys were collected for subsequent analysis. At 24 h post-infection, surviving mice were euthanized by cervical dislocation, and the blood, liver, spleen, and kidneys were collected for bacterial CFU analysis and histopathological evaluation. Furthermore, the same mouse mice were given a single dose of FLO or F8 at a dose of 60 mg/kg bw, and the survival status of the mice was observed within 72 h (n = 10). The control mice were given the same volume of solvent. Histopathology After decapitation, the tissues were immediately fixed in 4% paraformaldehyde in phosphate buffer and stored at 4°C. For paraffin embedding, the organs were washed and dehydrated through a series of graded ethanol baths, followed by embedding in paraffin wax. Serial sections of 5 µm thickness were cut using a microtome and stained with haematoxylin-eosin (H&E). Sections were observed using a light microscope. TUNEL reaction Serial sections of 5 µm thickness were cut using a microtome, and sections were deparaffinized and rehydrated. Proteins were digested by placing tissue sections in 20 mg/mL of proteinase K (Merck, Germany) and incubated at 37°C for 15 min. Endogenous peroxidase was inactivated with 3% H 2 O 2 in methanol for 10 min at room temperature, then the sections were incubated with 50 µL of the TUNEL reaction mixture at 37°C for 1 h. They were protected from light, rinsed three times with PBS, and visualized under a fluorescence microscope with excitation wavelengths in the range of 450–500 nm and detection wavelengths in the range of 515–565 nm (green). Enzyme-linked immunosorbent assay (ELISA) The tissues were weighed and homogenized in PBS. After centrifugation at 3,000 rpm for 30 min, the supernatant was collected to quantify IL-6, IL-2, and Hsp70 levels with ELISA kits (MSKBIO, China), used according to the instructions. Statistical analyses Each reaction was performed in triplicate, and the results are expressed as the mean ± standard deviation (SD). p < 0.05 was considered statistically significant (not significant [n.s], 0.01 ≤ p < 0.05 [*], p < 0.01 [**]). Statistical analyses and graphical presentations were performed using GraphPad Prism 8 (GraphPad Prism Inc., San Diego, CA, USA). Abbreviations arcB ornithine carbamoyltransferase arcA arginine deiminase arcC carbamate kinase ADI arginine deiminase pathway CADD computer assisted drug design CFU colony forming units DSF Differential Scanning Fluorimetry DBD drug binding domain DEGs differentially expressed genes DEPs differentially expressed proteins DCFH-DA 2’,7’-dichlorofluorescein diacetate ELISA enzyme-linked immunosorbent assay FLO florfenicol HGB hemoglobin H&E hematoxylin-eosin ITC Isothermal Titration Calorimetry Lym lymphocyte MIC minimum inhibitory concentration MBC minimum bactericidal concentration MRSA methicillin-resistant S. aureus Neu neutrophil PTC peptidyl transferase center PLT platelet PMF proton motive force PI propidium iodide RBCs red blood cells ROS reactive oxygen species SPF specific pathogen free WBCs white blood cells. Declarations Data availability The data that support the findings of this study are available from the corresponding author on request. Source data are provided with this paper. Acknowledgements This work was supported by the National Key Research and Development Program of China (2018YFC1603005), National Natural Science Foundation of China (NSFC) (32072925), and Fundamental Research Funds for the Central Universities (2662020DKPY020). Author contributions Jin Feng conducted in vitro and in vivo antibacterial experiments and multi-omics analysis experiments. Youle Zheng synthesized small molecule compounds. Youle Zheng and Wanqing Ma conducted protein expression and purification experiments. Jin Feng conducted and analysed results from all additional experiments. Yindi Xu, Defeng Weng and Zhifang Wang provided reagents. Xu Wang supervised the research, coordination and strategy. All authors provided critical revisions and approved the final manuscript. 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Supplementalfigures26112023.docx Supplemental Fig. 1-13 Supportinginformation26112022.docx Supporting information Cite Share Download PDF Status: Under Review Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3667988","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":254919973,"identity":"445738b7-341d-45dd-8e22-5386d7d9ca0f","order_by":0,"name":"Xu 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Wang","email":"","orcid":"","institution":"[email protected]","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Zhifang","middleName":"","lastName":"Wang","suffix":""}],"badges":[],"createdAt":"2023-11-26 15:11:04","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3667988/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3667988/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":48154080,"identity":"3a72c23c-2660-448d-bbb9-b707a3d66283","added_by":"auto","created_at":"2023-12-13 20:06:49","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1746995,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDiscovery of F8. a,\u003c/strong\u003e Simulated image of the 4Å drug-binding domain (DBD) of FLO in the peptidyl transferase centre (PTC) of the 50s subunit generated by PyMOL software (PDB:6c4h), with residues GLU-236, A-2590, and C-2606 displayed in magenta. \u003cstrong\u003eb,\u003c/strong\u003e Simulated image of the 4Å DBD of F8 in the PTC of the 50s subunit produced by PyMOL software, showing residues U-2585, U-2586, and A-2590 in green. \u003cstrong\u003ec,\u003c/strong\u003e SYBYL simulation docking between 12 hybrid molecular structures and the PTC region of the 50s subunit. \u003cstrong\u003ed,\u003c/strong\u003e Structures of compounds generated based on structural hybridization strategy.\u003c/p\u003e","description":"","filename":"Fig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-3667988/v1/c17b8c18f59eb94f615082c7.png"},{"id":48153100,"identity":"218e39b3-6e85-4dbe-b62d-17b73103cdbf","added_by":"auto","created_at":"2023-12-13 19:58:49","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1840328,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAntimicrobial activity of F8 \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vitro\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vivo\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e. a,\u003c/strong\u003eAgar diffusion assay of F8 against \u003cem\u003eS. aureus\u003c/em\u003e and \u003cem\u003eE. coli\u003c/em\u003e. \u003cstrong\u003eb, c,\u003c/strong\u003e Growth curves of \u003cem\u003eS. aureus\u003c/em\u003e and \u003cem\u003eE. coli\u003c/em\u003ecultured with F8 at 1/2 × MIC concentration or positive control FLO. \u003cstrong\u003ed, e,\u003c/strong\u003eKill-time curves of \u003cem\u003eS. aureus\u003c/em\u003e and \u003cem\u003eE. coli\u003c/em\u003e with different concentrations of F8. \u003cstrong\u003ef–i,\u003c/strong\u003e Bacterial load in the blood, liver, spleen, and kidney in the different treatment groups in a mouse \u003cem\u003eS. aureus\u003c/em\u003e bacteraemia model (n = 6). ns, not significant, *, \u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05, **, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001. \u003cstrong\u003ej,\u003c/strong\u003eHistopathological assessment of the liver, spleen, and kidney in the different treatment groups in a mouse \u003cem\u003eS. aureus\u003c/em\u003e bacteraemia model. Scale bar, 100 μm.\u003c/p\u003e","description":"","filename":"Fig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-3667988/v1/86e1a701475b23d92588dbf7.png"},{"id":48154081,"identity":"4bd721bd-a5dd-4571-88fb-831e9605e20d","added_by":"auto","created_at":"2023-12-13 20:06:50","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":3154596,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSafety evaluation of F8. a,\u003c/strong\u003e Record of mouse body weight after treatment with F8 at a dose of 1,500 mg/kg or positive control FLO (n = 6). \u003cstrong\u003eb,\u003c/strong\u003e Spleen index. \u003cstrong\u003ec,\u003c/strong\u003e Thymus index. \u003cstrong\u003ed,\u003c/strong\u003e Concentration of IL-6 in serum. \u003cstrong\u003ee,\u003c/strong\u003eConcentration of IL-2 in serum.\u003cstrong\u003e f,\u003c/strong\u003e Concentration of Hsp70 in serum. \u003cstrong\u003eg–i,\u003c/strong\u003eConcentration of Hsp70 in the spleen, thymus, and bone marrow. \u003cstrong\u003ej,\u003c/strong\u003eHistopathological assessment of the spleen, thymus, and bone marrow. Scale bar, 100 μm. \u003cstrong\u003ek–p,\u003c/strong\u003e Count of white blood cells (WBCs), neutrophils (Neu), lymphocytes (Lym), red blood cells (RBCs), haemoglobin (HGB), and platelets (PLT) in different groups. ns, not significant, *, \u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05, **, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001. \u003cstrong\u003eq,\u003c/strong\u003e Apoptotic cells in the spleen, thymus, and bone marrow in different groups (TUNEL staining, under a fluorescence microscope at 200×).\u003c/p\u003e","description":"","filename":"Fig.3.png","url":"https://assets-eu.researchsquare.com/files/rs-3667988/v1/f697b0259ba582f323e186f8.png"},{"id":48153102,"identity":"693c489f-ab5e-4286-905b-fc70e21441f8","added_by":"auto","created_at":"2023-12-13 19:58:50","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1688645,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eF8 overcomes resistance and is effective \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vivo\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e. a,\u003c/strong\u003eThe development of drug resistance to F8 over 30 days of consecutive subculturing. \u003cem\u003eS. aureus\u003c/em\u003e ATCC 29213 is sub-cultured daily under sub-inhibitory concentrations of F8 or positive control FLO. \u003cstrong\u003eb,\u003c/strong\u003e The development of drug resistance to F8 over 30 days of consecutive subculturing. \u003cem\u003eE. coli\u003c/em\u003e ATCC 25922 is sub-cultured daily under sub-inhibitory concentrations of F8 or positive control FLO. \u003cstrong\u003ec,\u003c/strong\u003e Inhibition of flurofenicol-resistant \u003cem\u003eS. aureus\u003c/em\u003e and flurofenicol-resistant \u003cem\u003eE. coli\u003c/em\u003e by different concentrations of F8. \u003cstrong\u003ed–g,\u003c/strong\u003e Bacterial load in the blood, liver, spleen, and kidneys of different treatment groups in a mouse model of \u003cem\u003eS. aureus\u003c/em\u003e bacteraemia (n = 6). ns, not significant, *, \u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05, **, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001. \u003cstrong\u003eh,\u003c/strong\u003eHistopathological assessment of the liver, spleen, and kidneys in different treatment groups in a mouse model of flurofenicol-resistant \u003cem\u003eS. aureus\u003c/em\u003e bacteraemia. Scale bar, 100 μm. \u003cstrong\u003ei,\u003c/strong\u003e Survival curves of mice in an anti-\u003cem\u003eS. aureus\u003c/em\u003estudy (n = 10).\u003c/p\u003e","description":"","filename":"Fig.4.png","url":"https://assets-eu.researchsquare.com/files/rs-3667988/v1/9b0550cb160da38e955c874b.png"},{"id":48153104,"identity":"2fbe9e90-b7f0-4f89-898f-5729e7f235c4","added_by":"auto","created_at":"2023-12-13 19:58:50","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":966245,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMult-omics analysis of F8 treatment on \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eS. aureus\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e ATCC 29213. a,\u003c/strong\u003eExperimental schematic for the multi-omics experiment. \u003cstrong\u003eb,\u003c/strong\u003e Volcano plot of differentially-expressed genes (DEGs). \u003cstrong\u003ec,\u003c/strong\u003e Volcano plot of differentially-expressed proteins (DEPs). \u003cstrong\u003ed,\u003c/strong\u003e Correlation analysis between DEGs and DEPs using a nine-quadrant diagram.\u003c/p\u003e","description":"","filename":"Fig.5.png","url":"https://assets-eu.researchsquare.com/files/rs-3667988/v1/e3fbeca749a90377a0bd79f7.png"},{"id":48153111,"identity":"e4dabc5f-7679-4969-aeb9-f173ea0bdded","added_by":"auto","created_at":"2023-12-13 19:58:50","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":4171305,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTranscriptomic analysis of F8 treatment on \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eS. aureus\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e ATCC 29213. a,\u003c/strong\u003e GO annotation analysis. \u003cstrong\u003eb, c,\u003c/strong\u003e GO enrichment analysis. \u003cstrong\u003ed, \u003c/strong\u003eKEGG enrichment analysis. The x-axis and y-axis represent expression changes and the corresponding statistical significance, respectively. \u003cstrong\u003ee,\u003c/strong\u003e Clustering heatmap of some important DEGs. \u003cstrong\u003ef,\u003c/strong\u003e GO functional enrichment chord diagram.\u003c/p\u003e","description":"","filename":"Fig.6.png","url":"https://assets-eu.researchsquare.com/files/rs-3667988/v1/eea1826a055ad697302f3d1b.png"},{"id":48153110,"identity":"52363e3c-58e9-4692-a28e-f0f71dfbaad2","added_by":"auto","created_at":"2023-12-13 19:58:50","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":3042441,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eProteomic analysis of F8 treatment on \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eS. aureus\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e ATCC 29213. a,\u003c/strong\u003e KEGG annotation analysis. \u003cstrong\u003eb, c,\u003c/strong\u003e GO enrichment analysis. \u003cstrong\u003ed,\u003c/strong\u003eKEGG enrichment analysis. \u003cstrong\u003ee,\u003c/strong\u003e Clustering heatmap of some important DEPs.\u003c/p\u003e","description":"","filename":"Fig.7.png","url":"https://assets-eu.researchsquare.com/files/rs-3667988/v1/3949c57f3eaf37142d40cbde.png"},{"id":48155635,"identity":"44771d71-911a-4412-b829-7fa5bcc54b3e","added_by":"auto","created_at":"2023-12-13 20:14:50","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":2280474,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMetabolomic analysis of F8 treatment on \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eS. aureus\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eATCC 29213. a,\u003c/strong\u003ePrincipal Component Analysis (PCA). \u003cstrong\u003eb,\u003c/strong\u003e Volcano plot of differentially-expressed metabolites. \u003cstrong\u003ec,\u003c/strong\u003e Clustering heatmap of metabolic profiles. \u003cstrong\u003ed, \u003c/strong\u003eKEGG enrichment analysis. The x-axis and y-axis represent expression changes and the corresponding statistical significance, respectively.\u003c/p\u003e","description":"","filename":"Fig.8.png","url":"https://assets-eu.researchsquare.com/files/rs-3667988/v1/8997c3ef27da564362fd4452.png"},{"id":48153109,"identity":"92c7b76b-8997-4535-b2d7-5ec88843434e","added_by":"auto","created_at":"2023-12-13 19:58:50","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":298963,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBinding of F8 with arcB. a,\u003c/strong\u003e Simulated image of the F8 4Å DBD in arcB (PDB:2ksd) generated by PyMOL software, with residues LEU-31 shown in green. \u003cstrong\u003eb,\u003c/strong\u003e The superposition of the crystal structure of the DBDs of F8 and FLO bound to arcB, with residues LEU-31 shown in green, FLO in light purple, and F8 in blue. \u003cstrong\u003ec,\u003c/strong\u003e Gene-level differences of arcB in \u003cem\u003eS. aureus\u003c/em\u003e treated with F8 or positive control FLO. \u003cstrong\u003ed,\u003c/strong\u003e The expression of arcB was analysed by SDS-PAGE, and compared with the negative control (C). The target protein was expressed in the form of total protein (T), soluble (S), and insoluble (P) of the lysate.\u003cstrong\u003e e, f,\u003c/strong\u003e ITC detection of ligand target-binding affinity. \u003cstrong\u003eg,\u003c/strong\u003e Detection of thermal displacement (T\u003csub\u003em\u003c/sub\u003e change) by DSF.\u003c/p\u003e","description":"","filename":"Fig.9.png","url":"https://assets-eu.researchsquare.com/files/rs-3667988/v1/12b3da6c1957e2f038911603.png"},{"id":48153103,"identity":"e75b24d2-c6c3-4c3d-bcc2-5cbd5a16ae25","added_by":"auto","created_at":"2023-12-13 19:58:50","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":499368,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBactericidal mechanism of F8. a, b,\u003c/strong\u003e Concentrations of extracellular DNA, RNA, and protein at different time intervals after treatment with various concentrations (4×MIC, 2×MIC, and MIC) of F8 in \u003cem\u003eS. aureus\u003c/em\u003eand \u003cem\u003eE. coli\u003c/em\u003e. \u003cstrong\u003ec, d,\u003c/strong\u003e Increased membrane permeability in \u003cem\u003eS. aureus\u003c/em\u003e and \u003cem\u003eE. coli\u003c/em\u003e after treatment with 4×MIC of F8. Membrane permeability measured by propidium iodide (PI), with excitation/emission wavelengths at 535 nm/615 nm. \u003cstrong\u003ee, f,\u003c/strong\u003e Detection of ΔpH in \u003cem\u003eS. aureus\u003c/em\u003eand \u003cem\u003eE. coli\u003c/em\u003e treated with 4×MIC of F8, measured using the fluorescent probe, BCECF-AM, with excitation/emission wavelengths at 488 nm/535 nm. \u003cstrong\u003eg, h,\u003c/strong\u003e Accumulation of intracellular ROS in \u003cem\u003eS. aureus\u003c/em\u003e and \u003cem\u003eE. coli\u003c/em\u003eafter treatment with 4×MIC of F8. The positive control for ROS accumulation was ROSUP. \u003cstrong\u003ei, j,\u003c/strong\u003e Changes in intracellular ATP levels in \u003cem\u003eS. aureus\u003c/em\u003e and \u003cem\u003eE. coli\u003c/em\u003e after treatment with 4×MIC of F8. ns, not significant, *, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, \u003cem\u003e**,\u003c/em\u003e \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, \u003cem\u003e***,\u003c/em\u003e \u003cem\u003ep\u003c/em\u003e\u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"Fig.10.png","url":"https://assets-eu.researchsquare.com/files/rs-3667988/v1/4921f2a31e6d328a2240b0d4.png"},{"id":48157403,"identity":"a75913fd-1a1f-4bbd-9455-01a5a3e73c52","added_by":"auto","created_at":"2023-12-13 20:22:52","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6096786,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3667988/v1/dcdccf39-1e1f-4e7d-9398-f874e31caa3e.pdf"},{"id":48153099,"identity":"6fd861d4-7050-4677-8a79-03430816d4f9","added_by":"auto","created_at":"2023-12-13 19:58:49","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":37655,"visible":true,"origin":"","legend":"\u003cp\u003eTable S1, Table S2.\u003c/p\u003e","description":"","filename":"SupplementaryTables26112023.docx","url":"https://assets-eu.researchsquare.com/files/rs-3667988/v1/2cc72696b53e6f5cb688cf50.docx"},{"id":48153105,"identity":"a285e6ba-6aca-487d-a19c-496bfd7b50d9","added_by":"auto","created_at":"2023-12-13 19:58:50","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":7779013,"visible":true,"origin":"","legend":"\u003cp\u003eSupplemental Fig. 1-13\u003c/p\u003e","description":"","filename":"Supplementalfigures26112023.docx","url":"https://assets-eu.researchsquare.com/files/rs-3667988/v1/d1120f7696a5b71f86e1d650.docx"},{"id":48155636,"identity":"e09bd708-24db-41ee-b0a4-435f0a7a0a5d","added_by":"auto","created_at":"2023-12-13 20:14:50","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":3453422,"visible":true,"origin":"","legend":"\u003cp\u003eSupporting information\u003c/p\u003e","description":"","filename":"Supportinginformation26112022.docx","url":"https://assets-eu.researchsquare.com/files/rs-3667988/v1/b9d6fd6b0e56f2b1780813c1.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"A synthetic antibiotic class with a deeply-optimized design for overcoming bacterial resistance","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe rapid increase of drug-resistant pathogens, particularly the emergence of superbugs, poses a significant threat to public health\u003csup\u003e\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. The discovery and clinical application of antibiotics undoubtedly mark a milestone in human and modern medical history. They have driven advancements in various medical practices and are indispensable life-saving weapons\u003csup\u003e\u003cspan additionalcitationids=\"CR5 CR6\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e; however, the rise and spread of bacteria that have developed resistance to most or all existing antibiotics have raised concerns about an impending global infectious disease crisis\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan additionalcitationids=\"CR9 CR10\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. In the United States alone, there are over 2.8\u0026nbsp;million antibiotic-resistant infections each year, resulting in more than 35,000 deaths\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. In Europe, antibiotic resistance leads to about 33,000 deaths per year, with estimated hospital costs exceeding \u0026euro;900 million\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Any plan to address this issue relies on the discovery of new antibiotics that are effective against modern bacterial pathogens, making the development of antibiotics with unique mechanisms crucial\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. The development of antibiotics is continuously challenging nowadays, leading to the rarity of new antibiotics being introduced clinically. Therefore, there is an urgent need to identify new targets and drugs to fill the gaps in antibiotic discovery and development, and to combat bacterial infections.\u003c/p\u003e\u003cp\u003eArcB is a catabolic ornithine carbamoyl transferase involved in the degradation of arginine, and is crucial for bacterial arginine biosynthesis and catabolism metabolism. It also has a key role in gene regulation, cell growth, biofilm formation, and biological mechanisms\u003csup\u003e\u003cspan additionalcitationids=\"CR16 CR17 CR18 CR19\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Arginine metabolism is tightly regulated in bacteria, as it is vital for cell growth and biofilm formation\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. The expression of arcB is known to be affected by treatment with antibiotics such as gentamicin and polymyxin B, indicating that cell membrane stress affects arcB activity\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. In addition, arcB is related to nitrogen metabolism, which is an essential nutrient for synthetic metabolism and is crucial for the synthesis of amino acids. Cells must balance the activity of synthetic metabolic pathways with the overlapping pathways used for energy production through chemical interactions\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. It is worth noting that arcB not only participates in the regulation of cellular processes, but is also involved in cell death regulation due to the modulation of reactive oxygen species levels. Importantly, mutants of arcB may affect bacterial growth\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e, therefore, arcB appears to be a promising target for antibacterial drug development.\u003c/p\u003e\u003cp\u003eIn recent years, hybrid antimicrobial agents have garnered significant attention as a novel strategy for combating bacterial resistance. They utilize the principle of structural hybridization, which is a rational drug design method where new chemical entities are formed by combining two or more drugs or pharmacophores from different bioactive compounds into a single entity\u003csup\u003e\u003cspan additionalcitationids=\"CR26\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. The resulting compounds have multiple potential advantages, such as broadening the spectrum of antimicrobial activity, enhancing activity against resistant strains, and reducing the emergence of bacterial resistance. Compared to combination therapy, drug structural hybridization better addresses issues of differential bioavailability, pharmacokinetics, and metabolism, resulting in improved therapeutic safety and avoidance of drug-drug interactions.\u003c/p\u003e\u003cp\u003eCurrently, several hybrid antimicrobial molecules have been synthesized and tested, with some entering clinical trial stages\u003csup\u003e\u003cspan additionalcitationids=\"CR29 CR30\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e, showing promise as prospects for development. Given the clear advantages of structural hybrids, the concept of structural hybridization is attractive; however, the complexity of molecular structures, challenging chemical synthesis, and the rigorous work required to establish modes of action and benefits of structural hybrids compared to conventional drugs can make the method daunting. Cell permeability is also a key problem, as antibacterials with high molecular weight will not pass through non-selective protein channels, limiting their cell uptake to receptor-mediated endocytosis or passive diffusion\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. In fact, permeability barriers due to high molecular weight are the main reason for the limited activity of most antibiotics against bacteria\u003csup\u003e\u003cspan additionalcitationids=\"CR33\" citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. Despite the challenges, the structural hybridization strategy remains a viable approach to expand our existing antimicrobial drug library.\u003c/p\u003e\u003cp\u003eTo find compounds with potent antimicrobial activity and a reduced propensity for resistance, this study employed the approach of drug structure hybridization, leading to the discovery of the optimized modified compound, F8. We found that F8 exhibited excellent \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e anti-resistant bacterial activity, and through multi-omics analysis (transcriptomics, proteomics, and metabolomics), we inferred that the antibacterial target of F8 may be the key enzyme of arcB in the arginine degradation pathway. This study evaluated the therapeutic effects of F8 both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e. F8 demonstrates significant antibacterial activity against both gram-positive and gram-negative bacteria, with a minimum inhibitory concentration (MIC) range of 2 to 8 \u0026micro;M, which is notably superior to the positive control florfenicol (FLO) (4\u0026times;MIC, and 2\u0026times;MIC). In a mouse model of FLO-resistant \u003cem\u003eS. aureus\u003c/em\u003e bacteraemia, the bacterial burden in tissues was significantly reduced (Log\u003csub\u003e10\u003c/sub\u003e CFU/mL as low as 2\u0026thinsp;~\u0026thinsp;3), and the survival rate of the mice within 72 h is as high as 50% (all deaths within 24 h were in the control group). Furthermore, the antibacterial mechanism of F8 targeting arcB was systematically investigated. Overall, our research findings suggest that F8 competitively binds to arcB, thereby activating multiple pathways to exert its antimicrobial effects. The results of this study provides a foundation and strategy for the development of novel antimicrobial drugs, and reaffirmed the ability of chemical synthesis to supplement our antibiotic arsenal, providing broad-spectrum drugs that can overcome increasingly severe drug resistance mechanisms.\u003c/p\u003e"},{"header":"Results","content":"\u003ch3\u003eDesign and synthesis of broad-spectrum antibacterial small molecule compounds based on structural hybridization strategy\u003c/h3\u003e\n\u003cp\u003eIn order to find compounds with effective antibacterial activity and that are less likely to develop resistance, we endeavoured to design and synthesize broad-spectrum antibacterial hybrid small-molecule compounds using a drug structure hybridization strategy and computer assisted drug design (CADD)\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. Guided by the hybrid strategy, we chose FLO, a compound with simple structure, low molecular weight, and easy synthesis, as our main skeleton structure. With peptidyl transferase centre (PTC) of the bacterial 50S ribosome subunit as the target, the key area of FLO, we applied CADD and developed a modular synthesis route. By forming an ester bond to connect these components, we produced a variety of antibacterial candidate drugs. Therefore, we studied a wide range of structural changes and evaluated their antibacterial activities.\u003c/p\u003e \u003cp\u003eFirst, we conducted molecular docking studies using the crystal structure of the PTC region of the 50S subunit from the protein data bank (PDB: 6c4h)\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. We then modelled the binding pocket of the PTC region of the 50S subunit (method) and established an atomic property field (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). The structure-activity relationship of FLO has shown that its main scaffold occupies the conserved region of the binding pocket of the 50S subunit's PTC region, therefore, we have chosen to keep this region static and instead structurally modified the β-hydroxy position. The hydroxyl group provides a considerable advantage for the synthesis of small molecules. Even for the novel compounds generated, it must conform to a mechanism that can be easily synthesized, otherwise, it would have minimal significant broad influences\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eNext, we selected various simple small molecule structures, including tricyclic, tetracyclic, pentacyclic, and hexacyclic structures, to hybridize with the skeleton structure (ChemDraw software), generating over a hundred novel hybrid structures for automated molecular docking. According to the scoring of SYBYL-X, some of the lead compounds exhibited improved binding power, reduced intermolecular collision force, and tighter binding with the receptor protein when compared to the skeleton structure (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb, c). Based on docking simulations, ligands with smaller fragments have lower crash values (a crash value close to 0 is beneficial), suggesting that structures with smaller spatial conformation within the binding pocket can lower the level of internal self-collisions that the ligand might experience, thereby enhancing the stability of binding. Among these studies defined within relatively narrow parameters, we comprehensively selected 11 compounds with the best scores (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec), total scores and polarity all higher than the total scores and polarity of the scaffold compounds (score\u0026thinsp;=\u0026thinsp;5.1467), and with good binding specificity and affinity. These were then visually compared and synthesized against the PTC region of the 50S subunit. The structure was confirmed by LCMS-IT-TOF, \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003eH-NMR, and \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC-NMR measurements (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed, Supplementary Table\u0026nbsp;1, and Supporting information).\u003c/p\u003e \u003cp\u003eWe identified a hybrid structure 8 (F8, 3-(1-Piperidinyl) propanoic acid) as the optimal modification, which further enhanced the antibacterial effects against pathogens such as \u003cem\u003eE. coli\u003c/em\u003e, \u003cem\u003eS. aureus\u003c/em\u003e, \u003cem\u003eS. typhi\u003c/em\u003e, \u003cem\u003eP. multocida\u003c/em\u003e, and \u003cem\u003eH. parasuis\u003c/em\u003e, and imparted measurable activity against the particularly challenging \u003cem\u003eP. aeruginosa\u003c/em\u003e. The antibacterial activity test results are shown in Tables\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and \u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. As shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, hybrid structures 1, 2, 6, 7, 8, and 14 exhibit good antibacterial effects. When hybrid small molecule fragments consist of tetracyclic and pentacyclic structures, including thiazole and cyclobutane classes (hybrid structures 1, 2, and 7), their antibacterial activity is weaker compared to the skeleton structure. When the hybrid small molecule fragments feature hexacyclic structures (hybrid structure 14), the antibacterial activity is similar to the skeleton structure. Further using hexacyclic structures, we found that quinoline and morpholine structures (hybrid structures 11 and 13) virtually lose their antibacterial activities. We found that in general, short chain small fragments are more active than their homologues. Furthermore, it was found that the piperidine structure (hybrid structure 8) is sensitive to antibacterial activity and antibacterial spectra, and its effect is superior to that of the skeleton structure. Docking simulation shows that F8 can extend to a deeper position at the end of the PTC region of the 50S subunit (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). We observed changes in the binding amino acid sites, with a greater number of hydrogen bonds between F8 and the target involved in ligand-protein binding than the skeleton structure, which may directly lead to an increase in binding affinity. Moreover, F8 binds near U2585 (key site), which may compete with the substrate of the a-site (one of the core functional regions of the ribosome), interrupting the binding of the a-site with tRNA and preventing the formation of peptide bonds, thereby playing a crucial role. The software also scored F8 (score\u0026thinsp;=\u0026thinsp;6.1738) higher than the scaffold structure (score\u0026thinsp;=\u0026thinsp;5.1467) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec), providing support for our design strategy.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eMIC of broad-spectrum antibacterial small molecule compounds based on structural hybridization strategy.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"14\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c12\" colnum=\"12\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c13\" colnum=\"13\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c14\" colnum=\"14\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eOrganism\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"13\" nameend=\"c14\" namest=\"c2\"\u003e \u003cp\u003eMIC (\u0026micro;M)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eF\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eF1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eF2\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eF3\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eF4\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eF6\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eF7\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eF8\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c10\"\u003e \u003cp\u003eF11\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c11\"\u003e \u003cp\u003eF13\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c12\"\u003e \u003cp\u003eF14\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c13\"\u003e \u003cp\u003eF15\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"1\" nameend=\"c14\" namest=\"c14\"\u003e\u0026nbsp;\u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eE. coli\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c14\" namest=\"c14\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eS. typhi\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c14\" namest=\"c14\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eP. aeruginosa\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c14\" namest=\"c14\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eS. aureus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c14\" namest=\"c14\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eB. subtilis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c14\" namest=\"c14\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eE. faecalis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c14\" namest=\"c14\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eP. multocida\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c14\" namest=\"c14\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eA. pleuropneumoniae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c14\" namest=\"c14\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eS. suis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c14\" namest=\"c14\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eH. parasuis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c14\" namest=\"c14\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eMBC of broad-spectrum antibacterial small molecule compounds based on structural hybridization strategy.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"14\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c12\" colnum=\"12\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c13\" colnum=\"13\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c14\" colnum=\"14\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eOrganism\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"13\" nameend=\"c14\" namest=\"c2\"\u003e \u003cp\u003eMBC (\u0026micro;M)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eF\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eF1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eF2\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eF3\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eF4\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eF6\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eF7\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eF8\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c10\"\u003e \u003cp\u003eF11\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c11\"\u003e \u003cp\u003eF13\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c12\"\u003e \u003cp\u003eF14\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c13\"\u003e \u003cp\u003eF15\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"1\" nameend=\"c14\" namest=\"c14\"\u003e\u0026nbsp;\u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eE. coli\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c14\" namest=\"c14\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eS. typhi\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c14\" namest=\"c14\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eP. aeruginosa\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c14\" namest=\"c14\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eS. aureus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c14\" namest=\"c14\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eB. subtilis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c14\" namest=\"c14\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eE. faecalis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c14\" namest=\"c14\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eP. multocida\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c14\" namest=\"c14\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eA. pleuropneumoniae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c14\" namest=\"c14\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eS. suis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c14\" namest=\"c14\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eH. parasuis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c14\" namest=\"c14\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e\n\u003ch3\u003eF8 is an excellent candidate antibiotic\u003c/h3\u003e\n\u003cp\u003eThe chemical structure of F8 is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed. To evaluate its \u003cem\u003ein vitro\u003c/em\u003e antibacterial activity, we determined the MIC against various bacteria, including both gram-negative and gram-positive bacteria. F8 exhibited significant antibacterial activity against both gram-positive and gram-negative bacteria, with MIC values ranging from 2 to 8 \u0026micro;M, superior to the positive control FLO (4\u0026times;MIC and 2\u0026times;MIC). Measurable activity was noted against the particularly challenging \u003cem\u003eP. aeruginosa\u003c/em\u003e, with an MIC of 128 \u0026micro;M (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). F8 exhibits significant bactericidal activity against methicillin-resistant \u003cem\u003eS. aureus\u003c/em\u003e (MRSA), polymyxin B-resistant \u003cem\u003eE. hormaechei\u003c/em\u003e, FLO-resistant \u003cem\u003eS. suis\u003c/em\u003e, FLO-resistant \u003cem\u003eH. parasuis\u003c/em\u003e, doxycycline-resistant \u003cem\u003eS. typhi\u003c/em\u003e, ampicillin-resistant \u003cem\u003eS. typhi\u003c/em\u003e and sulfamethoxazole-resistant \u003cem\u003eS. typhi\u003c/em\u003e, among other drug-resistant bacteria (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Additionally, we used a representative gram-positive bacterium, \u003cem\u003eS. aureus\u003c/em\u003e, and a gram-negative bacterium, \u003cem\u003eE. coli\u003c/em\u003e, as models to investigate the antibacterial activity and mechanism of action of F8.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eMIC of F8 against antibiotic-resistant strains.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOrganism\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMIC (\u0026micro;M)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMethicillin-resistant \u003cem\u003eS. aureus\u003c/em\u003e B1-1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePolymyxin B-resistant \u003cem\u003eE. hormaechei\u003c/em\u003e wb 4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFlorfenicol-resistant \u003cem\u003eS. suis\u003c/em\u003e 1136\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFlorfenicol-resistant \u003cem\u003eS. suis\u003c/em\u003e 1194\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFlorfenicol-resistant \u003cem\u003eS. suis\u003c/em\u003e 1197\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFlorfenicol-resistant \u003cem\u003eS. suis\u003c/em\u003e 1655\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFlorfenicol-resistant \u003cem\u003eS. suis\u003c/em\u003e 1658\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFlorfenicol-resistant \u003cem\u003eS. suis\u003c/em\u003e 1669\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFlorfenicol-resistant \u003cem\u003eH. parasuis\u003c/em\u003e 1565\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFlorfenicol-resistant \u003cem\u003eH. parasuis\u003c/em\u003e 1614\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFlorfenicol-resistant \u003cem\u003eH. parasuis\u003c/em\u003e 1651\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAmpicillin-resistant \u003cem\u003eS. typhi\u003c/em\u003e BYG 9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSulfamethoxazole-resistant \u003cem\u003eS. typhi\u003c/em\u003e BYG 21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDoxycycline-resistant \u003cem\u003eS. typhi\u003c/em\u003e BYG 11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDoxycycline-resistant \u003cem\u003eS. typhi\u003c/em\u003e BYG 25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDoxycycline-resistant \u003cem\u003eS. typhi\u003c/em\u003e BYG 31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eTo confirm its biological activity, the agar diffusion method was employed to test the antibacterial activity of F8 against \u003cem\u003eS. aureus\u003c/em\u003e and \u003cem\u003eE. coli\u003c/em\u003e. We observed that F8 consistently inhibited the growth of the pathogens, displaying distinct inhibition zones (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). The growth inhibition effect of F8 on \u003cem\u003eS. aureus\u003c/em\u003e and \u003cem\u003eE. coli\u003c/em\u003e was further assessed (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb, c), and growth curves demonstrated that F8 effectively suppressed \u003cem\u003eS. aureus\u003c/em\u003e and \u003cem\u003eE. coli\u003c/em\u003e at 4\u0026times;MIC, 2\u0026times;MIC, and MIC, notably diminishing the growth of \u003cem\u003eS. aureus\u003c/em\u003e at half the MIC concentration. Additionally, we monitored the bacterial viability of \u003cem\u003eS. aureus\u003c/em\u003e and \u003cem\u003eE. coli\u003c/em\u003e exposed to different concentrations of F8 at various time points (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed, e and Supplementary Fig.\u0026nbsp;1a, b), and found that F8 at 4\u0026times;MIC, 2\u0026times;MIC, and MIC could kill the bacteria within 8 hours and eliminate the majority of bacteria for an extended period. Based on the minimum bactericidal concentration (MBC) and MIC values (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), we confirmed F8 as an effective candidate antibacterial agent.\u003c/p\u003e \u003cp\u003eTo further evaluate the \u003cem\u003ein vivo\u003c/em\u003e therapeutic potential of F8, we used a murine sepsis model induced by the intraperitoneal injection of \u003cem\u003eS. aureus\u003c/em\u003e to test the therapeutic effect of F8 on bacterial infection. Bacterial colony counts revealed that F8 at 60 mg/kg could effectively reduce bacterial load in the liver, spleen, kidneys, and blood (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef-i). The \u003cem\u003eS. aureus\u003c/em\u003e infection group displayed extensive tubular necrosis with an indistinct structure in kidney tissues (cortical and medullary) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ej). Inflammatory cell infiltration centred on granulocytes within the tubules, along with necrosis and conglomeration of inflammatory cells, significant tubular dilation, and focal haemorrhages were also notable in the infected group. Hepatic cells demonstrated vacuolar degeneration and inflammatory cell infiltration, with visible bleeding within vessels. The boundary between the red pulp and white pulp in the spleen was indistinct, with an increase in the number of neutrophils and macrophages within the red pulp. However, in the F8 treatment group, nearly no significant pathological changes were observed.\u003c/p\u003e \u003cp\u003eTo further characterize the potential of F8 as an antimicrobial agent, we also investigated the effect of F8 on organ damage. Results showed that no notable abnormalities were observed in the liver, kidneys, and intestines in the F8-treated group at 1500 mg/kg, with no significant pathological changes in the spleen, kidneys, and bone marrow (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea-c, j and Supplementary Fig.\u0026nbsp;2); however, the positive control FLO group exhibited minor vacuolar degeneration and inflammatory cell infiltration in hepatic cells, with mild bleeding visible inside vessels. Kidney structure was abnormal with indistinct boundaries, a disappearance of tubular lumens, and glomerular atrophic degeneration. Extensive fibrosis with slight necrotic degeneration and shedding was observed in the intestinal tissues. Thymic medullary lymphocytes showed relatively loose arrangements with serum exudation, the number of lymphocytes was lower than in the control group, and minor inflammatory cell infiltration was observed within the medulla. The boundary between the red pulp and white pulp in the spleen was indistinct, with increased numbers of neutrophils and macrophages in the red pulp (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ej). The ELISA results showed no significant differences in the levels of IL-6, IL-2, and Hsp70 in the blood between the F8 treatment group and the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed-f). As a highly conserved stress protein, Hsp70 significantly increases in expression level when exposed to harmful stimuli or stress factors, playing a crucial role in immune regulation. Compared with the control group, the concentration of Hsp70 in the thymus and spleen of the F8 group showed no significant difference, but significantly increased in the bone marrow (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eg-i). In addition, no significant differences in the number of white blood cells (WBCs), neutrophils (Neu), lymphocytes (Lym), red blood cells (RBCs), haemoglobin (HGB), and platelets (PLT) were observed between the F8-treated group and the control group, indicating that F8 has low toxicity to mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ek-p). In TUNEL staining analysis, the immune organs (thymus, spleen, and bone marrow) treated with F8 did not show intense fluorescence compared with the positive control FLO, indicating fewer instances of cell death (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eq).\u003c/p\u003e\u003cp\u003eAdditionally, F8 exhibited excellent tolerance in a 14-day study of acute toxicity in mice. At a maximum dose of 5000 mg/kg, no abnormal clinical signs were observed (Supplementary Fig.\u0026nbsp;3a, b). The cytotoxicity of F8 to mammalian cells was also low (Supplementary Fig.\u0026nbsp;4a-c). Furthermore, we found that F8 has an extremely low haemolysis rate on sheep red blood cells, showing negligible haemolysis even at 256 \u0026micro;M (Supplementary Fig.\u0026nbsp;5a, b). In conclusion, these results suggest that F8 possesses good pharmacological efficacy \u003cem\u003ein vivo\u003c/em\u003e; however, further studies should be carried out to investigate its pharmacokinetic characters and impacts on a wider range of clinical infections.\u003c/p\u003e \u003cp\u003e \u003cb\u003eF8 overcomes resistance and is effective\u003c/b\u003e \u003cb\u003ein vivo\u003c/b\u003e\u003c/p\u003e \u003cp\u003eBacterial resistance is also an important indicator of the potential clinical application of antimicrobial drugs. To evaluate the development of F8 resistance, we conducted an \u003cem\u003ein vitro\u003c/em\u003e study, continuously exposing \u003cem\u003eS. aureus\u003c/em\u003e and \u003cem\u003eE. coli\u003c/em\u003e to sub-inhibitory concentrations of F8 for 30 consecutive days. Compared with the positive control FLO, the trend of bacteria developing resistance to F8 was lower (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea, b). Subsequently, the inhibitory effect of F8 on induced FLO-resistant \u003cem\u003eS. aureus\u003c/em\u003e and FLO-resistant \u003cem\u003eE. coli\u003c/em\u003e was also tested, and it was found that F8 significantly enhanced the inhibitory effect on these FLO-resistant bacteria, which was significantly superior to the positive control FLO (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). These results suggest that F8 holds greater advantages in avoiding bacterial resistance.\u003c/p\u003e\u003cp\u003eTo further evaluate the \u003cem\u003ein vivo\u003c/em\u003e antibacterial action of F8, a mouse sepsis model with FLO-resistant \u003cem\u003eS. aureus\u003c/em\u003e was established. Excitingly, the survival curve showed that the survival rate of all mice infected with drug-resistant bacteria was as high as 50% within 72 h after treatment with 60 mg/kg of F8, while under the same conditions, the survival rate of the positive control FLO decreased to 0 within 48 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ei). This survival analysis showed that the F8 treatment group was able to significantly inhibit drug-resistant bacteria \u003cem\u003ein vivo\u003c/em\u003e and had a significant survival advantage.\u003c/p\u003e \u003cp\u003eSubsequent evaluation of bacterial load measurements was consistent with the survival rate analysis. After 24 h of F8 treatment, the bacterial load in the liver, spleen, kidneys, and blood showed significant reduction (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed-g), and furthermore, F8 exhibited a considerable inhibitory effect on the invasion of FLO-resistant \u003cem\u003eS. aureus\u003c/em\u003e. Moreover, histopathological examinations revealed noticeable tissue necrosis and damage in the FLO-resistant \u003cem\u003eS. aureus\u003c/em\u003e group, while F8 treatment significantly alleviated the necrosis and damage in the liver, kidney, and spleen tissues caused by bacterial infection (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eh). Overall, our results suggest that F8 effectively alleviates the severity of FLO-resistant \u003cem\u003eS. aureus\u003c/em\u003e infection.\u003c/p\u003e\n\u003ch3\u003eDiscovery of arcB as the antibacterial target of F8 from multi-omics analysis\u003c/h3\u003e\n\u003cp\u003eTo uncover the potential targets of F8 in its anti-\u003cem\u003eS. aureus\u003c/em\u003e activity, we analysed the bacterial changes post-F8 treatment using transcriptomics, proteomics, and metabolomics (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea-d). From transcriptomic analysis, a total of 1,212 differentially-expressed genes (DEGs) (598 up-regulated and 614 down-regulated) showed significant expression patterns before and after F8 treatment (FC\u0026thinsp;\u0026lt;\u0026thinsp;0.5 or \u0026gt;\u0026thinsp;2, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). Kyoto encyclopedia of genes and genomes (KEGG) and gene ontology (GO) analysis revealed that pathways for cellular components (cytoplasmic, membrane, and plastid parts), molecular function (amino acid biosynthesis process, small molecule metabolic process, and oxidoreductase activity), and energy metabolism (arginine biosynthesis, nitrogen metabolism, histidine metabolism, glycolysis, and alanine, aspartate and glutamate metabolism) were down-regulated, whereas activities of RNA processing, RNA metabolic processes, and cellular macromolecule biosynthetic processes were up-regulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb-f). In conclusion, these up-regulated DEGs are largely associated with the metabolic disorder and degradation of secondary metabolites, whereas processes like amino acid biosynthesis, nitrogen source metabolism, and glycolysis are significantly suppressed.\u003c/p\u003e \u003cp\u003eProteomic analysis revealed that after F8 treatment, there were 457 differentially-expressed proteins (DEPs) (224 up-regulated and 233 down-regulated), with significantly down-regulated genes mainly concentrated in the pathways of histidine metabolism, nitrogen metabolism, arginine biosynthesis, and alanine, aspartate, and glutamate metabolism (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). In the metabolomics data, there were 326 different metabolites between the control group and F8 group (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). KEGG pathway analysis revealed a significant down-regulation in tricarboxylic acid (TCA) cycle and cofactor biosynthesis. Furthermore, after F8 treatment, molecules associated with purine and pyrimidine metabolism, as well as ornithine, pyruvate, aspartate, glutamate, and arginine metabolism were significantly downregulated.\u003c/p\u003e \u003cp\u003eSubsequently, multi-omics pathway analysis was utilized to further scrutinize the data across the three omics (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). In the integrated transcriptomics-proteomics analysis, a strong correlation was observed in nitrogen metabolism, amino acid synthesis, and the stress response. Similarly, in the integrated transcriptomics-metabolomics analysis, amino acid synthesis, nitrogen metabolism, and energy metabolism were significantly downregulated. After conducting KEGG enrichment analysis of significantly down-regulated proteins and metabolites, we noticed a significant down-regulation in arginine metabolism, alanine, aspartate, and glutamate metabolism. Finally, multi-omics analysis suggested that F8 down-regulates the biosynthesis of nitrogen metabolism, arginine biosynthesis, and alanine, aspartate, and glutamate metabolism. Arginine metabolism in bacteria is tightly regulated, and arginine is crucial for cell growth and biomembrane formation\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e, thus, we infer that F8 exerts its antibacterial effects through multifaceted pathways including participation in the arginine degradation metabolic pathway, bacterial cell membranes, and energy metabolism.\u003c/p\u003e \u003cp\u003eBased on the multi-omics analysis, we speculate that the antibacterial activity of F8 is associated with multiple pathways, including the arginine degradation metabolic pathway, the bacterial cell membrane, and energy metabolism. Among these, downregulation of nitrogen metabolism and arginine biosynthesis was observed in all three omics datasets, hence, we hypothesize that an important antibacterial target of F8 against \u003cem\u003eS. aureus\u003c/em\u003e could potentially involve the arginine degradation metabolic pathway. Within the arginine degradation metabolic pathway, arginine deiminase (arcA), arcB, and carbamate kinase (arcC) are categorized as an arginine deiminase pathway (ADI)\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. The RT-PCR results are consistent with the transcriptomics data (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003ec, Supplementary Fig.\u0026nbsp;6a-i and Supplementary Table\u0026nbsp;2). Notably, since arcB is involved in arginine degradation and is also indispensable for arginine biosynthesis, studies have shown that arcB levels might limit arginine biosynthesis of \u003cem\u003eS. gordonii\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Expression of arcB is also known to be affected following treatment with antibiotics, including gentamicin and polymyxin B\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Therefore, we preliminarily infer arcB as a possible target of F8.\u003c/p\u003e\n\u003ch3\u003eArcB binding by F8\u003c/h3\u003e\n\u003cp\u003eGiven that F8 significantly impacts the expression of arcB in multi-omics analysis, we posit that F8 exerts its antibacterial activity by targeting the arcB protein and affecting its function. To further detail the binding mode of F8 and arcB, we searched for the crystal structure of the arcB protein (PDB: 2ksd) in the PDB and performed molecular docking using the SYBYL-X software. We found that F8 is capable of binding with the arcB protein and occupying the binding pocket (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003ea, b and Supplementary Fig.\u0026nbsp;7), and that the cyclic structure of F8 nestles into the cavity of the receptor protein, enhancing the tightness of the binding. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003ea, F8 binds directly to arcB through hydrogen bonding interactions with LEU-31 of arcB.\u003c/p\u003e \u003cp\u003eTo further validate that F8 can directly bind to arcB, we expressed the arcB protein and then examined their interactions (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003ed and Supplementary Fig.\u0026nbsp;8a). Isothermal titration calorimetry (ITC) measurements further confirmed the predicted interaction between F8 and the active pocket of arcB. The results showed that F8 directly and specifically binds to the arcB protein (Kd\u0026thinsp;=\u0026thinsp;1.081\u0026thinsp;\u0026plusmn;\u0026thinsp;e\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e M) (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003ee, f and Supplementary Fig.\u0026nbsp;8b, c). Subsequently, differential scanning fluorimetry (DSF) was employed to assess protein thermal stability induced by compound binding to evaluate the interaction. A thermal shift (ΔT\u003csub\u003em\u003c/sub\u003e change) was observed when the arcB protein was incubated with F8 compared to the blank control (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eg), providing evidence for the binding of F8 to arcB protein. These results collectively demonstrate that F8 can directly bind to arcB with high affinity \u003cem\u003ein vitro\u003c/em\u003e.\u003c/p\u003e\n\u003ch3\u003eBactericidal mechanism of F8\u003c/h3\u003e\n\u003cp\u003eGiven the wide-ranging regulatory ability of arcB, which is crucial for cell growth and biofilm formation, we hypothesize that F8 may exert its antibacterial effect through bacterial cell membrane damage. To this end, we first examined the bacterial extracellular contents, showing an increasing trend in the concentration of DNA, RNA, and protein at various time intervals after F8 treatment at different concentrations (4\u0026times;MIC, 2\u0026times;MIC, and MIC) (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003ea, b and Supplementary Fig.\u0026nbsp;9). The reason for this observation may be that F8 can alter bacterial cell membrane permeability, resulting in an increased efflux of these three substances. Alternatively, F8 might have a destructive effect on the bacterial cell membrane structure, resulting in a direct efflux of bacterial contents. Regardless of the reason, the reduction in bacterial DNA, RNA, and protein content can disrupt its normal physiological activities, leading to bacteriostatic or bactericidal effects. Further examination of bacterial cytoplasmic membrane permeability showed that F8 induced increased membrane permeability in \u003cem\u003eS. aureus\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003ec, d and Supplementary Fig.\u0026nbsp;10). This demonstrates that F8 can alter bacterial cell membrane permeability or disrupt cell membrane structure, subsequently exerting bacteriostatic or bactericidal effects.\u003c/p\u003e\u003cp\u003eConsistent with this, changes in membrane rigidity disrupted the bacterial homeostasis, leading to fundamental metabolic disarray, including the dissipation of proton motive force (PMF)\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. Thus, we evaluated the ΔpH, a key component of PMF, in \u003cem\u003eS. aureus\u003c/em\u003e using the fluorescent probe BCECF-AM\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. The experiment was conducted by treating the bacteria with 4\u0026times;MIC of F8. Within 1 h after the medication was added, the ΔpH significantly dissipated (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003ee, f and Supplementary Fig.\u0026nbsp;11), which is consistent with the bactericidal effect. This may indicate that changes in bacterial membrane rigidity disrupt the bacterial steady state, causing fundamental metabolic disarray and leading to the dissipation of proton motive force.\u003c/p\u003e \u003cp\u003eOn the other hand, disruption of membrane homeostasis often contributes to the accumulation of reactive oxygen species (ROS)\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. Moreover, previous studies have shown that the deletion of arcB in \u003cem\u003eE. coli\u003c/em\u003e leads to an increased sensitivity to hydrogen peroxide under aerobic conditions\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. Our investigations revealed that F8 increased intracellular ROS content (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eg, h and Supplementary Fig.\u0026nbsp;12), which may correspondingly exacerbate membrane damage and further disrupt bacterial homeostasis. Endogenous ROS plays a crucial role in bactericidal activity\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. F8 is expected to stimulate the production of ROS in various ways, while also inhibiting the activity of arcB, leading to a decrease in antioxidant capacity. This dual approach may lead to more severe oxidative damage, thereby achieving better bactericidal effects and potentially avoiding the resistance effect caused by a single factor. Interestingly, we observed that ATP levels increased in a dose-dependent manner following F8 treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003ei, j and Supplementary Fig.\u0026nbsp;13). Such results are in line with previous research, suggesting that bactericidal antibiotics are associated with accelerated respiration\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. These findings provide convincing phenotypic support for the notion that F8 disrupts bacterial cell membranes and induces a certain degree of oxidative damage.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eAntimicrobial drug resistance has evolved into a global health crisis. Antibiotics have been indispensable in the battle against bacteria, saving millions of lives\u003csup\u003e\u003cspan additionalcitationids=\"CR46 CR47\" citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e; however, the overuse of antibiotics has led to a rapid increase in the number and types of resistant bacteria, particularly the emergence of superbugs, making it crucial to develop new and more effective strategies to treat bacterial infections\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e,\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. Nowadays, the development of antibiotics is constantly challenged, resulting in few new antibiotics being introduced into clinical practice\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Despite considerable efforts, almost no new antibiotics have been approved for clinical use in recent years, therefore, there is an urgent need for feasible approaches to increase the number of therapeutic options.\u003c/p\u003e \u003cp\u003eBased on the structural hybridization method, we determined that F8 is the optimal modifying compound. It further enhanced its antibacterial effect against pathogenic bacteria such as \u003cem\u003eE. coli\u003c/em\u003e, \u003cem\u003eS. aureus\u003c/em\u003e, \u003cem\u003eS. typhi\u003c/em\u003e, \u003cem\u003eP. multocida\u003c/em\u003e, and \u003cem\u003eH. parasuis\u003c/em\u003e, and imparted measurable activity against the particularly challenging \u003cem\u003eP. aeruginosa\u003c/em\u003e. The most notorious drug-resistant bacterium, MRSA, exhibits resistance to a multitude of antibiotics and is spreading at an alarming rate worldwide. Currently, there is a significant lack of antibiotics capable of combating MRSA\u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e. Our research findings indicate that F8 has a strong inhibitory effect on MRSA, with a MIC value of 8 \u0026micro;M, consistent with the results observed in the standard strains. Polymyxins, particularly polymyxin B, have become the last line of therapy against multi-drug resistant gram-negative bacteria\u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. However, the rapid development of resistance has led to the emergence of more and more polymyxin B resistant bacteria. The MIC value of F8 against polymyxin B-resistant \u003cem\u003eE. hormaechei\u003c/em\u003e is 16 \u0026micro;M, which is a promising lead compound for resisting polymyxin B resistance. Sulfamethoxazole is reported to be one of the most widely used sulfonamide antibiotics in the world; however, the increasingly drug resistance greatly limits its utility\u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e. The MIC value of F8 against sulfamethoxazole-resistant \u003cem\u003eS. typhi\u003c/em\u003e only 4 \u0026micro;M. In addition, F8 also showed excellent bactericidal effects on FLO-resistant \u003cem\u003eS. suis\u003c/em\u003e, FLO-resistant \u003cem\u003eH. parasuis\u003c/em\u003e and doxycycline-resistant \u003cem\u003eS. typhi\u003c/em\u003e. Recently, many structurally-hybrid antibiotics have entered trials, but only a few have reportedly entered clinical trials\u003csup\u003e\u003cspan additionalcitationids=\"CR55\" citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e. Structural hybridization has obvious advantages, but the complexity of molecular structure, difficult-to-handle chemical synthesis, and the rigorous work required to establish the mode of action and benefits of structural hybridization compared to conventional drugs make structural hybridization methods daunting\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e,\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e,\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn this study, we first chose structural hybridization to perform at the β-hydroxy position of the bioactive scaffold. The hydroxyl group offers significant advantages for the fusion of small molecules, enabling the generation of novel structures with straightforward synthetic routes and conforming to easily-synthesizable mechanisms. The increase in molecular weight is a major limitation of the structural hybridization approach. In fact, the permeability barriers caused by a high molecular weight are the primary reasons for the limited antibacterial activity exhibited by most antibiotics\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Given the bacterial cell wall and membrane structure, antibacterial compounds with a high molecular weight (\u0026gt;\u0026thinsp;600 g/mol) do not pass through non-selective protein channels, restricting uptake by bacteria to receptor-mediated endocytosis or passive diffusion\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e,\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e. In this study, we have selected the simple, low-molecular-weight, and easily-hybridisable FLO as the primary bioactive scaffold. Targeting the key bacterial ribosome 50S subunit PTC region of FLO, we employed a computer-aided drug design to develop a modular synthetic route, and investigated a broad range of structural variations. We discovered that, in general, short fragments have higher activity compared to their homologs. Smaller spatial conformations within the binding pocket reduce the level of inherent self-collision incidences the ligand may experience, leading to increased stability in binding. This also aligns with the lower molecular weight required for cell permeability. Although the conceptualization of an ideal blueprint may seem trivial, the discovery of F8 undoubtedly reaffirms that structural hybridization and chemical synthesis remain powerful means to supplement our arsenal of antibiotics.\u003c/p\u003e \u003cp\u003eF8 slows down the development of drug resistance and exhibits significant \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e antibacterial effects on resistant bacteria, which means that compared to traditional antibiotics, F8 has a novel mechanism of action or exerts its antibacterial effect through the combination of several mechanisms of action. Although the structural hybridization strategy is crucial and effective, the resulting compounds are ultimately influenced by the same type of resistance mechanism, which evolves and spreads in response to the resistance mechanism of previously-used compounds. In contrast, F8, due to its novel mechanism of action, bypasses existing drug resistance mechanisms and exhibits excellent \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e activity against drug resistance. Multi-omics analysis indicates that F8 affects multiple metabolic routes, including nitrogen metabolism, arginine biosynthesis, and alanine, aspartic acid, and glutamate biosynthesis. Arginine metabolism is strictly regulated in bacteria and arginine is essential for cell growth and biofilm formation\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. In the arginine catabolism pathway, arcA, arcB, and arcC are classified as the arginine deiminase pathway\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. Research indicates that in \u003cem\u003eS. aureus\u003c/em\u003e, the activation of the arginine deiminase pathway confers resistance to vancomycin\u003csup\u003e\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e, which can explain why \u003cem\u003eS. aureus\u003c/em\u003e showed a lower resistance to F8 in the process of continuous passage. Meanwhile, in ITC and DSF assays, we discovered and validated the target protein arcB of F8. This finding is consistent with our multi-omics analysis.\u003c/p\u003e \u003cp\u003eAs ArcB is involved in arginine degradation metabolism biosynthesis\u003csup\u003e\u003cspan additionalcitationids=\"CR18 CR19\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e, the report by Jakubovics et al.\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e suggests that arcB levels may limit the biosynthesis of arginine in \u003cem\u003eS. gordonii\u003c/em\u003e. Our research results also indicate that F8 can alter bacterial cell membrane permeability or disrupt the cell membrane structure, thus exerting an antibacterial or bactericidal effect. The expression of arcB is also known to be affected after treatment with antibiotics, including gentamicin and polymyxin B, suggesting that cell membrane stress influences ArcB activity\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. In addition, our research indicates that F8 increases the content of intracellular ROS, which may correspondingly exacerbate membrane damage and further disrupt bacterial homeostasis. Cells are sensitive to ROS, which is composed of oxygen and its derivatives (peroxides, superoxides, and hydroxyl radicals), as they can cause damage to bacterial DNA, proteins, and lipids\u003csup\u003e\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e. Therefore, bacteria have evolved methods to clear ROS, reducing them to less harmful byproducts\u003csup\u003e\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u003c/sup\u003e. Several studies have determined that arcA/arcB play a role in this ROS resistance, and in fact, the deletion of arcA or arcB has been shown to lead to increased sensitivity to hydrogen peroxide under aerobic conditions in \u003cem\u003eE. coli\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. It has been demonstrated that the arcA/arcB can regulate the synthesis and transport of proteins and amino acids, thereby affecting the adaptability of \u003cem\u003eE. coli\u003c/em\u003e under ROS stress\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e, thus, it is hypothesized that bactericidal antibiotics lead to metabolic instability and toxic ROS formation as part of their lethal action\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e,\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e,\u003cspan additionalcitationids=\"CR64\" citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e\u003c/sup\u003e. Considering the potential significant role of arcB in antimicrobial activities, further extensive work is required focusing on the study of arcB, including target knockout and key amino acid residue mutations, among other approaches. The role of F8 in arcB (a simple and immutable target) has expanded our understanding of antibiotic development to avoid drug resistance, and has guided the rational design of compounds with long-term clinical lifespan.\u003c/p\u003e \u003cp\u003eIn summary, our research findings suggest that F8 is a broad-spectrum antibacterial small molecule compound, exhibiting potent \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e antibacterial activity against both standard and drug-resistant strains, with a low propensity for the development of resistance. This mechanism of antibacterial action is thought to be based on competitive binding with arcB, thereby activating multiple pathways. Together, our results suggest that F8 may be a promising candidate drug for developing new antibiotic formulations to combat antibiotic-resistant bacteria-associated infections.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eStrains, media, and growth conditions\u003c/h2\u003e \u003cp\u003e \u003cem\u003eE. coli\u003c/em\u003e (ATCC 25922), \u003cem\u003eS. aureus\u003c/em\u003e (ATCC 29213), \u003cem\u003eS. typhi\u003c/em\u003e (CICC 110420), \u003cem\u003eP. aeruginosa\u003c/em\u003e (ATCC 9027), \u003cem\u003eB. subtilis\u003c/em\u003e (CMCC 63501), \u003cem\u003eE. faecalis\u003c/em\u003e (ATCC 29212), \u003cem\u003eP. multocida\u003c/em\u003e (ATCC 43137), \u003cem\u003eA. pleuropneumoniae\u003c/em\u003e (ATCC 27088), \u003cem\u003eS. suis\u003c/em\u003e (CVCC 606), \u003cem\u003eH. parasuis\u003c/em\u003e (ATCC 19417), methicillin-resistant \u003cem\u003eS. aureus\u003c/em\u003e (B1-1), polymyxin B-resistant \u003cem\u003eE. hormaechei\u003c/em\u003e (wb 4), FLO-resistant \u003cem\u003eS. suis\u003c/em\u003e (1136, 1194, 1197, 1655, 1658, 1669), FLO-resistant \u003cem\u003eH. parasuis\u003c/em\u003e (1565, 1614, 1651), ampicillin-resistant \u003cem\u003eS. typhi\u003c/em\u003e (BYG 9), sulfamethoxazole-resistant \u003cem\u003eS. typhi\u003c/em\u003e (BYG 21) and doxycycline-resistant \u003cem\u003eS. typhi\u003c/em\u003e (BYG 11, BYG 25, BYG 31) were used in this study. Trypticase soy broth (TSB), Mueller-Hinton broth (MHB), Muller-Hinton agar (MHA), LB, and LB nutrient agar were bought from Haibo Biotechnology (Qingdao, China). Cells were grown at 37\u0026deg;C on a rotating shaker at 300 rpm in flasks, or at 900 rpm in plate shakers.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMolecular docking studies\u003c/h3\u003e\n\u003cp\u003ePreparation of the binding site: The X-Ray crystal structure of the 50S subunit PTC during translation termination (the PTC region) with the PDB code 6c4h was used for the virtual screening. The binding pockets were confirmed using SYBYL-X Suite with default parameters. Hydrogen atoms were added to the structure, and considerations were made regarding correct orientation of Asn and Gln sidechains, ligands, and protein charges. All waters were removed except for the iron/sulphur complex. A diverse subset of the compound library (i.e., SPECS with 1,084,348 small-molecule compounds and a Topscience compound library with 16,594 natural products) was prepared to generate three-dimensional configurations by Surflex for searching SYBYL-X with all options set as default. Docking projects were conducted using Surflex-Dock GeomX in the SYBYL-X Suite. Scoring calculations were calculated by CScore in the SYBYL-X Suite.\u003c/p\u003e\n\u003ch3\u003eDesign and synthesis of broad-spectrum antibacterial small molecule compounds\u003c/h3\u003e\n\u003cp\u003eThe synthesis work was performed as follows: N,N-dimethylformamide (DMF, 20 mL) was added to a round-bottom flask, followed by the addition of 3-(1-Piperidinyl)propanoic acid (CAS: 26371-07-3, 2 g) until complete dissolution. Subsequently, 4-dimethylaminopyridine (DMAP, 1.5 g), 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride (EDC, 3 g), and FLO (2,2-Dichloro-N-[(1R,2S)-3-fluoro-1-hydroxy-1-(4-methylsulfonylphenyl)propan-2-yl]acetamide) (3 g) (Longxiang, China) were sequentially added. The reaction mixture was stirred at 25\u0026deg;C for 24 h. The output was monitored by thin-layer chromatography (TLC) (hexyl hydride/ethyl acetate [hexane/EtOAc]\u0026thinsp;=\u0026thinsp;1/1, retention factor [Rf]\u0026thinsp;=\u0026thinsp;0.25) and visualized by a 5% vanillin sulfuric acid/ethanol solution. The product was purified by column chromatography on a silica gel (hexane/EtOAc\u0026thinsp;=\u0026thinsp;2/1). Other derivatives, such as F1, F2, F3, F4, F6, F7, F11, F13, F14, and F15, were synthesized using the same procedure. Broad-spectrum antibacterial small molecule compounds were characterized by various techniques, including MS, \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003eH-NMR, and \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC-NMR (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed, Supplementary Table\u0026nbsp;1 and Supporting information).\u003c/p\u003e \u003cp\u003e \u003cb\u003eIn vitro\u003c/b\u003e \u003cb\u003esusceptibility\u003c/b\u003e\u003c/p\u003e \u003cp\u003e The MICs of antibiotics were determined by the broth microdilution method following the Clinical and Laboratory Standards Institute (CLSI) guidelines. Briefly, single bacterial colonies were cultured in MHB at 37\u0026deg;C at 220 rpm for 8\u0026ndash;12 h. Subsequently, the potential lead candidates or other antibiotics were diluted two-fold in MHB and mixed with an equal volume of bacterial suspensions in MHB containing approximately 1 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e CFU/mL in a clear UV-sterilized 96‐well microtiter plate. The plate was placed in the incubator for 18 h at 37\u0026deg;C, then the MIC values were read. MIC values were defined as the lowest concentrations of antibiotics with no visible growth of bacteria.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eGrowth curves of bacteria\u003c/h2\u003e \u003cp\u003eA single colony of \u003cem\u003eE. coli\u003c/em\u003e ATCC 25922 or \u003cem\u003eS. aureus\u003c/em\u003e ATCC 29213 was selected and inoculated into MHB for cultivation. The cultures were incubated at 37\u0026deg;C with shaking at 200 rpm for overnight growth. The overnight culture was standardized to a 0.5 McFarland turbidity standard. It was then diluted at 1:100 in MHB, and adjusted to approximately 1 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e CFU/mL. Different concentrations of FLO or F8 were added to a 96-well microplate and mixed with an equal volume of bacterial dilution, then sealed with a plate lid. The plate was incubated at 37\u0026deg;C, and the wavelength of 600 nm was measured every 1 h using an Infinite M200 Microplate reader (Tecan).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eTime-kill curves\u003c/h2\u003e \u003cp\u003eA single colony of \u003cem\u003eE. coli\u003c/em\u003e ATCC 25922 or \u003cem\u003eS. aureus\u003c/em\u003e ATCC 29213 was selected and inoculated into MHB for cultivation. The cultures were incubated at 37\u0026deg;C with shaking at 200 rpm for overnight growth. The overnight cultured bacteria were diluted at 1:100 in MHB and adjusted to approximately 1 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e CFU/mL. Different concentrations of FLO and F8 (4 \u0026times; MIC, 2 \u0026times; MIC, MIC, 1/2 \u0026times; MIC) were added to the diluted bacterial suspension. The cultures were then incubated at 37\u0026deg;C with shaking at 200 rpm. After incubation for 30 min, 1, 2, 4, 8, and 24 h, 100 \u0026micro;L aliquots were taken, serially diluted 10-fold, then inoculated on MHA plates (with the inclusion of three parallel plates), and the colony forming units (CFU) were calculated after incubation for 24 h at 37\u0026deg;C.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eMembrane integrity assay\u003c/h2\u003e \u003cp\u003eOvernight cultures of the bacterial fluid were centrifuged and resuspended in 0.01 mol/L PBS (pH 7.4), and the bacterial suspensions were adjusted to an OD600 nm of approximately 0.5. Subsequently, FLO or F8 was added at a final concentration of 4 \u0026times; MIC, and the bacterial suspensions were incubated at 37\u0026deg;C for 2 h without light. The suspensions were then incubated with propidium iodide (PI, Thermo Scientific, P1304MP) at a final concentration of 10 nmol/L for 20 min before the fluorescence values were quantified using an Infinite M200 Microplate reader (Tecan) at an excitation wavelength of 535 nm and an emission wavelength of 615 nm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eMembrane permeability measurement\u003c/h2\u003e \u003cp\u003eOvernight cultures of the bacterial fluid were centrifuged and resuspended in 0.01 mol/L PBS (pH 7.4), and the bacterial suspensions were adjusted to approximately 1 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e CFU/mL. The diluted bacterial cultures were supplemented with different concentrations of FLO and F8 (4 \u0026times; MIC, 2 \u0026times; MIC, MIC) and incubated at 37\u0026deg;C with shaking at 150 rpm. After incubation for 2, 4, and 8 h, the bacterial suspensions were collected, and the supernatant was obtained by centrifugation at 12,000 rpm for 2 min. The DNA, RNA, and protein contents were measured using a Nano Drop microspectrophotometer.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eΔpH measurement\u003c/h2\u003e \u003cp\u003eOvernight cultures of the bacterial fluid were centrifuged and resuspended in 0.01 mol/L PBS (pH 7.4), and the bacterial suspensions were adjusted to an OD600 nm of approximately 0.5. Subsequently, 10 \u0026micro;L of FLO or F8 (final concentration of 4 \u0026times; MIC) was added, followed by the addition of a pH-sensitive fluorescent probe, BCECF-AM, at a final concentration of 10 \u0026micro;mol/L. The mixture was then incubated at 37\u0026deg;C and fluorescence measurements were taken every 5 min using the Infinite M200 Microplate reader (Tecan) with an excitation wavelength of 488 nm and an emission wavelength of 535 nm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eATP determination\u003c/h2\u003e \u003cp\u003eExtracellular and intracellular ATP levels were determined using the Enhanced ATP Assay Kit (Beyotime, Cat. No. S29213). Overnight cultures of the bacterial fluid were centrifuged and resuspended in 0.01 mol/L PBS (pH 7.4), and the bacterial suspensions were adjusted to an OD600 nm of approximately 0.5. After treatment with 4 \u0026times; MIC of FLO or F8 for 2 h, the bacterial cultures were centrifuged at 12,000 rpm for 5 min at 4\u0026deg;C, and the supernatant was collected to determine extracellular ATP levels. Meanwhile, the bacterial precipitate was lysed with lysozyme, centrifuged, and the supernatant was prepared to determine the intracellular ATP levels. The assay solution was added to a 96-well plate and incubated for another 5 min at room temperature. The supernatants were added to the well and mixed quickly before recording in the model of luminescence using the Infinite M200 Microplate reader (Tecan).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eROS measurement\u003c/h2\u003e \u003cp\u003eOvernight cultures of the bacterial fluid were centrifuged and resuspended in 0.01 mol/L PBS (pH 7.4), and the bacterial suspensions were adjusted to an OD600 nm of approximately 0.5. The fluorescent probe, 2\u0026rsquo;,7\u0026rsquo;-dichlorofluorescein diacetate (DCFH-DA) (10 \u0026micro;mol/L), was used to detect ROS accumulation in the bacterial fluids after FLO or F8 treatment, following the manufacturer's instructions (Beyotime, Cat. No. S0033). Briefly, DCFH-DA was added to the bacterial suspension and incubated at 37\u0026deg;C for 20 min. After washing three times with 0.01 mol/L PBS, 190 \u0026micro;L of the bacterial suspension was added to a 96-well microplate and mixed with 10 \u0026micro;L of FLO or F8 (final concentration of 4 \u0026times; MIC). After incubation for 2 h, the fluorescent values were measured using the Infinite M200 Microplate reader (Tecan) with an excitation wavelength of 488 nm and an emission wavelength of 525 nm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eTranscriptomics\u003c/h2\u003e \u003cp\u003eUntreated and FLO- or F8-treated \u003cem\u003eS. aureus\u003c/em\u003e ATCC 29213 were used for transcriptomics studies. FLO or F8 (final concentration of 4 \u0026times; MIC) was added to the bacterial suspension and incubated for 2 h. The bacterial cultures were then washed three times with 0.01 mol/L PBS (pH 7.4) and centrifuged at 8,000 rpm for 10 min at 4\u0026deg;C to collect the bacterial precipitates. RNA-Seq was performed using the Illumina platform at Shanghai Majorbio Bio-pharm Technology Co., Ltd. (Shanghai, China). Total RNA was extracted from the samples, and the concentration and purity of the extracted RNA were determined using Nanodrop 2000. The integrity of the RNA was assessed by agarose gel electrophoresis, and the RNA integrity was further evaluated using the Agilent 2100 Bioanalyzer. Genes with adjusted \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, identified by DESeq, were considered differentially expressed. According to the KEGG analysis, differentially-significant genes were assigned to different functional groups. The data was uploaded to the Majorbio cloud platform (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://cloud.majorbio.com\u003c/span\u003e\u003cspan address=\"https://cloud.majorbio.com\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) after the database search for data analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eMetabolomics\u003c/h2\u003e \u003cp\u003eUntreated and FLO- or F8-treated \u003cem\u003eS. aureus\u003c/em\u003e ATCC 29213 were used for metabolomics studies. FLO or F8 (final concentration of 4 \u0026times; MIC) was added to the bacterial suspension and incubated for 2 h. The bacterial cultures were then washed three times with 0.01 mol/L PBS (pH 7.4) and centrifuged at 8,000 rpm for 10 min at 4\u0026deg;C to collect the bacterial precipitates. 50 mg bacterial precipitates were accurately weighed, and the metabolites were extracted using a 400 \u0026micro;L methanol:water (4:1, v/v) solution with 0.02 mg/mL L-2-chlorophenylalanin as an internal standard. The mixture was allowed to settle at -10\u0026deg;C, and was treated using the high throughput tissue crusher, Wonbio-96c (Shanghai Wanbo Biotechnology Co., LTD), at 50 Hz for 6 min, followed by sonication at 40 kHz for 30 min at 5\u0026deg;C. The samples were placed at -20\u0026deg;C for 30 min to precipitate the proteins. After centrifugation at 13,000 g at 4\u0026deg;C for 15 min, the supernatant was carefully transferred to sample vials for LC-MS/MS analysis. The instrument platform for this LC-MS analysis was UHPLC-Q Exactive HF-X system of Thermo Fisher Scientific. The data was uploaded to the Majorbio cloud platform (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://cloud.majorbio.com\u003c/span\u003e\u003cspan address=\"https://cloud.majorbio.com\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) after the database search for data analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eProteomics\u003c/h2\u003e \u003cp\u003eUntreated and FLO- or F8-treated \u003cem\u003eS. aureus\u003c/em\u003e ATCC 29213 were used for proteomics studies. FLO or F8 (final concentration of 4 \u0026times; MIC) was added to the bacterial suspension and incubated for 2 h. The bacterial cultures were then washed three times with 0.01 mol/L PBS (pH 7.4) and centrifuged at 8,000 rpm for 10 min at 4\u0026deg;C to collect the bacterial precipitates. The bacterial precipitates were removed and put on ice. An appropriate amount of protein lysate was then added, (8 M urea, 1% SDS) and the precipitates were sonicated for 2 min at a low temperature, then split for 30 min. After centrifugation at 12,000g at 4\u0026deg;C for 30min, the concentration of protein supernatant was determined through the Bicinchoninic acid (BCA) method using the BCA Protein Assay Kit (Pierce, Thermo, USA). Protein samples at 100 \u0026micro;g, TEAB (Triethylammonium bicarbonate buffer), and TCEP (tris (2-carboxyethyl) phosphine) was combined for reaction for 60 min at 37\u0026deg;C. IAM (Iodoacetamide) was added to the final concentration at 40 mM, and reacted for 40 min at room temperature under dark conditions. A certain percentage (acetone: sample v/v\u0026thinsp;=\u0026thinsp;6:1) of pre-cooled acetone was added to each sample, then allowed to settle for 4 h at -20\u0026deg;C. After centrifugation for 20 min at 10,000 g, the sediment was collected, and 100 \u0026micro;L of 100mM TEAB solution was added. Finally, the mixture was digested with Trypsin overnight at 37\u0026deg;C, and added at a 1:50 trypsin-to-protein mass ratio. Trypsin-digested peptides were analysed using online nano flow liquid chromatography tandem mass spectrometry performed on an EASY-nLC 1200 system (Thermo, USA) connected to a Q Exactive HF-X quadrupole orbitrap mass spectrometer (Thermo, USA) through a nanoelectrospray ion source. The data was uploaded to the Majorbio cloud platform (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://cloud.majorbio.com\u003c/span\u003e\u003cspan address=\"https://cloud.majorbio.com\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) after the database search for data analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eProtein expression and purification\u003c/h2\u003e \u003cp\u003ePlasmids pET28a-AcrA and pET28a-AcrB were constructed after codon optimization in \u003cem\u003eE. coli\u003c/em\u003e. The plasmid was amplified in DH5α-competent cells, then transformed into BL21 (DE3)- competent cells. After overnight incubation, single colonies were picked and transferred to LB medium. The cultures were shaken at 37\u0026deg;C for 4\u0026ndash;6 h until the OD600 reached approximately 0.8, before induction of fusion protein expression by 0.1 mM isopropyl thio-β-d-galactoside (IPTG) at 37\u0026deg;C for 5 h. Then, the cells were harvested by centrifugation and purified. Briefly, the cells were sonicated in PBS, the homogenate was centrifuged (12,000 g, 10 min, 4\u0026deg;C), and the pellet was collected. The supernatant was passed through the Ni-NTA column at a flow rate of 1.0 mL/min by a peristaltic pump at 4\u0026deg;C overnight, then passed onto the binding buffer (50 mM sodium phosphate buffer pH 8, 500 mM NaCl and 10 mM imidazole). The bacterial proteins were then removed with elution buffer A, which contained 30 mM imidazole (50 mM sodium phosphate buffer pH 8, 500 mM NaCl, and 30 mM imidazole) in 20 mL. Next, the column was eluted with elution buffer B and 200 mM imidazole (50 mM sodium phosphate buffer pH 8, 500 mM NaCl, and 200 mM imidazole) for 20 mL to obtain the target protein. The crude product was washed with PBS via a 10 kDa millipore centrifugal ultrafiltration tube to remove imidazole. The fractions of pure product, total protein, supernatant, and precipitate were then analysed by SDS-PAGE and WB using an anti-His tag antibody (AE003, ABclonal, China).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eReal-time quantitative polymerase chain reaction (RT-qPCR)\u003c/h2\u003e \u003cp\u003eCellular total RNA was extracted using the RNA Isolater Total RNA Extraction Reagent (Vazyme, Cat. No. RC112-01), following the manufacturer's instructions. Complementary DNA (cDNA) was synthesized using HiScript II Q Select RT SuperMix for qPCR with gDNA wiper (Vazyme, Cat. No. R233-01) in a total volume of 10 \u0026micro;L. RT-qPCR was performed using 2X Universal SYBR Green Rapid qPCR Mix (Abclonal, Cat. No. RK21203) with 100 ng of cDNA and 5 nM primer pairs at a time. The results were monitored using the CFX96 Real-Time PCR Assay System (Bio-Rad, USA). Supplementary Table\u0026nbsp;2 lists all primers used in the quantitative PCR.\u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eIsothermal titration calorimetry (ITC)\u003c/h2\u003e \u003cp\u003eTo assess the interaction between the ligand and the protein, ITC experiments were conducted using MicroCal ITC at 25\u0026deg;C. All titrations were performed at 25\u0026deg;C while stirring at 300 rpm in PBS. A control experiment of titrant into buffer was performed to account for the heat of dilution. All titrations were repeated at least three times with similar results. For ligand-protein titrations, an approximate protein concentration of 50 \u0026micro;M was used. The concentration of the ligand is approximately ten times higher than that of the protein. All ITC experiments were carried out and analysed using Launch NanoAnalyze Software.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eDifferential scanning fluorimetry (DSF)\u003c/h2\u003e \u003cp\u003eFor the DSF experiments, 20 \u0026micro;L samples were prepared in duplicate using 100 \u0026micro;M of protein and a compound concentration of 50 \u0026micro;M. The samples were heated from 20 to 95\u0026deg;C with increments of 1\u0026deg;C/min before incubation for 20 min, and fluorescence was measured at each step in nanoDSF (Nano Temper Prometheus NT.48, Germany). The change in melting temperature (T\u003csub\u003em\u003c/sub\u003e) values was calculated and recorded by the instrument. Data analysis and image generation were performed using PR. ChemControl Software.\u003c/p\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003ch2\u003eHaemolytic activity\u003c/h2\u003e \u003cp\u003eSterile defibrinated sheep haemocytes were washed three times with PBS, then 100 uL of 8% haemocytes was added to 100 uL of FLO or F8 at different concentrations (0, 1, 2, 4, 8, 16, 32, 64, 128, 256, and 512 \u0026micro;M). Meanwhile, 0.2% Triton X-100 and PBS were used as positive and negative controls, respectively. The solution was placed in a 96-well microtiter plate and was incubated at 37\u0026deg;C for 1 h before centrifugation at 3,000 g for 10 min. Then, 100 \u0026micro;L of the supernatant was taken, and its absorbance was determined at 576 nm by an Infinity M200 Microplate reader (Tecan).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section3\"\u003e \u003ch2\u003eCytotoxicity\u003c/h2\u003e \u003cp\u003eThe cytotoxicity of FLO or F8 on pk15, Vero, and L-02 cells was estimated using the CCK-8 Cell Proliferation and Cytotoxicity Assay Kit (CCK-8, Solarbio, Cat. No. CA1210). Cells were seeded, counted, and evenly distributed into 96-well microtiter plates at a cell density of 10\u003csup\u003e5\u003c/sup\u003e cells per well, and incubated for 1 day at 37\u0026deg;C and 5% CO\u003csub\u003e2\u003c/sub\u003e to promote cell growth. Subsequently, cells were treated with different concentrations of FLO or F8, with six replicates for each condition. After incubating at 37\u0026deg;C and 5% CO\u003csub\u003e2\u003c/sub\u003e for 24 h, 10\u0026micro;L of CCK-8 solution was added to each well, then further incubated for 2 h. The absorbance was then measured at 450 nm using an Infinite M200 Microplate reader (Tecan).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section3\"\u003e \u003ch2\u003eResistance-development studies\u003c/h2\u003e \u003cp\u003eA single colony of \u003cem\u003eE. coli\u003c/em\u003e ATCC 25922 or \u003cem\u003eS. aureus\u003c/em\u003e ATCC 29213 was picked and inoculated into MHB for overnight cultivation. Overnight cultures were inoculated in fresh MHB containing 1/2, 1, 2, and 4 \u0026times; MIC of FLO or F8. The bacterial cultures were then incubated at 37\u0026deg;C for 24 h at 200 rpm with continuous shaking. Subsequently, the MIC of bacteria from the second-highest concentrations with visible growth (OD600 nm\u0026thinsp;\u0026ge;\u0026thinsp;0.3) was determined by broth microdilution in fresh MHB media containing different concentrations of FLO or F8. The cultures were serially passaged for 30 days.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec28\" class=\"Section2\"\u003e \u003ch2\u003eAnimal studies\u003c/h2\u003e \u003cp\u003e All experimental procedures were conducted in accordance with animal welfare guidelines, and were previously approved by the Animal Welfare and Ethics Committee of the Huazhong Agricultural University Wuhan, China (approval permit numbers: 202311010007 and 202311010008). All specific pathogen-free (SPF) Kunming mice (6\u0026ndash;7 weeks old, weighing approximately 25 g) and SPF C57BL/6 mice (6\u0026ndash;7 weeks old, weighing 18\u0026thinsp;\u0026plusmn;\u0026thinsp;2 g) were purchased from Hubei Provincial Laboratory Animal Center (Wuhan, China). Animals were housed under standard humidity (50\u0026thinsp;\u0026plusmn;\u0026thinsp;10%), temperature (25\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C), and light-dark cycle (12 h each) conditions with free access to food and water.\u003c/p\u003e \u003cp\u003eTo study the toxicity of FLO and F8, SPF Kunming mice were randomly divided into three groups and weighed once a day. Each group was given a dose of 1,500 mg/kg of body weight (bw) of FLO or F8 for seven consecutive days, and their health conditions were observed daily. The control mice were given the same volume of solvent. These doses did not cause any deaths and were considered safe in this experiment. The mice were euthanized and dissected seven days later, and the blood, spleen, kidneys, thymus, liver, small intestine, and femur were removed and collected for further analysis. For acute toxicity assays of FLO and F8, survival and mortality status were recorded after a single administration of 5,000 mg/kg bw, followed by 14 days of observation of the surviving mice once a day.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec29\" class=\"Section2\"\u003e \u003ch2\u003eMouse systemic infection study\u003c/h2\u003e \u003cp\u003eTo evaluate the anti-infective effects of FLO and F8, a mouse systemic infection model was established. Briefly, the experiments were performed using SPF C57BL/6 mice (6\u0026ndash;7 weeks old, weighing 18\u0026thinsp;\u0026plusmn;\u0026thinsp;2 g), and the mice were infected by intraperitoneal injection with \u003cem\u003eS. aureus\u003c/em\u003e ATCC 29213 at a dose of 5 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e CFU, or a pre-induced FLO-resistant \u003cem\u003eS. aureus\u003c/em\u003e suspension (n\u0026thinsp;=\u0026thinsp;6). One hour after the infection, mice were administered a dose of 60 mg/kg bw of either FLO or F8. The control mice were given an equivalent volume of the solvent. The health status of the mice was observed, and once an infected mouse died, the blood, liver, spleen, and kidneys were collected for subsequent analysis. At 24 h post-infection, surviving mice were euthanized by cervical dislocation, and the blood, liver, spleen, and kidneys were collected for bacterial CFU analysis and histopathological evaluation. Furthermore, the same mouse mice were given a single dose of FLO or F8 at a dose of 60 mg/kg bw, and the survival status of the mice was observed within 72 h (n\u0026thinsp;=\u0026thinsp;10). The control mice were given the same volume of solvent.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eHistopathology\u003c/h3\u003e\n\u003cp\u003eAfter decapitation, the tissues were immediately fixed in 4% paraformaldehyde in phosphate buffer and stored at 4\u0026deg;C. For paraffin embedding, the organs were washed and dehydrated through a series of graded ethanol baths, followed by embedding in paraffin wax. Serial sections of 5 \u0026micro;m thickness were cut using a microtome and stained with haematoxylin-eosin (H\u0026amp;E). Sections were observed using a light microscope.\u003c/p\u003e \u003cdiv id=\"Sec31\" class=\"Section2\"\u003e \u003ch2\u003eTUNEL reaction\u003c/h2\u003e \u003cp\u003eSerial sections of 5 \u0026micro;m thickness were cut using a microtome, and sections were deparaffinized and rehydrated. Proteins were digested by placing tissue sections in 20 mg/mL of proteinase K (Merck, Germany) and incubated at 37\u0026deg;C for 15 min. Endogenous peroxidase was inactivated with 3% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e in methanol for 10 min at room temperature, then the sections were incubated with 50 \u0026micro;L of the TUNEL reaction mixture at 37\u0026deg;C for 1 h. They were protected from light, rinsed three times with PBS, and visualized under a fluorescence microscope with excitation wavelengths in the range of 450\u0026ndash;500 nm and detection wavelengths in the range of 515\u0026ndash;565 nm (green).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec32\" class=\"Section2\"\u003e \u003ch2\u003eEnzyme-linked immunosorbent assay (ELISA)\u003c/h2\u003e \u003cp\u003eThe tissues were weighed and homogenized in PBS. After centrifugation at 3,000 rpm for 30 min, the supernatant was collected to quantify IL-6, IL-2, and Hsp70 levels with ELISA kits (MSKBIO, China), used according to the instructions.\u003c/p\u003e \u003cdiv id=\"Sec33\" class=\"Section3\"\u003e \u003ch2\u003eStatistical analyses\u003c/h2\u003e \u003cp\u003eEach reaction was performed in triplicate, and the results are expressed as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant (not significant [n.s], 0.01\u0026thinsp;\u0026le;\u0026thinsp;\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 [*], \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01 [**]). Statistical analyses and graphical presentations were performed using GraphPad Prism 8 (GraphPad Prism Inc., San Diego, CA, USA).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003earcB\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eornithine carbamoyltransferase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003earcA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003earginine deiminase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003earcC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ecarbamate kinase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eADI\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003earginine deiminase pathway\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCADD\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ecomputer assisted drug design\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCFU\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ecolony forming units\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDSF\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eDifferential Scanning Fluorimetry\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDBD\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003edrug binding domain\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDEGs\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003edifferentially expressed genes\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDEPs\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003edifferentially expressed proteins\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDCFH-DA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e2\u0026rsquo;,7\u0026rsquo;-dichlorofluorescein diacetate\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eELISA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eenzyme-linked immunosorbent assay\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eFLO\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eflorfenicol\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHGB\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ehemoglobin\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eH\u0026amp;E\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ehematoxylin-eosin\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eITC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eIsothermal Titration Calorimetry\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eLym\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003elymphocyte\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMIC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eminimum inhibitory concentration\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMBC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eminimum bactericidal concentration\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMRSA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003emethicillin-resistant \u003cem\u003eS. aureus\u003c/em\u003e\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNeu\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eneutrophil\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePTC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003epeptidyl transferase center\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePLT\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eplatelet\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePMF\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eproton motive force\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePI\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003epropidium iodide\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eRBCs\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ered blood cells\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eROS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ereactive oxygen species\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSPF\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003especific pathogen free\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eWBCs\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ewhite blood cells.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data that support the findings of this study are available from the corresponding author on request. Source data are provided with this paper.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Key Research and Development Program of China (2018YFC1603005), National Natural Science Foundation of China (NSFC) (32072925), and Fundamental Research Funds for the Central Universities (2662020DKPY020).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJin Feng conducted in vitro and in vivo antibacterial experiments and multi-omics analysis experiments. Youle Zheng synthesized small molecule compounds. Youle Zheng and Wanqing Ma conducted protein expression and purification experiments. Jin Feng conducted and analysed results from all additional experiments. Yindi Xu, Defeng Weng and Zhifang Wang provided reagents. Xu Wang supervised the research, coordination and strategy. All authors provided critical revisions and approved the final manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests. \u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eMitcheltree MJ et al (2021) A synthetic antibiotic class overcoming bacterial multidrug resistance. Nature 599:507\u0026ndash;512. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/s41586-021-04045-6\u003c/span\u003e\u003cspan address=\"10.1038/s41586-021-04045-6\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCassini A et al (2019) Attributable deaths and disability-adjusted life-years caused by infections with antibiotic-resistant bacteria in the EU and the European Economic Area in 2015: a population-level modelling analysis. 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Sci (New York N Y) 336:315\u0026ndash;319. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1126/science.1219192\u003c/span\u003e\u003cspan address=\"10.1126/science.1219192\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"antibiotic resistance, structural hybridization, arcB, bacteria, drug, target","lastPublishedDoi":"10.21203/rs.3.rs-3667988/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3667988/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe lack of new drugs that are effective against antibiotic-resistant bacteria has caused increasing concern in global public health. As antibiotic resistance continues to escalate worldwide, the development of new antibiotics that can effectively treat bacterial infections is crucial. Based on this study, we report the development of a hybrid antimicrobial drug that is rationally designed through drug structural hybridization-based structure-guided design and component-based synthesis. The optimal modified compound, F8, was identified, which demonstrated excellent \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e anti-resistant bacterial activity and effectively mitigated the development of resistance. F8 exhibits significant bactericidal activity against bacteria resistant to antibiotics such as methicillin, polymyxin B, florfenicol, doxycycline, ampicillin and sulfamethoxazole. In the mouse model of drug-resistant bacterial bacteremia, F8 was found to increase survival and significantly reduce bacterial load in infected mice. Multi-omics analysis (transcriptomics, proteomics, and metabolomics) have indicated that ornithine carbamoyl transferase (arcB) is a novel antimicrobial target of F8. Further molecular docking, Isothermal Titration Calorimetry (ITC), and Differential Scanning Fluorimetry (DSF) studies verified arcB as a novel and effective target for F8. Finally, mechanistic studies suggest that F8 competitively binds to arcB, disrupting the bacterial cell membrane and inducing a certain degree of oxidative damage. The findings of this study highlight F8 as a promising candidate drug for the development of novel antibiotic formulations to combat antibiotic-resistant bacteria-associated infections.\u003c/p\u003e","manuscriptTitle":"A synthetic antibiotic class with a deeply-optimized design for overcoming bacterial resistance","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-12-13 19:58:45","doi":"10.21203/rs.3.rs-3667988/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"b0e5fdbc-1bdd-4df6-b7b9-2d096f95d9dc","owner":[],"postedDate":"December 13th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":26929818,"name":"Biological sciences/Microbiology/Antimicrobials/Antimicrobial resistance"},{"id":26929819,"name":"Biological sciences/Drug discovery/Drug delivery"}],"tags":[],"updatedAt":"2023-12-13T19:58:45+00:00","versionOfRecord":[],"versionCreatedAt":"2023-12-13 19:58:45","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3667988","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3667988","identity":"rs-3667988","version":["v1"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}