Rational Design of Thrombin-Derived VFR12 Analogs with Enhanced Antimicrobial, Antibiofilm, and Anti-inflammatory Properties

Rational Design of Thrombin-Derived VFR12 Analogs with Enhanced Antimicrobial, Antibiofilm, and Anti-inflammatory Properties | 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 Rational Design of Thrombin-Derived VFR12 Analogs with Enhanced Antimicrobial, Antibiofilm, and Anti-inflammatory Properties Ishrat Jahan, S. Dinesh Kumar, Chelladurai Ajish, Chul Won Lee, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7306037/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 19 Nov, 2025 Read the published version in Scientific Reports → Version 1 posted 11 You are reading this latest preprint version Abstract The worsening crisis of antibiotic resistance demands innovative therapies to combat multidrug-resistant pathogens. In this study, we report the rational design and optimization of VFR12 (VFRLKKWIQKVI), a thrombin C-terminus-derived 12-mer peptide, via systematic amino acid substitutions and stereochemical modifications. A series of 12-mer analogs were synthesized and assessed for antimicrobial activity, cell selectivity, biofilm disruption, and immunomodulatory effects. Six lead candidates (VFR12-a7, VFR12-a8, VFR12-a7(L), VFR12-a8(L), VFR12-a7(L)-d, VFR12-a8(L)-d) demonstrated potent, broad-spectrum antimicrobial activity against both Gram-positive and Gram-negative bacteria, including multidrug-resistant Pseudomonas aeruginosa , while exhibiting minimal hemolytic activity. Notably, D-amino acid variants VFR12-a7(L)-d and VFR12-a8(L)-d showed significantly improved therapeutic indices, complete resistance to proteolysis, and enhanced serum stability compared to their L-isomers. These peptides effectively inhibited biofilm formation and disrupted preformed biofilms while maintaining excellent biocompatibility. Furthermore, anti-inflammatory assessments revealed a significant suppression of LPS-induced production of TNF-α, IL-6, and nitric oxide, along with strong endotoxin neutralization. Among the analogs, VFR12-a8(L)-d emerged as the most promising candidate, combining potent antimicrobial activity with excellent safety and multifaceted therapeutic properties. These findings provide a valuable framework for the development of next-generation host defense peptides with integrated antimicrobial, antibiofilm, and anti-inflammatory properties, offering a multifaceted approach to tackling antibiotic resistance and sepsis. Biological sciences/Drug discovery Biological sciences/Microbiology antimicrobial peptide thrombin-derived peptide D-isomer antibiofilm immunomodulation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Please have a look at courier new font provided for text in article. The escalating crisis of antibiotic resistance, coupled with a shrinking antimicrobial pipeline, has become one of the biggest threats to global health. This crisis stems from the widespread misuse and overuse of antibiotics, which has led to the rise of superbugs that can resist multiple drugs. The most dangerous of these are known as ESKAPE pathogens− Enterococcus faecium , Staphylococcus aureus , Klebsiella pneumoniae , Acinetobacter baumannii , Pseudomonas aeruginosa , and Enterobacter species 1 – 4 . These bacteria have been identified as high-priority targets because they resist most available treatments and cause serious infections that are becoming increasingly difficult to cure 1 , 2 , 4 – 6 . To address this urgent medical need, researchers have turned to antimicrobial peptides (AMPs), also known as host defense peptides (HDPs), which are natural infection-fighting molecules found in plants, animals, and humans. These bioactive molecules exhibit multifaceted therapeutic properties encompassing broad-spectrum antimicrobial efficacy, biofilm disruption capabilities, and immunomodulatory functions that distinguish them from conventional antibiotics. The antimicrobial mechanisms of peptides involve complex interactions with microbial cell membranes, where electrostatic attraction to negatively charged membrane phospholipids facilitates initial binding, followed by membrane insertion, pore formation, and subsequent cellular lysis 7 – 10 . Additionally, these peptides demonstrate the capacity for intracellular target engagement and biofilm matrix disruption, contributing to their comprehensive anti-pathogenic activity profile. Currently, AMPs are being developed and used in clinical settings primarily for treating bacterial infections, promoting wound healing, and managing inflammatory conditions, positions them as promising therapeutic alternatives in the post-antibiotic era 11 – 14 . Thrombin C-terminal peptides (TCPs) represent a novel and significant class of host defense peptides that emerge from the proteolytic cleavage of human thrombin, a central enzyme in the coagulation cascade. These peptides are generated in vivo during wound healing and inflammation through the action of neutrophil elastase and other proteases released at sites of tissue injury. The discovery of TCPs has revealed an unexpected connection between the coagulation system and innate immune defense, demonstrating that hemostatic and antimicrobial responses are more integrated than previously understood 15 – 17 . The prototypic thrombin-derived peptide GKY25 (GKYGFYTHVFRLKKWIQKVIDQFGE) exemplifies the multifunctional nature of these molecules, exhibiting classical antimicrobial peptide characteristics including cationicity, amphipathicity, and an α-helical structure. Under physiological conditions, these peptides demonstrate broad-spectrum antimicrobial activity against both Gram-positive and Gram-negative bacteria through membrane-disrupting mechanisms, while simultaneously providing immunomodulatory functions by inhibiting macrophage responses to bacterial lipopolysaccharide. In murine models, TCPs have shown protective effects against Pseudomonas aeruginosa sepsis and lipopolysaccharide-induced shock, validating their therapeutic potential 15 , 16 , 18 – 21 . Previous investigations demonstrated that the prototypic thrombin-derived peptide GKY25 exhibits optimal antimicrobial and anti-inflammatory activities, requiring a peptide length of at least 20 amino acids for maximum therapeutic efficacy 15 . However, structure-activity relationship studies revealed that shorter variants retain significant antimicrobial effects, indicating the potential for peptide truncation without loss of biological activity 9 , 22 , 23 . In this study, we selected the central core sequence VFR12 (VFRLKKWIQKVI) from thrombin C-terminal peptides (TCPs) as our lead fragment for rational peptide design. We synthesized a systematic series of analogs through strategic amino acid substitutions and stereochemical modifications with three primary objectives: (1) enhancement of antimicrobial potency against both planktonic bacteria and biofilm-associated infections, (2) improvement of cell selectivity to achieve superior therapeutic indices with reduced cytotoxicity toward mammalian cells, and (3) optimization of immunomodulatory properties to provide beneficial anti-inflammatory effects that complement direct antimicrobial activity. This comprehensive approach aims to develop next-generation host defense peptides that overcome the limitations of current antimicrobial agents while retaining the multifaceted therapeutic advantages inherent in thrombin-derived peptides. Results Peptide design A series of short amphipathic α-helical peptides was rationally designed based on VFR12, a truncated thrombin-derived peptide, to explore structure-activity relationships through systematic amino acid substitutions and stereochemical modifications. The helical wheel projections (Fig. 1 a) illustrate the structural organization of analogs with strategic modifications. The parent VFR12 underwent sequential optimization: VFR12-a1 featured a phenylalanine-to-lysine substitution, increasing net charge (+ 1) and hydrophobic moment, while VFR12-a2 incorporated a glutamine-to-lysine replacement, enhancing charge with minimal hydrophobicity changes. Subsequent analogs (VFR12-a3 to VFR12-a9) involved strategic modifications by removing phenylalanine and glutamine and substituting with lysine or arginine in multiple positions to systematically increase cationicity and amphipathicity, progressively enhancing hydrophobic moments from 0.610 to 0.877 (Table 1 ). Key structural modifications included: VFR12-a6 versus VFR12-a7 (arginine to lysine substitution), VFR12-a7 versus VFR12-a8 (single tryptophan to isoleucine replacement), and VFR12-a6 versus VFR12-a9 (single tryptophan to isoleucine replacement) in the same position. For stereochemical optimization, VFR12-a7 and VFR12-a8 were selected for D-amino acid incorporation, where all L-amino acids were replaced with D-isomers. Due to the prohibitive cost of Fmoc-D-Ile and Fmoc-D-allo-Ile for large-scale solid-phase synthesis, D-isoleucine was substituted with D-leucine, maintaining equivalent molecular weight and hydrophobic character while ensuring synthetic feasibility. Given the minimal structural variations among VFR12-a6 through VFR12-a9, these analogs were selected for comprehensive mechanistic evaluation. Table 1 Amino acid sequence and physicochemical properties of VFR-12 and its analogs. Peptides Amino acid sequence a MS analysis b Net charge R t c Hydrophobic moment (μH) d z m/z calculated m/z observed VFR12 VFR12-a1 VFR12-a2 VFR12-a3 VFR12-a4 VFR12-a5 VFR12-a6 VFR12-a7 VFR12-a7(L) VFR12-a8 VFR12-a8(L) VFR12-a9 VFRLKKWIQKVI-NH 2 V K RLKKWIQKVI-NH 2 VFRLKKWI K KVI-NH 2 V K RLKKWI K KVI-NH 2 V RR L RR WI RR VI-NH 2 V KK L KK WI K KVI-NH 2 V RR L WR WI RR VI-NH 2 V KK L W KWI K KVI-NH 2 V KK L W KW LK KV L -NH 2 V KK L I KWI K KVI-NH 2 V KK L L KW LK KV L -NH 2 V RR L IR WI RR VI-NH 2 [H+3H] 3+ [H+3H] 3+ [H+3H] 3+ [H+3H] 3+ [H+3H] 3+ [H+3H] 3+ [H+3H] 3+ [H+3H] 3+ [H+3H] 3+ [H+3H] 3+ [H+3H] 3+ [H+3H] 3+ 519.90 513.70 520.01 513.70 560.40 504.34 570.40 523.70 523.70 499.30 499.30 546.02 519.70 513.30 519.60 513.30 560.00 504.20 570.00 523.40 524.00 499.10 499.80 545.70 4 5 5 6 6 6 5 5 5 5 5 5 21.9 18.1 20.7 17.2 16.8 16.8 21.7 21.3 22.6 20.8 22.4 20.7 0.610 0.787 0.638 0.826 0.831 0.825 0.877 0.872 0.858 0.860 0.866 0.845 VFR12-a7-d VFR12-a8 -d v kk l w kw lk kv l -NH 2 v kk l l kw lk kv l -NH 2 [H+3H] 3+ [H+3H] 3+ 523.70 499.00 524.00 499.70 5 5 22.5 22.3 0.858 0.845 a The substituted amino acids are highlighted in bold and lower-case letters indicate d-isomers of analogs b Molecular masses were determined using electrospray ionization mass spectrometry (ESI-MS). z stands for the charge of the ion whereas m/z represents the ratio of mass to charge. c Retention time, R t , was determined by timing the elution with RP-HPLC, represents the relative hydrophobicity of the peptides. d Hydrophobic moment, μH was quantified using an online tool- Heliquest (https://heliquest.ipmc.cnrs.fr/cgi-bin/ComputParams.py) The parent peptide, VFR12, possessed a net charge of + 4 and moderate hydrophobicity (hydrophobic moment µH = 0.610; RP-HPLC retention time R t = 21.9 min), shown in Table 1 . Substitution of lysine residues in VFR12-a1 to a3 increased the net charge from + 4 to + 6, resulting in decreased retention times (21.9 → 18.1 → 17.2 min) and increased hydrophobic moments (0.610 → 0.826), suggesting an inverse correlation between charge density and lipophilicity. VFR12-a6 exhibited high retention (R t = 21.7 min) and hydrophobic moment (0.877) despite a + 5 charge, attributed to optimal tryptophan placement. Stereochemical variants revealed conformational flexibility: VFR12-a7(L) increased retention time (22.6 vs. 21.3 min) while the D-isomer a7(L)-d maintained similar properties (R t = 22.5 min, µH = 0.858). The VFR12-a8 series showed identical properties for both L- and D-forms, indicating stereochemical equivalence. VFR12-a9 achieved optimal amphiphilic balance with high charge (+ 5) and enhanced hydrophobic moment (0.866). All peptides were purified by preparative RP-HPLC with molecular weights and purities confirmed by LC-MS analysis (Figure S1 ) and retention times measured by RP-HPLC (Figure S2). Secondary structures of peptides Circular dichroism (CD) spectroscopy (Fig. 1 b) revealed distinct conformational behaviors of VFR12 and its analogs under varying environmental conditions that mimic physiological and membrane-interactive states. In aqueous buffer, the spectra were relatively flat, indicating disordered or loosely structured conformations in hydrophilic environments. Upon exposure to 50% trifluoroethanol (TFE), a helix-inducing solvent that mimics the hydrophobic membrane environment, the peptides underwent marked conformational transitions, as evidenced by negative ellipticity minima near 208 and 222 nm, characteristic of α-helical structures. In 30 mM SDS micelles, which simulate anionic bacterial membrane environments, the peptides maintained strong α-helical character with well-defined spectral features, confirming their membrane-active conformational preferences. Notably, the D-enantiomeric analogs (VFR12-a7(L)-d and VFR12-a8(L)-d) demonstrated mirror-image CD spectra with positive ellipticity at corresponding wavelengths, as expected for peptides with inverted chirality, while maintaining comparable helical propensities and membrane-binding capabilities. Antimicrobial activity and cell selectivity Antimicrobial susceptibility assays were conducted against six representative bacterial strains. The antimicrobial evaluation of VFR12 and its analogs revealed structure-activity relationships with variations in therapeutic efficacy and cell selectivity (Table 2 ). The parent peptide VFR12 demonstrated moderate antibacterial activity, with MIC values ranging from 16 to 128 µM and a therapeutic index (TI) of 3.68, which is suboptimal. VFR12-a1 to VFR12-a5 exhibited minimal hemolytic activity above 256 µM (Figure S3); nevertheless, their antibacterial efficacy remained insufficient. VFR12-a6 showed markedly enhanced antimicrobial activity, but this was accompanied by elevated hemolytic activity as low concentrations such as 40µM, leading to a reduced therapeutic index. Notably, VFR12-a7 and VFR12-a8 demonstrated remarkable cell selectivity, with 10% hemolysis values of 135 and 182 µM, respectively, while maintaining excellent antibacterial efficacy with TI values of 9.47 and 9.03. These findings prompted the synthesis of isomeric forms of VFR12-a7 and VFR12-a8. VFR12-a8(L)-d demonstrated the highest therapeutic index (11.36), indicating exceptional safety with minimal hemolytic activity (102 µM), though with moderate antimicrobial potency. The melittin control showed poor selectivity (TI = 0.62) with high hemolytic activity, confirming the validity of the experiment. These results demonstrate that strategic amino acid modifications successfully enhanced both antimicrobial potency and biocompatibility as indicated by low hemolytic activity, with VFR12-a7 and VFR12-a8 variants representing promising therapeutic candidates for further study. Table 2 Antimicrobial and hemolytic activities and therapeutic index of VFR-12 and its analogs against different bacteria Peptides Minimum Inhibitory Concentration (µM) a GM b MHC c TI d (MHC/GM) Gram-positive bacteria Gram-negative bacteria B. subtilis KCTC3068 S. aureus KCTC1621 S. epidermidis KCTC1917 E. coli KCTC1682 P. aureuginosa KCTC1637 S. tryphimurium KCTC1926 VFR12 128 16 32 64 128 64 57.02 210 3.68 VFR12-a1 256 128 128 128 > 256 256 203.19 > 256 2.52 VFR12-a2 128 32 128 64 128 128 90.51 > 256 5.66 VFR12-a3 256 128 128 128 > 256 256 203.19 > 256 2.52 VFR12-a4 > 256 128 128 128 > 256 > 256 222.86 > 256 2.30 VFR12-a5 > 256 128 128 128 > 256 > 256 222.86 > 256 2.30 VFR12-a6 8 4 32 64 16 8 14.25 40 2.81 VFR12-a7 8 4 32 32 16 16 14.25 135 9.47 VFR12-a7(L) 8 16 16 16 16 32 16 89 5.56 VFR12-a8 8 8 32 32 32 32 20.16 182 9.03 VFR12-a8(L) 8 16 16 16 16 32 17.96 124 6.90 VFR12-a9 8 4 32 64 16 16 16 85 5.31 VFR12-a7(L)-d 8 8 8 8 8 16 8.98 75 8.35 VFR12-a8(L)-d 8 8 8 8 8 16 8.98 102 11.36 Melittin 8 4 8 8 8 8 7.13 4.4 0.62 a Minimum inhibitory concentrations (MICs) were determined as the lowest concentration of the peptides that inhibited bacterial growth. b GM denotes the geometric mean of MIC values from all bacterial strains tested. When the MIC observed was > 256 µM, a value of 512 µM was used to calculate the GM. c MHC is the minimum hemolytic concentration that resulted in 10% hemolysis of sheep red blood cells. d Therapeutic index (TI) was calculated as the ratio of the MHC value to GM. When the hemolytic activity observed was > 256 µM, a value of 512 µM was used to calculate the TI. Mechanism of antimicrobial action The mechanism of action of VFR12 and its selected analogs was investigated through membrane interaction studies, including membrane depolarization, SYTOX Green uptake, NPN (N-phenyl-1-naphthylamine) assays and flow cytometric analysis to elucidate their antimicrobial mode of action (Figs. 2 & 3 ). The membrane depolarization assay against S. aureus revealed that all peptides induced rapid and sustained membrane potential disruption, with fluorescence intensity increasing rapidly and then plateauing (Fig. 2 a). VFR12-a7(L), VFR12-a8(L) and VFR12-a8(L)-d demonstrated the most pronounced depolarization effects, correlating with their superior antimicrobial potencies. The SYTOX Green uptake assay provided complementary evidence of membrane permeabilization (Fig. 2 b) and all tested peptides induced substantial SYTOX Green uptake compared to the intracellular control peptide, Buforin-2. Notably, the kinetics of uptake were rapid, indicating immediate membrane disruption rather than gradual pore formation. The dose-dependent NPN assay against E. coli further confirmed the membrane-active mechanism (Fig. 2 c), where NPN fluorescence enhancement reflects outer membrane permeabilization. All peptides exhibited concentration-dependent membrane interaction, with melittin and several VFR12 analogs showing saturation at higher concentrations (16–32 µM), confirming the membrane-targeting mechanism. Additionally, flow cytometric analysis using a FACScan provided a quantitative assessment of membrane integrity disruption at 2× MIC concentrations (Fig. 3 ). Against S. aureus (Fig. 3 a), the peptides induced significant membrane permeabilization with D-isomers showing over 90% cell permeabilization, which was significantly higher than that of the parent peptide VFR12 (57.96%). Similarly, against E. coli (Fig. 3 b), all peptides induced significant membrane permeabilization, with D-variants maintaining effective membrane disruption capabilities (VFR12-a7(L)-d: 92.53%, VFR12-a8(L)-d: 93.13%). These consistent results across multiple assays confirm that VFR12 and its selected analogs exert their antimicrobial effects primarily through rapid membrane disruption. Salt and serum sensitivity assay The physiological stability of selected VFR12 analogs was evaluated under physiological salt conditions and in human serum, using E. coli as the test organism (Table 3 ). Exposure to physiological salt concentrations led to varying degrees of activity reduction across the peptide series. Under various salt conditions, most peptides experienced a moderate reduction in activity. VFR12 showed a two-fold increase in MIC (64 → 128 µM), whereas VFR12-a8(L)-d retained consistent potency (8 µM), even in the presence of 10% human serum. The most challenging condition was 10% human serum, where VFR12-a7(L)-d demonstrated remarkable stability, significantly outperforming the other analogs and exhibiting activity comparable to that of rifampicin. VFR12-a8(L)-d also maintained its full antimicrobial activity (MIC = 8 µM) in serum, indicating excellent stability. In contrast, the parent peptide VFR12 and other analogs exhibited reduced activity in the presence of serum. These results indicate that analogs containing D-amino acids—particularly VFR12-a7(L)-d and VFR12-a8(L)-d—exhibit excellent physiological stability and enhanced resistance to proteolysis. Therefore, they represent promising candidates for systemic therapeutic applications, where conventional L-peptides typically suffer from poor stability. Table 3 MIC values of selected peptides in the presence of physiological salts and human serum (10%) against E.coli Peptide Control 150mM NaCl 4.5mM KCl 6 µM NH 4 Cl 1 mM MgCl 2 2.5 mM CaCl 2 4 µM FeCl 3 Human serum (10%) VFR12 64 128 32 64 64 128 32 128 VFR12-a7 32 32 16 16 32 32 16 64 VFR12-a7(L) 16 32 32 16 32 64 16 32 VFR12-a8 32 64 64 32 64 128 32 32 VFR12-a8(L) 32 64 64 32 64 64 32 64 VFR12-a7(L)-d 8 8 16 8 8 32 16 4 VFR12-a8(L)-d 8 8 8 8 8 16 16 8 Tetracycline 8 16 4 16 16 16 8 16 Rifampicin 16 8 16 16 16 32 8 8 Proteolytic stability assessment To assess resistance to enzymatic degradation, the proteolytic stability of VFR12 and its selected analogs was evaluated using trypsin digestion, followed by radial diffusion assays against E. coli (Fig. 4 a). Trypsin, a serine protease that cleaves peptide bonds at the carboxyl side of basic residues such as lysine and arginine, was chosen as a representative proteolytic enzyme commonly found in biological systems. In the absence of trypsin, all peptides retained their antimicrobial activity, as demonstrated by well-defined zones of inhibition surrounding the peptide discs. However, following trypsin treatment, marked differences in proteolytic resistance were observed between L-amino acid and D-amino acid–containing analogs. The parent peptide VFR12 and L-amino acid variants (VFR12-a7, VFR12-a7(L), VFR12-a8, and VFR12-a8(L)) completely lost their antimicrobial activity after trypsin treatment, as evidenced by the disappearance of inhibition zones. This vulnerability reflects trypsin’s inherent preference for cleaving L-amino acid peptide bonds, especially at the frequent lysine and arginine residues found in these cationic antimicrobial peptides. In contrast, the D-isomers VFR12-a7(L)-d and VFR12-a8(L)-d exhibited strong resistance to enzymatic degradation, retaining significant antimicrobial activity after trypsin exposure, as shown by the persistence of inhibition zones. These findings support the enhanced serum stability observed in the D-isomers and further validate their potential as therapeutically promising antimicrobial agents. Antibiofilm activity The antibiofilm efficacy of VFR12 and its selected analogs was evaluated using minimum biofilm inhibitory concentration (MBIC) and minimum biofilm eradication concentration (MBEC) assays against multidrug-resistant Pseudomonas aeruginosa (MDRPA) biofilms (Fig. 4 b). In biofilm inhibition studies, VFR12-a7(L)-d exhibited potent activity at concentrations as low as 4 µM, while the remaining VFR12-a7 stereoisomers demonstrated substantial inhibition (80–90%) at 8 µM. Notably, VFR12-a8 displayed limited inhibitory capacity, whereas VFR12-a8(L) and its D-isomer VFR12-a8(L)-d showed statistically significant biofilm inhibition activity (p < 0.0001). All selected analogs exhibited markedly enhanced antibiofilm performance compared to the parent peptide VFR12. In the biofilm eradication assays using preformed biofilms, VFR12-a7(L) and its stereochemical counterpart demonstrated significant disruption activity starting at 4 µM (> 60% eradication), while VFR12-a8(L) and VFR12-a8(L)-d achieved comparable biofilm elimination from 8 µM onwards. Eradication efficacy showed dose-dependent enhancement, with VFR12-a7(L), VFR12-a8(L), and their respective D-isomers achieving above 80% biofilm disruption at 16 µM. In contrast, the parent peptide VFR12 required substantially higher concentrations (64 µM) to attain equivalent eradication levels. Biocompatibility assay The cytotoxicity profiles of VFR12 and its selected analogs were evaluated in both RAW 264.7 macrophages (Fig. 5 a) and HaCaT keratinocytes (Fig. 5 b) to assess their safety toward mammalian cells across a concentration range of 1–32 µM. In RAW 264.7 cells, all peptides demonstrated excellent biocompatibility at therapeutic concentrations (1–4 µM), maintaining cell viability above 70%. Concentration-dependent effects on cell viability became apparent at higher concentrations, with notable reductions emerging at 8 µM and above. At 16 µM, most peptides showed a moderate impact on cell viability, with values dropping to 50–70%, while at 32 µM, significant viability reduction was observed with values falling below 50% for the parent VFR12 and some analogs. Similarly, in HaCaT cells, the peptides maintained favorable viability profiles at lower concentrations (1–4 µM) with cell viability consistently above 70%. The viability patterns in HaCaT cells were comparable to those in RAW 264.7 cells, showing concentration-dependent reductions with more pronounced effects at 16–32 µM concentrations. As all the selected peptides showed ≥ 70% viability at 4µM in both cell lines, this concentration was used for anti-inflammatory activity study. Anti-inflammatory activities The anti-inflammatory potential of VFR12 and its selected analogs was evaluated using RAW 264.7 macrophage cells to assess their effects on key inflammatory mediators including tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) secretion and nitrite production (Fig. 5 c-e). All peptides were tested at 4 µM concentration in LPS-stimulated RAW 264.7 cells, with LL-37 serving as a positive control and untreated cells (control) representing baseline inflammatory status. The results revealed that the selected VFR12 analogs (a7 and a8 isomers) demonstrated favorable anti-inflammatory profiles under LPS stimulation. TNF-α secretion, measured by ELISA, showed that all VFR12-a7 and VFR12-a8 analogs maintained minimal pro-inflammatory cytokine release in LPS-stimulated conditions, contrasting with the substantial TNF-α production observed with LPS alone and VFR12 treatment, demonstrating improved inflammatory safety profiles of the designed analogs. The IL-6 response pattern revealed that selected analogs VFR12-a7, VFR12-a7(L), VFR12-a7(L)-d, VFR12-a8, VFR12-a8(L) and VFR12-a8(L)-d maintained IL-6 levels similar to unstimulated control cells, indicating negligible inflammatory induction. Only LPS alone and the parent peptide VFR12 induced significant IL-6 production, while all the targeted analogs demonstrated excellent anti-inflammatory profiles. In addition, nitrite production remained consistently suppressed (≤ 10%) across all tested VFR 12 analogs (except the parent VFR12) compared to LPS-alone stimulation, which induced nearly 100% nitrite. The overall inflammatory assessment in LPS-stimulated RAW 264.7 macrophages demonstrates that the VFR12 analogs possess excellent immunomodulatory properties, effectively suppressing LPS-induced inflammatory responses, positioning them as promising therapeutic candidates with favorable biocompatibility profiles. LPS-binding capability The LPS-binding assay demonstrated dose-dependent endotoxin neutralization capabilities across all tested VFR12 analogs (Fig. 5 f). At low concentrations (1–2 µM), most peptides exhibited minimal LPS binding, whereas a progressive increase in binding efficiency was observed with increasing concentrations, with distinct patterns emerging at 4−8 µM. At the highest tested concentration (32 µM), most analogs achieved substantial LPS binding, with several peptides reaching 80–100% BC displacement efficiency comparable to that of the LL-37 positive control. Notably, the D-amino acid variants VFR12-a7(L)-d and VFR12-a8(L)-d demonstrated excellent LPS-binding capabilities while the parent VFR12 exhibited moderate LPS-binding activity with gradual increases across the concentration range. Discussion Thrombin C-terminal peptides represent a promising class of host defense peptides with demonstrated antimicrobial and anti-inflammatory properties derived from the proteolytic cleavage of the coagulation enzyme thrombin. These peptides have been shown to exhibit broad-spectrum activity against both Gram-positive and Gram-negative bacteria in vitro and in vivo through membrane disruption, while concurrently exhibiting immunomodulatory effects by suppressing macrophage activation in response to bacterial endotoxins 17 , 20 , 21 , 23 – 27 . Building upon this natural template, we employed rational design principles to develop VFR12 and its analogs through systematic amino acid substitutions and stereochemical modifications. The truncation strategy from the parent thrombin-derived sequence yielded VFR12 as a compact α-helical peptide with balanced amphiphilic properties. Strategic modifications focused on optimizing the charge-hydrophobicity relationship through lysine/arginine substitutions and tryptophan positioning to enhance membrane interaction capabilities. Among the designed peptides, VFR12-a7 and VFR12-a8 demonstrated the highest specificity toward bacterial cells, highlighting their potential for further exploration. To improve proteolytic resistance, we synthesized D-enantiomeric variants (VFR12-a7(L)-d and VFR12-a8(L)-d) by substituting their L-amino acids with D-forms, thereby enhancing peptide stability. VFR12-a7 and VFR12-a8 contain two and three isoleucine (Ile) residues, respectively. Owing to two chiral centers—located at the backbone and side chain—Ile exists as four stereoisomers: L-Ile (2S,3S), D-Ile (2R,3R), L-allo-Ile (2S,3R), and D-allo-Ile (2R,3S). Because Fmoc-D-Ile and Fmoc-D-allo-Ile are prohibitively expensive and unsuitable for solid-phase synthesis, we replaced D-Ile with hydrophobic D-leucine (D-Leu), which has the same molecular weight. Since these peptides exert antimicrobial action via membrane disruption rather than receptor-mediated mechanisms, this substitution was not expected to significantly affect their bioactivity. Consistent with this prediction, both VFR12-a7 and its analog VFR12-a7(L), as well as VFR12-a8 and its corresponding variant VFR12-a8(L), exhibited comparable antimicrobial and hemolytic profiles. The incorporation of D-amino acids, particularly in VFR12-a7(L)-d and VFR12-a8(L)-d, successfully preserved amphiphilic architecture while conferring enhanced hydrophobic character and proteolytic resistance. The systematic increase in hydrophobic moments (0.610 → 0.877) across the analog series, coupled with strategic planar positioning of tryptophan residues, facilitated optimal membrane insertion geometry essential for antimicrobial activity. These modifications resulted in improved cell selectivity profiles, with therapeutic indices ranging from 3.68 to 11.36, demonstrating enhanced discrimination between bacterial and mammalian cell membranes compared to the parent peptide. The biocompatibility assessment confirmed excellent safety profiles, with all optimized analogs maintaining approximately 70% cell viability in both RAW 264.7 macrophages and HaCaT keratinocytes. The antimicrobial evaluation revealed structure-activity relationships that validate the rational design approach. VFR12-a6 through a9 demonstrated potent broad-spectrum activity with lower MIC values against both Gram-positive and Gram-negative bacteria, representing significant improvements over the parent VFR12. Mechanistic studies confirmed a membrane-disrupting mode of action, as evidenced by rapid membrane depolarization, permeabilization assays, and flow cytometric analysis showing above 90% bacterial membrane disruption in the case of D-isomers. The D-amino acid-containing peptides maintained comparable antimicrobial potency while exhibiting superior stability in physiological conditions, including resistance to proteolytic degradation and enhanced activity in human serum. Notably, the LPS-binding capacity of these peptides provides an additional therapeutic advantage by neutralizing endotoxins released during bacterial lysis, potentially mitigating inflammatory responses associated with Gram-negative bacterial infections. The antibiofilm activity represents a critical advancement, as biofilm-associated infections pose significant therapeutic challenges due to enhanced antibiotic resistance and persistence. VFR12-a7(L)-d and VFR12-a8(L)-d demonstrated dual functionality, effectively preventing biofilm formation (MBIC) and disrupting established biofilms (MBEC) against multidrug-resistant P. aeruginosa . The concentration-dependent efficacy, achieving > 80% biofilm eradication at 32 µM, substantially outperformed parent peptide. This dual anti-biofilm capacity is particularly valuable for treating chronic infections where mature biofilms represent the primary therapeutic obstacle. The anti-inflammatory assessment revealed that optimized VFR12 selected analogs possess excellent immunomodulatory properties, effectively suppressing LPS-induced TNF-α, IL-6, and nitric oxide production in macrophage cellular models while maintaining minimal intrinsic inflammatory activity. Unlike the parent VFR12 which induced significant cytokine release, the selected analogs demonstrated anti-inflammatory profiles comparable to unstimulated controls, suggesting therapeutic benefits beyond direct antimicrobial activity. The dose-dependent LPS-binding capabilities, achieving 80–100% endotoxin neutralization at therapeutic concentrations, provide a probable mechanism for preventing cytokine storms and septic shock associated with Gram-negative bacterial infections. This multifaceted therapeutic approach – combining direct antimicrobial activity, biofilm disruption, and immunomodulation – positions these peptides as comprehensive therapeutic agents capable of addressing both pathogen elimination and host inflammatory responses. VFR12-a8(L)-d emerges as the most promising therapeutic candidate due to its exceptional combination of properties: potent broad-spectrum antimicrobial activity (MIC 8–16 µM), outstanding cell selectivity (therapeutic index of 11.36), complete proteolytic resistance, effective antibiofilm activity, and strong anti-inflammatory action. The D-amino acid configuration addresses the primary limitation of peptide therapeutics by providing stability in biological systems while maintaining full therapeutic efficacy, potentially enabling oral bioavailability 28 – 31 . The superior performance in human serum and resistance to enzymatic degradation positions VFR12-a8(L)-d as a viable candidate for systemic administration in treating serious infections caused by multidrug-resistant pathogens. Future development should focus on in vivo efficacy validation in relevant animal models to confirm therapeutic potential and safety profiles. Murine infection models−including systemic sepsis, skin and soft tissue infections, and biofilm-associated implant infections−will establish dose-response relationships and optimal therapeutic windows. Overall, this study provides valuable insights into the rational modification of host defense peptides, establishing a framework for developing new therapeutics to combat antimicrobial resistance and sepsis. Methods Peptide synthesis & characterization Peptides were synthesized using Fmoc solid-phase peptide synthesis (SPPS) with Rink amide MBHA resin, as previously described 32 . Detailed information is presented in the supporting information. Circular Dichroism Spectroscopic Analysis Circular dichroism (CD) spectroscopy was used to assess the secondary structure of the synthesized peptides, as described previously and explained in the supporting information 33 . Antimicrobial susceptibility assay The antimicrobial activity of the synthesized peptides against various bacterial strains was evaluated according to the guidelines established by the Clinical and Laboratory Standards Institute, following the previous manner 32 , 34 . The supplementary information provides a detailed outline of the antimicrobial properties of the compounds. Hemolytic activity Hemolytic activity was determined by measuring sheep red blood cells (sRBCs) according to the established protocol 35 . Comprehensive details regarding hemolysis are available in the supplementary materials. Membrane depolarization assay Bacterial membrane depolarization was assessed according to a prior study with minor modifications 36 . The supplementary information provides a detailed description of this method. SYTOX Green uptake assay Membrane permeabilization was assessed using SYTOX Green dye uptake, adapted from previous work and explained in the supplementary information 37 . Outer membrane permeability assay Outer membrane permeability in E. coli (KCTC1682) was assessed using the fluorescent probe NPN (1-N-phenylnaphthylamine) in a dose-dependent manner, as described previously 38 . Detailed information is provided in the supporting information. FACScan analysis Using FACScan equipment and established protocols, propidium iodide (PI) uptake was quantified to assess bacterial cell membrane integrity 32 , 39 . The supplementary information outlines this analysis in detail. Protease stability (Radial diffusion) The effect of peptides on protease stability was assessed using the radial diffusion method on agar plate, focusing on trypsin activity. Briefly, LB agar plates were prepared with E. coli strain (KCTC1682). Peptides were co-incubated with trypsin for several hours, then applied to agar plates using sterile discs alongside peptide-only controls. After a 24-hour incubation at 37°C, proteolytic degradation was evaluated by comparing inhibition zones, with reduced or absent zones indicating peptide degradation by trypsin activity. Antibiofilm activity (MBIC, MBEC) The antibiofilm properties were evaluated by determining minimum biofilm inhibition concentration (MBIC) and minimum biofilm eradication concentration (MBEC) against MDRPA (CCARM 2095), following previously described methods 32 , 39 . The supplementary information provides a detailed explanation of MBIC and MBEC procedures. Salt and serum stability assay Salt and serum stability tests were conducted to evaluate efficacy of the peptides under physiological conditions, as previously described 40 . Comprehensive details regarding this method are available in the supplementary materials. Cell viability assay Cell viability was evaluated using two cell lines: mouse macrophage RAW 264.7 cells and human keratinocyte HaCaT cells. Cells were seeded in 96-well plates and incubated for 24 h at 37°C, in 5% CO₂. After 24 h of peptide treatment, viability was assessed using the WST-8 kit. This assay relies on the reduction of the WST-8 reagent by metabolically active cells, resulting in the formation of an orange formazan product. WST-8 reagent (10 µL/well) was added and incubated for 2 h, and absorbance was measured at 450 nm to evaluate dose-dependent effects across various concentrations and cell types. Measurement of pro-inflammatory cytokines RAW 264.7 cells were plated in 96-well plates at 3 × 10 5 cells per well and incubated overnight at 37°C. Peptides were added to the cultures, followed by stimulation with 20 ng/mL lipopolysaccharide (LPS) and a 24-hour incubation at 37°C. The LPS-induced activation of the macrophages was expected to trigger the release of inflammatory cytokines, including production of TNF-α and IL-6. TNF-α and IL-6 cytokine was quantified using ELISA kits according to the manufacturer's instructions to evaluate the immunomodulatory effects of the peptides on LPS-stimulated macrophages. Measurement of NO production Nitric oxide (NO) production in LPS-stimulated RAW 264.7 cells was quantified using the Griess assay according to a previously described protocol 41 . RAW 264.7 cells (3 × 10 5 cells/well) were cultured in 96-well plates and stimulated with LPS (20 ng/mL) in the presence or absence of peptides for 24 hours. Culture supernatants were transferred to new plates, mixed with equal volumes of 1% Griess reagent (Sigma), and incubated for 15 minutes at room temperature. Nitrite levels were quantified by measuring absorbance at 540 nm using a microplate reader (KLAB, MRX A2000). LPS binding assay The binding ability of peptides to lipopolysaccharide (LPS) was assessed using a BODIPY-TR cadaverine (BC) displacement assay, as outlined in previous studies 32 , 42 . A detailed description of the procedure is provided in the supporting information. Statistical analysis All graphical representations and statistical analyses were performed using one-way analysis of variance (ANOVA) with SPSS 16.0 and SigmaPlot v12.0 software. Data are presented as the means ± standard deviation (SD) from three independent experiments conducted in triplicate. Statistical significance was determined using two-sample t-tests, with p-values < 0.05 considered statistically significant. Significance levels are indicated as follows: ** for p < 0.01, *** for p < 0.001, and **** for p < 0.0001. Declarations Acknowledgements This research was supported by the National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIT) (No. RS-2023-00210292 and No. 2022R1F1A1068595) and the Global-Learning & Academic Research Institution for Master's·PhD students, and Postdocs (LAMP) Program of the National Research Foundation of Korea (NRF) grant funded by the Ministry of Education (No. RS-2023-00285353). Author Contributions I.J.: Investigation, methodology, data analysis, and writing original draft, S.D.K.: data curation, C.A.: methodology, C.W.L.: writing-review and editing, S.Y.S.: conceptualization, supervision, writing-review and editing, S.Y.: supervision, funding acquisition, writing-review and editing. Competing interests The authors declare no competing interests. Data availability All data generated or analysed during this study are included in this published article and its supplementary information files. Additional Information Supplementary Information The online version contains supplementary material available at References Laxminarayan, R. et al. Antibiotic resistance—the need for global solutions. Lancet. Infect. Dis . 13 , 1057–1098 (2013). Santajit, S. & Indrawattana, N. Mechanisms of antimicrobial resistance in ESKAPE pathogens. BioMed research international 2475067 (2016). (2016). Resistance, G. A. Use Surveillance System (GLASS) Report 2022. World Health Organization 71 (2022). Miller, W. R. & Arias, C. A. ESKAPE pathogens: antimicrobial resistance, epidemiology, clinical impact and therapeutics. Nat. Rev. Microbiol. 22 , 598–616 (2024). Ayobami, O., Brinkwirth, S., Eckmanns, T. & Markwart, R. Antibiotic resistance in hospital-acquired ESKAPE-E infections in low-and lower-middle-income countries: a systematic review and meta-analysis. Emerg. microbes infections . 11 , 443–451 (2022). Venkateswaran, P. et al. Revisiting ESKAPE Pathogens: virulence, resistance, and combating strategies focusing on quorum sensing. Front. Cell. Infect. Microbiol. 13 , 1159798 (2023). Mwangi, J., Kamau, P. M., Thuku, R. C. & Lai, R. Design methods for antimicrobial peptides with improved performance. Zoological Res. 44 , 1095 (2023). Huan, Y., Kong, Q., Mou, H. & Yi, H. Antimicrobial peptides: classification, design, application and research progress in multiple fields. Front. Microbiol. 11 , 582779 (2020). Kumar, S. D. & Shin, S. Y. Antimicrobial and anti-inflammatory activities of short dodecapeptides derived from duck cathelicidin: Plausible mechanism of bactericidal action and endotoxin neutralization. Eur. J. Med. Chem. 204 , 112580 (2020). Ji, S. et al. Antimicrobial peptides: An alternative to traditional antibiotics. Eur. J. Med. Chem. 265 , 116072 (2024). Costa, F., Teixeira, C., Gomes, P. & Martins, M. C. L. Clinical application of AMPs. Antimicrobial peptides: basics Clin. application , 281–298 (2019). Mahlapuu, M., Håkansson, J., Ringstad, L. & Björn, C. Antimicrobial peptides: an emerging category of therapeutic agents. Front. Cell. Infect. Microbiol. 6 , 194 (2016). Findlay, F., Proudfoot, L., Stevens, C. & Barlow, P. G. Cationic host defense peptides; novel antimicrobial therapeutics against Category A pathogens and emerging infections. Pathogens global health . 110 , 137–147 (2016). Lee, H., Yang, S. & Shin, S. H. Effect of central PxxP motif in amphipathic alpha-helical peptides on antimicrobial activity and mode of action. J. Anal. Sci. Technol. 14 , 33 (2023). Kasetty, G. et al. Structure-activity studies and therapeutic potential of host defense peptides of human thrombin. Antimicrob. Agents Chemother. 55 , 2880–2890 (2011). Petrlova, J. et al. Aggregation of thrombin-derived C-terminal fragments as a previously undisclosed host defense mechanism. Proceedings of the National Academy of Sciences 114, E4213-E4222 (2017). Hansen, F. C., Strömdahl, A. C., Mörgelin, M., Schmidtchen, A. & van der Plas, M. J. Thrombin-derived host-defense peptides modulate monocyte/macrophage inflammatory responses to gram-negative bacteria. Front. Immunol. 8 , 843 (2017). Lim, C. H. et al. Thrombin-derived host defence peptide modulates neutrophil rolling and migration in vitro and functional response in vivo. Sci. Rep. 7 , 11201 (2017). Petrlova, J. et al. Thrombin-derived C-terminal fragments aggregate and scavenge bacteria and their proinflammatory products. J. Biol. Chem. 295 , 3417–3430 (2020). Kalle, M. et al. Host defense peptides of thrombin modulate inflammation and coagulation in endotoxin-mediated shock and Pseudomonas aeruginosa sepsis. PloS one . 7 , e51313 (2012). Papareddy, P. et al. Proteolysis of human thrombin generates novel host defense peptides. PLoS Pathog. 6 , e1000857 (2010). Kim, E. Y., Rajasekaran, G. & Shin, S. Y. LL-37-derived short antimicrobial peptide KR-12-a5 and its d-amino acid substituted analogs with cell selectivity, anti-biofilm activity, synergistic effect with conventional antibiotics, and anti-inflammatory activity. Eur. J. Med. Chem. 136 , 428–441 (2017). Kumar, S. D. et al. Antibacterial, Antibiofilm, and Anti-inflammatory Effects of a Novel Thrombin-Derived Peptide in Sepsis Models: Insights into Underlying Mechanisms. J. Med. Chem. 67 , 19791–19812 (2024). Saravanan, R. et al. Proteolytic signatures define unique thrombin-derived peptides present in human wound fluid in vivo. Sci. Rep. 7 , 13136 (2017). Saravanan, R. et al. Structural basis for endotoxin neutralisation and anti-inflammatory activity of thrombin-derived C-terminal peptides. Nat. Commun. 9 , 2762 (2018). Merza, M. et al. Human thrombin-derived host defense peptides inhibit neutrophil recruitment and tissue injury in severe acute pancreatitis. Am. J. Physiology-Gastrointestinal Liver Physiol. 307 , G914–G921 (2014). Hansen, F. C. et al. Differential internalization of thrombin-derived host defense peptides into monocytes and macrophages. J. Innate Immun. 14 , 418–432 (2022). Hamamoto, K., Kida, Y., Zhang, Y., Shimizu, T. & Kuwano, K. Antimicrobial activity and stability to proteolysis of small linear cationic peptides with D-amino acid substitutions. Microbiol. Immunol. 46 , 741–749 (2002). Kapil, S. & Sharma, V. d-Amino acids in antimicrobial peptides: A potential approach to treat and combat antimicrobial resistance. Can. J. Microbiol. 67 , 119–137 (2021). Genchi, G. An overview on D-amino acids. Amino acids . 49 , 1521–1533 (2017). Feng, Z. & Xu, B. Inspiration from the mirror: D-amino acid containing peptides in biomedical approaches. Biomol. concepts . 7 , 179–187 (2016). Jahan, I. et al. Multifunctional properties of BMAP-18 and its aliphatic analog against drug-resistant bacteria. Pharmaceuticals 16 , 1356 (2023). Lee, H. et al. Conjugation of cell-penetrating peptides to antimicrobial peptides enhances antibacterial activity. ACS omega . 4 , 15694–15701 (2019). Cockerill, F. R. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically: approved standard. (No Title) (2012). Yang, S. et al. Structural analysis and mode of action of BMAP-27, a cathelicidin-derived antimicrobial peptide. Peptides 118 , 170106 (2019). Lee, H. & Yang, S. Dimerization of cell-penetrating buforin II enhances antimicrobial properties. J. Anal. Sci. Technol. 12 , 9 (2021). Kim, E. Y., Kumar, S. D., Bang, J. K. & Shin, S. Y. Mechanisms of antimicrobial and antiendotoxin activities of a triazine-based amphipathic polymer. Biotechnol. Bioeng. 117 , 3508–3521 (2020). Radhakrishnan, N., Kumar, S. D., Shin, S. Y. & Yang, S. Enhancing selective antimicrobial and antibiofilm activities of melittin through 6-aminohexanoic acid substitution. Biomolecules 14 , 699 (2024). Jahan, I., Ganbaatar, B., Lee, C. W., Shin, S. H. & Yang, S. Antibacterial and antibiofilm features of mutSMAP-18 against Vibrio cholerae. Heliyon 10 (2024). Kumar, S. D. et al. Novel Leech Antimicrobial Peptides, Hirunipins: Real-Time 3D Monitoring of Antimicrobial and Antibiofilm Mechanisms Using Optical Diffraction Tomography. Adv. Sci. 12 , 2409803 (2025). Rajasekaran, G. et al. Antimicrobial and anti-inflammatory activities of chemokine CXCL14-derived antimicrobial peptide and its analogs. Biochim. Et Biophys. Acta (Bba)-Biomembranes . 1861 , 256–267 (2019). Wood, S. J., Miller, K. A. & David, S. A. Anti-endotoxin agents. 1. Development of a fluorescent probe displacement method optimized for the rapid identification of lipopolysaccharide-binding agents. Comb. Chem. High Throughput Screen. 7 , 239–249 (2004). Additional Declarations No competing interests reported. Supplementary Files VFR12Supplementary.docx Cite Share Download PDF Status: Published Journal Publication published 19 Nov, 2025 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 03 Sep, 2025 Reviews received at journal 25 Aug, 2025 Reviews received at journal 22 Aug, 2025 Reviewers agreed at journal 19 Aug, 2025 Reviewers agreed at journal 13 Aug, 2025 Reviewers agreed at journal 11 Aug, 2025 Reviewers invited by journal 11 Aug, 2025 Editor assigned by journal 11 Aug, 2025 Editor invited by journal 11 Aug, 2025 Submission checks completed at journal 10 Aug, 2025 First submitted to journal 10 Aug, 2025 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. 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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-7306037","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":500221692,"identity":"d8c735ae-29fa-4732-9aaa-277d4e316080","order_by":0,"name":"Ishrat Jahan","email":"","orcid":"","institution":"Chosun University","correspondingAuthor":false,"prefix":"","firstName":"Ishrat","middleName":"","lastName":"Jahan","suffix":""},{"id":500221693,"identity":"8dab62e2-a2eb-490a-85b9-47b1be164dfc","order_by":1,"name":"S. Dinesh Kumar","email":"","orcid":"","institution":"Chosun University","correspondingAuthor":false,"prefix":"","firstName":"S.","middleName":"Dinesh","lastName":"Kumar","suffix":""},{"id":500221694,"identity":"8395fedb-98ce-4725-87c8-b2d0ea72a4be","order_by":2,"name":"Chelladurai Ajish","email":"","orcid":"","institution":"Chosun University","correspondingAuthor":false,"prefix":"","firstName":"Chelladurai","middleName":"","lastName":"Ajish","suffix":""},{"id":500221695,"identity":"85f93797-008d-4b1d-9b10-d0aa03261f25","order_by":3,"name":"Chul Won Lee","email":"","orcid":"","institution":"Chonnam National University","correspondingAuthor":false,"prefix":"","firstName":"Chul","middleName":"Won","lastName":"Lee","suffix":""},{"id":500221696,"identity":"c7ad2094-58a2-440b-bb93-652f8bd80dae","order_by":4,"name":"Song Yub Shin","email":"","orcid":"","institution":"Chosun University","correspondingAuthor":false,"prefix":"","firstName":"Song","middleName":"Yub","lastName":"Shin","suffix":""},{"id":500221697,"identity":"0a11b2fd-5543-438a-b7bf-5d8454f7836c","order_by":5,"name":"Sungtae Yang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA3ElEQVRIiWNgGAWjYPACGwYDOJuHkGI2MJlGupbDJGiRn99j+Lng13l5c/b2Z9IFDHbyDDxnH+DVYnCMx1h6Zt9tw509Z8ykZzAkGzbwthvg18LGYyDN23M7weBGDps0DwNzAgM/GwGHtfEY/+btOQfUkv4MqKWesBaGYzxm0jw/DgC1JAAZDIcTGHjb8OswOJZWZs3bkGy44cwZY2seg+OGbTzHCDis+fDm2zx/7OQNjrc/vM1TUS3Pz5NGwGEMHAYMjHCnGMAiCi9gf8DA8IewslEwCkbBKBjBAACwkTpYNKfJ4gAAAABJRU5ErkJggg==","orcid":"","institution":"Chosun University","correspondingAuthor":true,"prefix":"","firstName":"Sungtae","middleName":"","lastName":"Yang","suffix":""}],"badges":[],"createdAt":"2025-08-06 05:53:23","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7306037/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7306037/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-24789-9","type":"published","date":"2025-11-19T15:57:42+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":89420561,"identity":"14869297-e73b-4eef-99d4-1409aae7c3ce","added_by":"auto","created_at":"2025-08-19 18:27:35","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1228024,"visible":true,"origin":"","legend":"\u003cp\u003eStructural illustrations\u003cstrong\u003e \u003c/strong\u003eof VFR12 and all its analogs. (a) Helical wheel representations, highlighting colour-coded residues: nonpolar hydrophobic (yellow) and polar basic (dark blue). The black arrow indicates the hydrophobic moment. (b) CD spectra of the peptides in buffer, 50% TFE and 30mM SDS.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7306037/v1/d838365e6c8fea8f1a52e8d3.jpeg"},{"id":89420566,"identity":"4c24128c-f8c5-4340-a317-10d40f5e729c","added_by":"auto","created_at":"2025-08-19 18:27:35","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":662991,"visible":true,"origin":"","legend":"\u003cp\u003eMechanism study of the targeted peptides. (a) Membrane depolarization assay and (b) SYTOX Green uptake assay against \u003cem\u003eS. aureus\u003c/em\u003eusing peptides at 2× MIC and controls. (c) Dose-dependent NPN assay against \u003cem\u003eE. coli \u003c/em\u003ein response to peptide treatment.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7306037/v1/ec0c73f51802c813c7abc0c0.jpeg"},{"id":89420698,"identity":"a090d036-5343-499b-8448-f9fa80af863c","added_by":"auto","created_at":"2025-08-19 18:35:35","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1073148,"visible":true,"origin":"","legend":"\u003cp\u003eFACScan analysis of the targeted peptides alongside control peptides using the respective 2× MIC. (a) \u003cem\u003eS. aureus\u003c/em\u003e and (b) \u003cem\u003eE. coli\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7306037/v1/99973a99d8a825fd09d00f67.jpeg"},{"id":89421272,"identity":"350026cb-1118-4ec3-a206-20c9f045fedd","added_by":"auto","created_at":"2025-08-19 18:43:35","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":836444,"visible":true,"origin":"","legend":"\u003cp\u003eProteolytic stability and anti-biofilm efficacy of selected VFR12 analogs. (a) Radial diffusion on agar plates illustrating the protease stability of the targeted peptides in response to trypsin. (b) Antibiofilm activities: biofilm inhibition and biofilm eradication against the MDRPA 2095 strain. Dotted lines represent 50% inhibition and eradication thresholds, with statistical significance indicated by asterisks (*), where *** represents p \u0026lt; 0.001 and **** denotes p \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"floatimage7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7306037/v1/6418042d85a917e3352ec68f.jpeg"},{"id":89420576,"identity":"5de8c597-4b48-48b2-ab9f-545539552a9c","added_by":"auto","created_at":"2025-08-19 18:27:35","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1007473,"visible":true,"origin":"","legend":"\u003cp\u003eCytotoxicity and anti-inflammatory activities of selected VFR12 peptides. Cell viability assessment on (a) RAW 264.7 macrophages and (b) HaCaT keratinocytes. (c), (d) and (e) Quantification of TNF-α, IL-6 production and nitrite level by RAW 264.7 cells following treatment with selected analogs compared with the parent peptide and control. (f) Lipopolysaccharide binding capacity of peptides on dose-dependent manner.\u003c/p\u003e","description":"","filename":"floatimage9.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7306037/v1/0c518f53c95d2b910fe1de9c.jpeg"},{"id":96650102,"identity":"08423a85-0b09-424a-889a-4a54bdcbaa9e","added_by":"auto","created_at":"2025-11-24 16:07:42","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6092818,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7306037/v1/45972977-d5d4-40da-b48f-a818d5bd64bd.pdf"},{"id":89421693,"identity":"dfb6d96f-47ea-4973-bd01-96d42ac2b6bd","added_by":"auto","created_at":"2025-08-19 18:59:35","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":836753,"visible":true,"origin":"","legend":"","description":"","filename":"VFR12Supplementary.docx","url":"https://assets-eu.researchsquare.com/files/rs-7306037/v1/5d62ab6c4afcd7a79b3c533b.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Rational Design of Thrombin-Derived VFR12 Analogs with Enhanced Antimicrobial, Antibiofilm, and Anti-inflammatory Properties","fulltext":[{"header":"Introduction","content":"\u003cp\u003e \u003cspan fontcategory=\"NonProportional\" class=\"\" name=\"Emphasis\"\u003ePlease have a look at courier new font provided for text in article.\u003c/span\u003e\u003c/p\u003e\u003cp\u003eThe escalating crisis of antibiotic resistance, coupled with a shrinking antimicrobial pipeline, has become one of the biggest threats to global health. This crisis stems from the widespread misuse and overuse of antibiotics, which has led to the rise of superbugs that can resist multiple drugs. The most dangerous of these are known as ESKAPE pathogens\u0026minus;\u003cem\u003eEnterococcus faecium\u003c/em\u003e, \u003cem\u003eStaphylococcus aureus\u003c/em\u003e, \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e, \u003cem\u003eAcinetobacter baumannii\u003c/em\u003e, \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e, and \u003cem\u003eEnterobacter\u003c/em\u003e species\u003csup\u003e\u003cspan additionalcitationids=\"CR2 CR3\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. These bacteria have been identified as high-priority targets because they resist most available treatments and cause serious infections that are becoming increasingly difficult to cure\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. To address this urgent medical need, researchers have turned to antimicrobial peptides (AMPs), also known as host defense peptides (HDPs), which are natural infection-fighting molecules found in plants, animals, and humans. These bioactive molecules exhibit multifaceted therapeutic properties encompassing broad-spectrum antimicrobial efficacy, biofilm disruption capabilities, and immunomodulatory functions that distinguish them from conventional antibiotics. The antimicrobial mechanisms of peptides involve complex interactions with microbial cell membranes, where electrostatic attraction to negatively charged membrane phospholipids facilitates initial binding, followed by membrane insertion, pore formation, and subsequent cellular lysis\u003csup\u003e\u003cspan additionalcitationids=\"CR8 CR9\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Additionally, these peptides demonstrate the capacity for intracellular target engagement and biofilm matrix disruption, contributing to their comprehensive anti-pathogenic activity profile. Currently, AMPs are being developed and used in clinical settings primarily for treating bacterial infections, promoting wound healing, and managing inflammatory conditions, positions them as promising therapeutic alternatives in the post-antibiotic era\u003csup\u003e\u003cspan additionalcitationids=\"CR12 CR13\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThrombin C-terminal peptides (TCPs) represent a novel and significant class of host defense peptides that emerge from the proteolytic cleavage of human thrombin, a central enzyme in the coagulation cascade. These peptides are generated in vivo during wound healing and inflammation through the action of neutrophil elastase and other proteases released at sites of tissue injury. The discovery of TCPs has revealed an unexpected connection between the coagulation system and innate immune defense, demonstrating that hemostatic and antimicrobial responses are more integrated than previously understood\u003csup\u003e\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. The prototypic thrombin-derived peptide GKY25 (GKYGFYTHVFRLKKWIQKVIDQFGE) exemplifies the multifunctional nature of these molecules, exhibiting classical antimicrobial peptide characteristics including cationicity, amphipathicity, and an α-helical structure. Under physiological conditions, these peptides demonstrate broad-spectrum antimicrobial activity against both Gram-positive and Gram-negative bacteria through membrane-disrupting mechanisms, while simultaneously providing immunomodulatory functions by inhibiting macrophage responses to bacterial lipopolysaccharide. In murine models, TCPs have shown protective effects against \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e sepsis and lipopolysaccharide-induced shock, validating their therapeutic potential\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan additionalcitationids=\"CR19 CR20\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003ePrevious investigations demonstrated that the prototypic thrombin-derived peptide GKY25 exhibits optimal antimicrobial and anti-inflammatory activities, requiring a peptide length of at least 20 amino acids for maximum therapeutic efficacy\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. However, structure-activity relationship studies revealed that shorter variants retain significant antimicrobial effects, indicating the potential for peptide truncation without loss of biological activity\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. In this study, we selected the central core sequence VFR12 (VFRLKKWIQKVI) from thrombin C-terminal peptides (TCPs) as our lead fragment for rational peptide design. We synthesized a systematic series of analogs through strategic amino acid substitutions and stereochemical modifications with three primary objectives: (1) enhancement of antimicrobial potency against both planktonic bacteria and biofilm-associated infections, (2) improvement of cell selectivity to achieve superior therapeutic indices with reduced cytotoxicity toward mammalian cells, and (3) optimization of immunomodulatory properties to provide beneficial anti-inflammatory effects that complement direct antimicrobial activity. This comprehensive approach aims to develop next-generation host defense peptides that overcome the limitations of current antimicrobial agents while retaining the multifaceted therapeutic advantages inherent in thrombin-derived peptides.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003ePeptide design\u003c/h2\u003e\n \u003cp\u003eA series of short amphipathic \u0026alpha;-helical peptides was rationally designed based on VFR12, a truncated thrombin-derived peptide, to explore structure-activity relationships through systematic amino acid substitutions and stereochemical modifications. The helical wheel projections (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ea) illustrate the structural organization of analogs with strategic modifications. The parent VFR12 underwent sequential optimization: VFR12-a1 featured a phenylalanine-to-lysine substitution, increasing net charge (+\u0026thinsp;1) and hydrophobic moment, while VFR12-a2 incorporated a glutamine-to-lysine replacement, enhancing charge with minimal hydrophobicity changes. Subsequent analogs (VFR12-a3 to VFR12-a9) involved strategic modifications by removing phenylalanine and glutamine and substituting with lysine or arginine in multiple positions to systematically increase cationicity and amphipathicity, progressively enhancing hydrophobic moments from 0.610 to 0.877 (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). Key structural modifications included: VFR12-a6 versus VFR12-a7 (arginine to lysine substitution), VFR12-a7 versus VFR12-a8 (single tryptophan to isoleucine replacement), and VFR12-a6 versus VFR12-a9 (single tryptophan to isoleucine replacement) in the same position. For stereochemical optimization, VFR12-a7 and VFR12-a8 were selected for D-amino acid incorporation, where all L-amino acids were replaced with D-isomers. Due to the prohibitive cost of Fmoc-D-Ile and Fmoc-D-allo-Ile for large-scale solid-phase synthesis, D-isoleucine was substituted with D-leucine, maintaining equivalent molecular weight and hydrophobic character while ensuring synthetic feasibility. Given the minimal structural variations among VFR12-a6 through VFR12-a9, these analogs were selected for comprehensive mechanistic evaluation.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eTable 1\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eAmino acid sequence and physicochemical properties of VFR-12 and its analogs.\u003c/p\u003e\n \u003ctable style=\"border-collapse: collapse;border: none;width: 889px;\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 91.9pt;border: 1pt solid windowtext;padding: 0cm 5.4pt;height: 11.65pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;line-height:normal;font-size:16px;font-family:\"Aptos\",sans-serif;text-align:center;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003ePeptides\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 149.1pt;border-top: 1pt solid windowtext;border-right: 1pt solid windowtext;border-bottom: 1pt solid windowtext;border-image: initial;border-left: none;padding: 0cm 5.4pt;height: 11.65pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;line-height:normal;font-size:16px;font-family:\"Aptos\",sans-serif;text-align:center;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eAmino acid sequence\u003cem\u003e\u003csup\u003ea\u003c/sup\u003e\u003c/em\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" style=\"width: 219.7pt;border-top: 1pt solid windowtext;border-right: 1pt solid windowtext;border-bottom: 1pt solid windowtext;border-image: initial;border-left: none;padding: 0cm 5.4pt;height: 11.65pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;line-height:normal;font-size:16px;font-family:\"Aptos\",sans-serif;text-align:center;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eMS analysis\u003cem\u003e\u003csup\u003eb\u003c/sup\u003e\u003c/em\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 56.45pt;border-top: 1pt solid windowtext;border-right: 1pt solid windowtext;border-bottom: 1pt solid windowtext;border-image: initial;border-left: none;padding: 0cm 5.4pt;height: 11.65pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;line-height:normal;font-size:16px;font-family:\"Aptos\",sans-serif;text-align:center;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eNet\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;line-height:normal;font-size:16px;font-family:\"Aptos\",sans-serif;text-align:center;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003echarge\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 2cm;border-top: 1pt solid windowtext;border-right: 1pt solid windowtext;border-bottom: 1pt solid windowtext;border-image: initial;border-left: none;padding: 0cm 5.4pt;height: 11.65pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;line-height:normal;font-size:16px;font-family:\"Aptos\",sans-serif;text-align:center;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eR\u003csub\u003et\u003c/sub\u003e\u003cem\u003e\u003csup\u003ec\u003c/sup\u003e\u003c/em\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 92.15pt;border-top: 1pt solid windowtext;border-right: 1pt solid windowtext;border-bottom: 1pt solid windowtext;border-image: initial;border-left: none;padding: 0cm 5.4pt;height: 11.65pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;line-height:normal;font-size:16px;font-family:\"Aptos\",sans-serif;text-align:center;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eHydrophobic moment (\u0026mu;H)\u003cem\u003e\u003csup\u003ed\u003c/sup\u003e\u003c/em\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 77.95pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0cm 5.4pt;height: 13.1pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;line-height:normal;font-size:16px;font-family:\"Aptos\",sans-serif;text-align:center;'\u003e\u003cem\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003ez\u003c/span\u003e\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 70.9pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0cm 5.4pt;height: 13.1pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;line-height:normal;font-size:16px;font-family:\"Aptos\",sans-serif;text-align:center;'\u003e\u003cem\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003em/z\u003c/span\u003e\u003c/em\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;calculated\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 70.85pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0cm 5.4pt;height: 13.1pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;line-height:normal;font-size:16px;font-family:\"Aptos\",sans-serif;text-align:center;'\u003e\u003cem\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003em/z\u003c/span\u003e\u003c/em\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;observed\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 91.9pt;border-top: none;border-left: 1pt solid windowtext;border-bottom: none;border-right: 1pt solid windowtext;padding: 0cm 5.4pt;height: 92.65pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;line-height:normal;font-size:16px;font-family:\"Aptos\",sans-serif;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eVFR12\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;line-height:normal;font-size:16px;font-family:\"Aptos\",sans-serif;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eVFR12-a1\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;line-height:normal;font-size:16px;font-family:\"Aptos\",sans-serif;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eVFR12-a2\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;line-height:normal;font-size:16px;font-family:\"Aptos\",sans-serif;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eVFR12-a3\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;line-height:normal;font-size:16px;font-family:\"Aptos\",sans-serif;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eVFR12-a4\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;line-height:normal;font-size:16px;font-family:\"Aptos\",sans-serif;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eVFR12-a5\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;line-height:normal;font-size:16px;font-family:\"Aptos\",sans-serif;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eVFR12-a6\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;line-height:normal;font-size:16px;font-family:\"Aptos\",sans-serif;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eVFR12-a7\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;line-height:normal;font-size:16px;font-family:\"Aptos\",sans-serif;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eVFR12-a7(L)\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;line-height:normal;font-size:16px;font-family:\"Aptos\",sans-serif;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eVFR12-a8\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;line-height:normal;font-size:16px;font-family:\"Aptos\",sans-serif;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eVFR12-a8(L)\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;line-height:normal;font-size:16px;font-family:\"Aptos\",sans-serif;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eVFR12-a9\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 149.1pt;border-top: none;border-bottom: none;border-left: none;border-image: initial;border-right: 1pt solid windowtext;padding: 0cm 5.4pt;height: 92.65pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;line-height:normal;font-size:16px;font-family:\"Aptos\",sans-serif;text-align:center;'\u003e\u003cspan style='font-family:\"Courier New\";'\u003eVFRLKKWIQKVI-NH\u003csub\u003e2\u003c/sub\u003e\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;line-height:normal;font-size:16px;font-family:\"Aptos\",sans-serif;text-align:center;'\u003e\u003cspan style='font-family:\"Courier New\";'\u003eV\u003cstrong\u003eK\u003c/strong\u003eRLKKWIQKVI-NH\u003csub\u003e2\u003c/sub\u003e\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;line-height:normal;font-size:16px;font-family:\"Aptos\",sans-serif;text-align:center;'\u003e\u003cspan style='font-family:\"Courier New\";'\u003eVFRLKKWI\u003cstrong\u003eK\u003c/strong\u003eKVI-NH\u003csub\u003e2\u003c/sub\u003e\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;line-height:normal;font-size:16px;font-family:\"Aptos\",sans-serif;text-align:center;'\u003e\u003cspan style='font-family:\"Courier New\";'\u003eV\u003cstrong\u003eK\u003c/strong\u003eRLKKWI\u003cstrong\u003eK\u003c/strong\u003eKVI-NH\u003csub\u003e2\u003c/sub\u003e\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;line-height:normal;font-size:16px;font-family:\"Aptos\",sans-serif;text-align:center;'\u003e\u003cspan style='font-family:\"Courier New\";'\u003eV\u003cstrong\u003eRR\u003c/strong\u003eL\u003cstrong\u003eRR\u003c/strong\u003eWI\u003cstrong\u003eRR\u003c/strong\u003eVI-NH\u003csub\u003e2\u003c/sub\u003e\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;line-height:normal;font-size:16px;font-family:\"Aptos\",sans-serif;text-align:center;'\u003e\u003cspan style='font-family:\"Courier New\";'\u003eV\u003cstrong\u003eKK\u003c/strong\u003eL\u003cstrong\u003eKK\u003c/strong\u003eWI\u003cstrong\u003eK\u003c/strong\u003eKVI-NH\u003csub\u003e2\u003c/sub\u003e\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;line-height:normal;font-size:16px;font-family:\"Aptos\",sans-serif;text-align:center;'\u003e\u003cspan style='font-family:\"Courier New\";'\u003eV\u003cstrong\u003eRR\u003c/strong\u003eL\u003cstrong\u003eWR\u003c/strong\u003eWI\u003cstrong\u003eRR\u003c/strong\u003eVI-NH\u003csub\u003e2\u003c/sub\u003e\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;line-height:normal;font-size:16px;font-family:\"Aptos\",sans-serif;text-align:center;'\u003e\u003cspan style='font-family:\"Courier New\";'\u003eV\u003cstrong\u003eKK\u003c/strong\u003eL\u003cstrong\u003eW\u003c/strong\u003eKWI\u003cstrong\u003eK\u003c/strong\u003eKVI-NH\u003csub\u003e2\u003c/sub\u003e\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;line-height:normal;font-size:16px;font-family:\"Aptos\",sans-serif;text-align:center;'\u003e\u003cspan style='font-family:\"Courier New\";'\u003eV\u003cstrong\u003eKK\u003c/strong\u003eL\u003cstrong\u003eW\u003c/strong\u003eKW\u003cstrong\u003eLK\u003c/strong\u003eKV\u003cstrong\u003eL\u003c/strong\u003e-NH\u003csub\u003e2\u003c/sub\u003e\u003c/span\u003e\u003c/p\u003e\n \u003cp 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91.9pt;border-right: 1pt solid windowtext;border-bottom: 1pt solid windowtext;border-left: 1pt solid windowtext;border-image: initial;border-top: none;padding: 0cm 5.4pt;height: 23.45pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;line-height:normal;font-size:16px;font-family:\"Aptos\",sans-serif;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;color:black;'\u003eVFR12-a7-d\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;line-height:normal;font-size:16px;font-family:\"Aptos\",sans-serif;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;color:black;'\u003eVFR12-a8\u003c/span\u003e\u003cspan style='font-family: \"Times New Roman\",serif;color:black;'\u003e-d\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 149.1pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0cm 5.4pt;height: 23.45pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;line-height:normal;font-size:16px;font-family:\"Aptos\",sans-serif;text-align:center;'\u003e\u003cspan style='font-family:\"Courier New\";color:black;'\u003ev\u003cstrong\u003ekk\u003c/strong\u003el\u003cstrong\u003ew\u003c/strong\u003ekw\u003cstrong\u003elk\u003c/strong\u003ekv\u003cstrong\u003el\u003c/strong\u003e-NH\u003csub\u003e2\u003c/sub\u003e\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;line-height:normal;font-size:16px;font-family:\"Aptos\",sans-serif;text-align:center;'\u003e\u003cspan style='font-family:\"Courier 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none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0cm 5.4pt;height: 23.45pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;line-height:normal;font-size:16px;font-family:\"Aptos\",sans-serif;text-align:center;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;color:black;'\u003e524.00\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;line-height:normal;font-size:16px;font-family:\"Aptos\",sans-serif;text-align:center;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;color:black;'\u003e499.70\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 56.45pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0cm 5.4pt;height: 23.45pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;line-height:normal;font-size:16px;font-family:\"Aptos\",sans-serif;text-align:center;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;color:black;'\u003e5\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;line-height:normal;font-size:16px;font-family:\"Aptos\",sans-serif;text-align:center;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;color:black;'\u003e5\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 2cm;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0cm 5.4pt;height: 23.45pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;line-height:normal;font-size:16px;font-family:\"Aptos\",sans-serif;text-align:center;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;color:black;'\u003e22.5\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;line-height:normal;font-size:16px;font-family:\"Aptos\",sans-serif;text-align:center;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;color:black;'\u003e22.3\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 92.15pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0cm 5.4pt;height: 23.45pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;line-height:normal;font-size:16px;font-family:\"Aptos\",sans-serif;text-align:center;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;color:black;'\u003e0.858\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;line-height:normal;font-size:16px;font-family:\"Aptos\",sans-serif;text-align:center;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;color:black;'\u003e0.845\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003e\u003cem\u003e\u003csup\u003ea\u0026nbsp;\u003c/sup\u003e\u003c/em\u003eThe substituted amino acids are highlighted in bold and lower-case letters indicate d-isomers of analogs\u003c/p\u003e\n \u003cp\u003e\u003cem\u003e\u003csup\u003eb\u0026nbsp;\u003c/sup\u003e\u003c/em\u003eMolecular masses were determined using electrospray ionization mass spectrometry (ESI-MS). z stands for the charge of the ion whereas m/z represents the ratio of mass to charge.\u003c/p\u003e\n \u003cp\u003e\u003cem\u003e\u003csup\u003ec\u0026nbsp;\u003c/sup\u003e\u003c/em\u003eRetention time, R\u003csub\u003et\u003c/sub\u003e, was determined by timing the elution with RP-HPLC, represents the relative hydrophobicity of the peptides.\u003c/p\u003e\n \u003cp\u003e\u003cem\u003e\u003csup\u003ed\u0026nbsp;\u003c/sup\u003e\u003c/em\u003eHydrophobic moment, \u0026mu;H was quantified using an online tool- Heliquest (https://heliquest.ipmc.cnrs.fr/cgi-bin/ComputParams.py)\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eThe parent peptide, VFR12, possessed a net charge of +\u0026thinsp;4 and moderate hydrophobicity (hydrophobic moment \u0026micro;H\u0026thinsp;=\u0026thinsp;0.610; RP-HPLC retention time R\u003csub\u003et\u003c/sub\u003e = 21.9 min), shown in Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e. Substitution of lysine residues in VFR12-a1 to a3 increased the net charge from +\u0026thinsp;4 to +\u0026thinsp;6, resulting in decreased retention times (21.9 \u0026rarr; 18.1 \u0026rarr; 17.2 min) and increased hydrophobic moments (0.610 \u0026rarr; 0.826), suggesting an inverse correlation between charge density and lipophilicity. VFR12-a6 exhibited high retention (R\u003csub\u003et\u003c/sub\u003e = 21.7 min) and hydrophobic moment (0.877) despite a\u0026thinsp;+\u0026thinsp;5 charge, attributed to optimal tryptophan placement. Stereochemical variants revealed conformational flexibility: VFR12-a7(L) increased retention time (22.6 vs. 21.3 min) while the D-isomer a7(L)-d maintained similar properties (R\u003csub\u003et\u003c/sub\u003e = 22.5 min, \u0026micro;H\u0026thinsp;=\u0026thinsp;0.858). The VFR12-a8 series showed identical properties for both L- and D-forms, indicating stereochemical equivalence. VFR12-a9 achieved optimal amphiphilic balance with high charge (+\u0026thinsp;5) and enhanced hydrophobic moment (0.866). All peptides were purified by preparative RP-HPLC with molecular weights and purities confirmed by LC-MS analysis (Figure \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e) and retention times measured by RP-HPLC (Figure S2).\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eSecondary structures of peptides\u003c/h3\u003e\n\u003cp\u003eCircular dichroism (CD) spectroscopy (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eb) revealed distinct conformational behaviors of VFR12 and its analogs under varying environmental conditions that mimic physiological and membrane-interactive states. In aqueous buffer, the spectra were relatively flat, indicating disordered or loosely structured conformations in hydrophilic environments. Upon exposure to 50% trifluoroethanol (TFE), a helix-inducing solvent that mimics the hydrophobic membrane environment, the peptides underwent marked conformational transitions, as evidenced by negative ellipticity minima near 208 and 222 nm, characteristic of \u0026alpha;-helical structures. In 30 mM SDS micelles, which simulate anionic bacterial membrane environments, the peptides maintained strong \u0026alpha;-helical character with well-defined spectral features, confirming their membrane-active conformational preferences. Notably, the D-enantiomeric analogs (VFR12-a7(L)-d and VFR12-a8(L)-d) demonstrated mirror-image CD spectra with positive ellipticity at corresponding wavelengths, as expected for peptides with inverted chirality, while maintaining comparable helical propensities and membrane-binding capabilities.\u003c/p\u003e\n\u003ch3\u003eAntimicrobial activity and cell selectivity\u003c/h3\u003e\n\u003cp\u003eAntimicrobial susceptibility assays were conducted against six representative bacterial strains. The antimicrobial evaluation of VFR12 and its analogs revealed structure-activity relationships with variations in therapeutic efficacy and cell selectivity (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). The parent peptide VFR12 demonstrated moderate antibacterial activity, with MIC values ranging from 16 to 128 \u0026micro;M and a therapeutic index (TI) of 3.68, which is suboptimal. VFR12-a1 to VFR12-a5 exhibited minimal hemolytic activity above 256 \u0026micro;M (Figure S3); nevertheless, their antibacterial efficacy remained insufficient. VFR12-a6 showed markedly enhanced antimicrobial activity, but this was accompanied by elevated hemolytic activity as low concentrations such as 40\u0026micro;M, leading to a reduced therapeutic index. Notably, VFR12-a7 and VFR12-a8 demonstrated remarkable cell selectivity, with 10% hemolysis values of 135 and 182 \u0026micro;M, respectively, while maintaining excellent antibacterial efficacy with TI values of 9.47 and 9.03. These findings prompted the synthesis of isomeric forms of VFR12-a7 and VFR12-a8. VFR12-a8(L)-d demonstrated the highest therapeutic index (11.36), indicating exceptional safety with minimal hemolytic activity (102 \u0026micro;M), though with moderate antimicrobial potency. The melittin control showed poor selectivity (TI\u0026thinsp;=\u0026thinsp;0.62) with high hemolytic activity, confirming the validity of the experiment. These results demonstrate that strategic amino acid modifications successfully enhanced both antimicrobial potency and biocompatibility as indicated by low hemolytic activity, with VFR12-a7 and VFR12-a8 variants representing promising therapeutic candidates for further study.\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n \u003ctable id=\"Tab2\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eAntimicrobial and hemolytic activities and therapeutic index of VFR-12 and its analogs against different bacteria\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" rowspan=\"3\"\u003e\n \u003cp\u003ePeptides\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"6\"\u003e\n \u003cp\u003eMinimum Inhibitory Concentration (\u0026micro;M)\u003csup\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" rowspan=\"3\"\u003e\n \u003cp\u003eGM\u003csup\u003e\u003cem\u003eb\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" rowspan=\"3\"\u003e\n \u003cp\u003eMHC\u003csup\u003e\u003cem\u003ec\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" rowspan=\"3\"\u003e\n \u003cp\u003eTI\u003csup\u003e\u003cem\u003ed\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e\n \u003cp\u003e(MHC/GM)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003eGram-positive bacteria\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003eGram-negative bacteria\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eB. subtilis\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003eKCTC3068\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eS. aureus\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003eKCTC1621\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eS. epidermidis\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003eKCTC1917\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eE. coli\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003eKCTC1682\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eP. aureuginosa\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003eKCTC1637\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eS. tryphimurium\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003eKCTC1926\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eVFR12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e128\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e64\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e128\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e64\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e57.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e210\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3.68\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eVFR12-a1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e256\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e128\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e128\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e128\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026gt;\u0026thinsp;256\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e256\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e203.19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026gt;\u0026thinsp;256\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.52\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eVFR12-a2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e128\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e128\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e64\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e128\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e128\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e90.51\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026gt;\u0026thinsp;256\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e5.66\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eVFR12-a3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e256\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e128\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e128\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e128\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026gt;\u0026thinsp;256\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e256\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e203.19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026gt;\u0026thinsp;256\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.52\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eVFR12-a4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026gt;\u0026thinsp;256\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e128\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e128\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e128\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026gt;\u0026thinsp;256\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026gt;\u0026thinsp;256\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e222.86\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026gt;\u0026thinsp;256\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.30\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eVFR12-a5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026gt;\u0026thinsp;256\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e128\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e128\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e128\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026gt;\u0026thinsp;256\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026gt;\u0026thinsp;256\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e222.86\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026gt;\u0026thinsp;256\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.30\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eVFR12-a6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e64\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e14.25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.81\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eVFR12-a7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e14.25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e135\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e9.47\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eVFR12-a7(L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e89\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e5.56\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eVFR12-a8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e20.16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e182\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e9.03\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eVFR12-a8(L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e17.96\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e124\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e6.90\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eVFR12-a9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e64\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e85\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e5.31\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eVFR12-a7(L)-d\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8.98\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e8.35\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eVFR12-a8(L)-d\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8.98\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e102\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e11.36\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMelittin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.62\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"10\"\u003e\u003csup\u003ea\u003c/sup\u003e Minimum inhibitory concentrations (MICs) were determined as the lowest concentration of the peptides that inhibited bacterial growth.\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"10\"\u003e\u003csup\u003eb\u003c/sup\u003e GM denotes the geometric mean of MIC values from all bacterial strains tested. When the MIC observed was \u0026gt;\u0026thinsp;256 \u0026micro;M, a value of 512 \u0026micro;M was used to calculate the GM.\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"10\"\u003e\u003csup\u003ec\u003c/sup\u003e MHC is the minimum hemolytic concentration that resulted in 10% hemolysis of sheep red blood cells.\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"10\"\u003e\u003csup\u003ed\u003c/sup\u003e Therapeutic index (TI) was calculated as the ratio of the MHC value to GM. When the hemolytic activity observed was \u0026gt;\u0026thinsp;256 \u0026micro;M, a value of 512 \u0026micro;M was used to calculate the TI.\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003ch3\u003eMechanism of antimicrobial action\u003c/h3\u003e\n\u003cp\u003eThe mechanism of action of VFR12 and its selected analogs was investigated through membrane interaction studies, including membrane depolarization, SYTOX Green uptake, NPN (N-phenyl-1-naphthylamine) assays and flow cytometric analysis to elucidate their antimicrobial mode of action (Figs. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e \u0026amp; \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). The membrane depolarization assay against \u003cem\u003eS. aureus\u003c/em\u003e revealed that all peptides induced rapid and sustained membrane potential disruption, with fluorescence intensity increasing rapidly and then plateauing (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ea). VFR12-a7(L), VFR12-a8(L) and VFR12-a8(L)-d demonstrated the most pronounced depolarization effects, correlating with their superior antimicrobial potencies. The SYTOX Green uptake assay provided complementary evidence of membrane permeabilization (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eb) and all tested peptides induced substantial SYTOX Green uptake compared to the intracellular control peptide, Buforin-2. Notably, the kinetics of uptake were rapid, indicating immediate membrane disruption rather than gradual pore formation. The dose-dependent NPN assay against \u003cem\u003eE. coli\u003c/em\u003e further confirmed the membrane-active mechanism (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ec), where NPN fluorescence enhancement reflects outer membrane permeabilization. All peptides exhibited concentration-dependent membrane interaction, with melittin and several VFR12 analogs showing saturation at higher concentrations (16\u0026ndash;32 \u0026micro;M), confirming the membrane-targeting mechanism. Additionally, flow cytometric analysis using a FACScan provided a quantitative assessment of membrane integrity disruption at 2\u0026times; MIC concentrations (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). Against \u003cem\u003eS. aureus\u003c/em\u003e (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ea), the peptides induced significant membrane permeabilization with D-isomers showing over 90% cell permeabilization, which was significantly higher than that of the parent peptide VFR12 (57.96%). Similarly, against \u003cem\u003eE. coli\u003c/em\u003e (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eb), all peptides induced significant membrane permeabilization, with D-variants maintaining effective membrane disruption capabilities (VFR12-a7(L)-d: 92.53%, VFR12-a8(L)-d: 93.13%). These consistent results across multiple assays confirm that VFR12 and its selected analogs exert their antimicrobial effects primarily through rapid membrane disruption.\u003c/p\u003e\n\u003ch3\u003eSalt and serum sensitivity assay\u003c/h3\u003e\n\u003cp\u003eThe physiological stability of selected VFR12 analogs was evaluated under physiological salt conditions and in human serum, using \u003cem\u003eE. coli\u003c/em\u003e as the test organism (Table \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). Exposure to physiological salt concentrations led to varying degrees of activity reduction across the peptide series. Under various salt conditions, most peptides experienced a moderate reduction in activity. VFR12 showed a two-fold increase in MIC (64 \u0026rarr; 128 \u0026micro;M), whereas VFR12-a8(L)-d retained consistent potency (8 \u0026micro;M), even in the presence of 10% human serum. The most challenging condition was 10% human serum, where VFR12-a7(L)-d demonstrated remarkable stability, significantly outperforming the other analogs and exhibiting activity comparable to that of rifampicin. VFR12-a8(L)-d also maintained its full antimicrobial activity (MIC\u0026thinsp;=\u0026thinsp;8 \u0026micro;M) in serum, indicating excellent stability. In contrast, the parent peptide VFR12 and other analogs exhibited reduced activity in the presence of serum. These results indicate that analogs containing D-amino acids\u0026mdash;particularly VFR12-a7(L)-d and VFR12-a8(L)-d\u0026mdash;exhibit excellent physiological stability and enhanced resistance to proteolysis. Therefore, they represent promising candidates for systemic therapeutic applications, where conventional L-peptides typically suffer from poor stability.\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n \u003ctable id=\"Tab3\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eMIC values of selected peptides in the presence of physiological salts and human serum (10%) against \u003cem\u003eE.coli\u003c/em\u003e\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePeptide\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eControl\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e150mM\u003c/p\u003e\n \u003cp\u003eNaCl\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e4.5mM\u003c/p\u003e\n \u003cp\u003eKCl\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e6 \u0026micro;M\u003c/p\u003e\n \u003cp\u003eNH\u003csub\u003e4\u003c/sub\u003eCl\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e1 mM MgCl\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e2.5 mM CaCl\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e4 \u0026micro;M FeCl\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eHuman serum (10%)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eVFR12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e64\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e128\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e64\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e64\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e128\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e128\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eVFR12-a7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e64\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eVFR12-a7(L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e64\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e32\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eVFR12-a8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e64\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e64\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e64\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e128\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e32\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eVFR12-a8(L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e64\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e64\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e64\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e64\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e64\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eVFR12-a7(L)-d\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eVFR12-a8(L)-d\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTetracycline\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eRifampicin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n \u003ch2\u003eProteolytic stability assessment\u003c/h2\u003e\n \u003cp\u003eTo assess resistance to enzymatic degradation, the proteolytic stability of VFR12 and its selected analogs was evaluated using trypsin digestion, followed by radial diffusion assays against \u003cem\u003eE. coli\u003c/em\u003e (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ea). Trypsin, a serine protease that cleaves peptide bonds at the carboxyl side of basic residues such as lysine and arginine, was chosen as a representative proteolytic enzyme commonly found in biological systems. In the absence of trypsin, all peptides retained their antimicrobial activity, as demonstrated by well-defined zones of inhibition surrounding the peptide discs. However, following trypsin treatment, marked differences in proteolytic resistance were observed between L-amino acid and D-amino acid\u0026ndash;containing analogs. The parent peptide VFR12 and L-amino acid variants (VFR12-a7, VFR12-a7(L), VFR12-a8, and VFR12-a8(L)) completely lost their antimicrobial activity after trypsin treatment, as evidenced by the disappearance of inhibition zones. This vulnerability reflects trypsin\u0026rsquo;s inherent preference for cleaving L-amino acid peptide bonds, especially at the frequent lysine and arginine residues found in these cationic antimicrobial peptides. In contrast, the D-isomers VFR12-a7(L)-d and VFR12-a8(L)-d exhibited strong resistance to enzymatic degradation, retaining significant antimicrobial activity after trypsin exposure, as shown by the persistence of inhibition zones. These findings support the enhanced serum stability observed in the D-isomers and further validate their potential as therapeutically promising antimicrobial agents.\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eAntibiofilm activity\u003c/h3\u003e\n\u003cp\u003eThe antibiofilm efficacy of VFR12 and its selected analogs was evaluated using minimum biofilm inhibitory concentration (MBIC) and minimum biofilm eradication concentration (MBEC) assays against multidrug-resistant \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e (MDRPA) biofilms (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eb). In biofilm inhibition studies, VFR12-a7(L)-d exhibited potent activity at concentrations as low as 4 \u0026micro;M, while the remaining VFR12-a7 stereoisomers demonstrated substantial inhibition (80\u0026ndash;90%) at 8 \u0026micro;M. Notably, VFR12-a8 displayed limited inhibitory capacity, whereas VFR12-a8(L) and its D-isomer VFR12-a8(L)-d showed statistically significant biofilm inhibition activity (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). All selected analogs exhibited markedly enhanced antibiofilm performance compared to the parent peptide VFR12. In the biofilm eradication assays using preformed biofilms, VFR12-a7(L) and its stereochemical counterpart demonstrated significant disruption activity starting at 4 \u0026micro;M (\u0026gt;\u0026thinsp;60% eradication), while VFR12-a8(L) and VFR12-a8(L)-d achieved comparable biofilm elimination from 8 \u0026micro;M onwards. Eradication efficacy showed dose-dependent enhancement, with VFR12-a7(L), VFR12-a8(L), and their respective D-isomers achieving above 80% biofilm disruption at 16 \u0026micro;M. In contrast, the parent peptide VFR12 required substantially higher concentrations (64 \u0026micro;M) to attain equivalent eradication levels.\u003c/p\u003e\n\u003ch3\u003eBiocompatibility assay\u003c/h3\u003e\n\u003cp\u003eThe cytotoxicity profiles of VFR12 and its selected analogs were evaluated in both RAW 264.7 macrophages (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ea) and HaCaT keratinocytes (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eb) to assess their safety toward mammalian cells across a concentration range of 1\u0026ndash;32 \u0026micro;M. In RAW 264.7 cells, all peptides demonstrated excellent biocompatibility at therapeutic concentrations (1\u0026ndash;4 \u0026micro;M), maintaining cell viability above 70%. Concentration-dependent effects on cell viability became apparent at higher concentrations, with notable reductions emerging at 8 \u0026micro;M and above. At 16 \u0026micro;M, most peptides showed a moderate impact on cell viability, with values dropping to 50\u0026ndash;70%, while at 32 \u0026micro;M, significant viability reduction was observed with values falling below 50% for the parent VFR12 and some analogs. Similarly, in HaCaT cells, the peptides maintained favorable viability profiles at lower concentrations (1\u0026ndash;4 \u0026micro;M) with cell viability consistently above 70%. The viability patterns in HaCaT cells were comparable to those in RAW 264.7 cells, showing concentration-dependent reductions with more pronounced effects at 16\u0026ndash;32 \u0026micro;M concentrations. As all the selected peptides showed\u0026thinsp;\u0026ge;\u0026thinsp;70% viability at 4\u0026micro;M in both cell lines, this concentration was used for anti-inflammatory activity study.\u003c/p\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003ch2\u003eAnti-inflammatory activities\u003c/h2\u003e\n \u003cp\u003eThe anti-inflammatory potential of VFR12 and its selected analogs was evaluated using RAW 264.7 macrophage cells to assess their effects on key inflammatory mediators including tumor necrosis factor-\u0026alpha; (TNF-\u0026alpha;), interleukin-6 (IL-6) secretion and nitrite production (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ec-e). All peptides were tested at 4 \u0026micro;M concentration in LPS-stimulated RAW 264.7 cells, with LL-37 serving as a positive control and untreated cells (control) representing baseline inflammatory status. The results revealed that the selected VFR12 analogs (a7 and a8 isomers) demonstrated favorable anti-inflammatory profiles under LPS stimulation. TNF-\u0026alpha; secretion, measured by ELISA, showed that all VFR12-a7 and VFR12-a8 analogs maintained minimal pro-inflammatory cytokine release in LPS-stimulated conditions, contrasting with the substantial TNF-\u0026alpha; production observed with LPS alone and VFR12 treatment, demonstrating improved inflammatory safety profiles of the designed analogs. The IL-6 response pattern revealed that selected analogs VFR12-a7, VFR12-a7(L), VFR12-a7(L)-d, VFR12-a8, VFR12-a8(L) and VFR12-a8(L)-d maintained IL-6 levels similar to unstimulated control cells, indicating negligible inflammatory induction. Only LPS alone and the parent peptide VFR12 induced significant IL-6 production, while all the targeted analogs demonstrated excellent anti-inflammatory profiles. In addition, nitrite production remained consistently suppressed (\u0026le;\u0026thinsp;10%) across all tested VFR 12 analogs (except the parent VFR12) compared to LPS-alone stimulation, which induced nearly 100% nitrite. The overall inflammatory assessment in LPS-stimulated RAW 264.7 macrophages demonstrates that the VFR12 analogs possess excellent immunomodulatory properties, effectively suppressing LPS-induced inflammatory responses, positioning them as promising therapeutic candidates with favorable biocompatibility profiles.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n \u003ch2\u003eLPS-binding capability\u003c/h2\u003e\n \u003cp\u003eThe LPS-binding assay demonstrated dose-dependent endotoxin neutralization capabilities across all tested VFR12 analogs (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ef). At low concentrations (1\u0026ndash;2 \u0026micro;M), most peptides exhibited minimal LPS binding, whereas a progressive increase in binding efficiency was observed with increasing concentrations, with distinct patterns emerging at 4\u0026minus;8 \u0026micro;M. At the highest tested concentration (32 \u0026micro;M), most analogs achieved substantial LPS binding, with several peptides reaching 80\u0026ndash;100% BC displacement efficiency comparable to that of the LL-37 positive control. Notably, the D-amino acid variants VFR12-a7(L)-d and VFR12-a8(L)-d demonstrated excellent LPS-binding capabilities while the parent VFR12 exhibited moderate LPS-binding activity with gradual increases across the concentration range.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThrombin C-terminal peptides represent a promising class of host defense peptides with demonstrated antimicrobial and anti-inflammatory properties derived from the proteolytic cleavage of the coagulation enzyme thrombin. These peptides have been shown to exhibit broad-spectrum activity against both Gram-positive and Gram-negative bacteria in vitro and in vivo through membrane disruption, while concurrently exhibiting immunomodulatory effects by suppressing macrophage activation in response to bacterial endotoxins\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan additionalcitationids=\"CR24 CR25 CR26\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Building upon this natural template, we employed rational design principles to develop VFR12 and its analogs through systematic amino acid substitutions and stereochemical modifications. The truncation strategy from the parent thrombin-derived sequence yielded VFR12 as a compact α-helical peptide with balanced amphiphilic properties. Strategic modifications focused on optimizing the charge-hydrophobicity relationship through lysine/arginine substitutions and tryptophan positioning to enhance membrane interaction capabilities. Among the designed peptides, VFR12-a7 and VFR12-a8 demonstrated the highest specificity toward bacterial cells, highlighting their potential for further exploration. To improve proteolytic resistance, we synthesized D-enantiomeric variants (VFR12-a7(L)-d and VFR12-a8(L)-d) by substituting their L-amino acids with D-forms, thereby enhancing peptide stability. VFR12-a7 and VFR12-a8 contain two and three isoleucine (Ile) residues, respectively. Owing to two chiral centers\u0026mdash;located at the backbone and side chain\u0026mdash;Ile exists as four stereoisomers: L-Ile (2S,3S), D-Ile (2R,3R), L-allo-Ile (2S,3R), and D-allo-Ile (2R,3S). Because Fmoc-D-Ile and Fmoc-D-allo-Ile are prohibitively expensive and unsuitable for solid-phase synthesis, we replaced D-Ile with hydrophobic D-leucine (D-Leu), which has the same molecular weight. Since these peptides exert antimicrobial action via membrane disruption rather than receptor-mediated mechanisms, this substitution was not expected to significantly affect their bioactivity. Consistent with this prediction, both VFR12-a7 and its analog VFR12-a7(L), as well as VFR12-a8 and its corresponding variant VFR12-a8(L), exhibited comparable antimicrobial and hemolytic profiles.\u003c/p\u003e\u003cp\u003eThe incorporation of D-amino acids, particularly in VFR12-a7(L)-d and VFR12-a8(L)-d, successfully preserved amphiphilic architecture while conferring enhanced hydrophobic character and proteolytic resistance. The systematic increase in hydrophobic moments (0.610 \u0026rarr; 0.877) across the analog series, coupled with strategic planar positioning of tryptophan residues, facilitated optimal membrane insertion geometry essential for antimicrobial activity. These modifications resulted in improved cell selectivity profiles, with therapeutic indices ranging from 3.68 to 11.36, demonstrating enhanced discrimination between bacterial and mammalian cell membranes compared to the parent peptide. The biocompatibility assessment confirmed excellent safety profiles, with all optimized analogs maintaining approximately 70% cell viability in both RAW 264.7 macrophages and HaCaT keratinocytes.\u003c/p\u003e\u003cp\u003eThe antimicrobial evaluation revealed structure-activity relationships that validate the rational design approach. VFR12-a6 through a9 demonstrated potent broad-spectrum activity with lower MIC values against both Gram-positive and Gram-negative bacteria, representing significant improvements over the parent VFR12. Mechanistic studies confirmed a membrane-disrupting mode of action, as evidenced by rapid membrane depolarization, permeabilization assays, and flow cytometric analysis showing above 90% bacterial membrane disruption in the case of D-isomers. The D-amino acid-containing peptides maintained comparable antimicrobial potency while exhibiting superior stability in physiological conditions, including resistance to proteolytic degradation and enhanced activity in human serum. Notably, the LPS-binding capacity of these peptides provides an additional therapeutic advantage by neutralizing endotoxins released during bacterial lysis, potentially mitigating inflammatory responses associated with Gram-negative bacterial infections. The antibiofilm activity represents a critical advancement, as biofilm-associated infections pose significant therapeutic challenges due to enhanced antibiotic resistance and persistence. VFR12-a7(L)-d and VFR12-a8(L)-d demonstrated dual functionality, effectively preventing biofilm formation (MBIC) and disrupting established biofilms (MBEC) against multidrug-resistant \u003cem\u003eP. aeruginosa\u003c/em\u003e. The concentration-dependent efficacy, achieving\u0026thinsp;\u0026gt;\u0026thinsp;80% biofilm eradication at 32 \u0026micro;M, substantially outperformed parent peptide. This dual anti-biofilm capacity is particularly valuable for treating chronic infections where mature biofilms represent the primary therapeutic obstacle.\u003c/p\u003e\u003cp\u003eThe anti-inflammatory assessment revealed that optimized VFR12 selected analogs possess excellent immunomodulatory properties, effectively suppressing LPS-induced TNF-α, IL-6, and nitric oxide production in macrophage cellular models while maintaining minimal intrinsic inflammatory activity. Unlike the parent VFR12 which induced significant cytokine release, the selected analogs demonstrated anti-inflammatory profiles comparable to unstimulated controls, suggesting therapeutic benefits beyond direct antimicrobial activity. The dose-dependent LPS-binding capabilities, achieving 80\u0026ndash;100% endotoxin neutralization at therapeutic concentrations, provide a probable mechanism for preventing cytokine storms and septic shock associated with Gram-negative bacterial infections. This multifaceted therapeutic approach \u0026ndash; combining direct antimicrobial activity, biofilm disruption, and immunomodulation \u0026ndash; positions these peptides as comprehensive therapeutic agents capable of addressing both pathogen elimination and host inflammatory responses.\u003c/p\u003e\u003cp\u003eVFR12-a8(L)-d emerges as the most promising therapeutic candidate due to its exceptional combination of properties: potent broad-spectrum antimicrobial activity (MIC 8\u0026ndash;16 \u0026micro;M), outstanding cell selectivity (therapeutic index of 11.36), complete proteolytic resistance, effective antibiofilm activity, and strong anti-inflammatory action. The D-amino acid configuration addresses the primary limitation of peptide therapeutics by providing stability in biological systems while maintaining full therapeutic efficacy, potentially enabling oral bioavailability\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. The superior performance in human serum and resistance to enzymatic degradation positions VFR12-a8(L)-d as a viable candidate for systemic administration in treating serious infections caused by multidrug-resistant pathogens. Future development should focus on in vivo efficacy validation in relevant animal models to confirm therapeutic potential and safety profiles. Murine infection models\u0026minus;including systemic sepsis, skin and soft tissue infections, and biofilm-associated implant infections\u0026minus;will establish dose-response relationships and optimal therapeutic windows. Overall, this study provides valuable insights into the rational modification of host defense peptides, establishing a framework for developing new therapeutics to combat antimicrobial resistance and sepsis.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003cdiv id=\"Sec15\" class=\"Section3\"\u003e\u003ch2\u003ePeptide synthesis \u0026amp; characterization\u003c/h2\u003e\u003cp\u003ePeptides were synthesized using Fmoc solid-phase peptide synthesis (SPPS) with Rink amide MBHA resin, as previously described\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. Detailed information is presented in the supporting information.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eCircular Dichroism Spectroscopic Analysis\u003c/h2\u003e\u003cp\u003eCircular dichroism (CD) spectroscopy was used to assess the secondary structure of the synthesized peptides, as described previously and explained in the supporting information\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eAntimicrobial susceptibility assay\u003c/h2\u003e\u003cp\u003eThe antimicrobial activity of the synthesized peptides against various bacterial strains was evaluated according to the guidelines established by the Clinical and Laboratory Standards Institute, following the previous manner\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e,\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. The supplementary information provides a detailed outline of the antimicrobial properties of the compounds.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003eHemolytic activity\u003c/h2\u003e\u003cp\u003eHemolytic activity was determined by measuring sheep red blood cells (sRBCs) according to the established protocol\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. Comprehensive details regarding hemolysis are available in the supplementary materials.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003eMembrane depolarization assay\u003c/h2\u003e\u003cp\u003eBacterial membrane depolarization was assessed according to a prior study with minor modifications\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. The supplementary information provides a detailed description of this method.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003eSYTOX Green uptake assay\u003c/h2\u003e\u003cp\u003eMembrane permeabilization was assessed using SYTOX Green dye uptake, adapted from previous work and explained in the supplementary information\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003eOuter membrane permeability assay\u003c/h2\u003e\u003cp\u003eOuter membrane permeability in \u003cem\u003eE. coli\u003c/em\u003e (KCTC1682) was assessed using the fluorescent probe NPN (1-N-phenylnaphthylamine) in a dose-dependent manner, as described previously\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. Detailed information is provided in the supporting information.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\u003ch2\u003eFACScan analysis\u003c/h2\u003e\u003cp\u003eUsing FACScan equipment and established protocols, propidium iodide (PI) uptake was quantified to assess bacterial cell membrane integrity\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e,\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. The supplementary information outlines this analysis in detail.\u003c/p\u003e\u003cdiv id=\"Sec23\" class=\"Section3\"\u003e\u003ch2\u003eProtease stability (Radial diffusion)\u003c/h2\u003e\u003cp\u003eThe effect of peptides on protease stability was assessed using the radial diffusion method on agar plate, focusing on trypsin activity. Briefly, LB agar plates were prepared with \u003cem\u003eE. coli\u003c/em\u003e strain (KCTC1682). Peptides were co-incubated with trypsin for several hours, then applied to agar plates using sterile discs alongside peptide-only controls. After a 24-hour incubation at 37\u0026deg;C, proteolytic degradation was evaluated by comparing inhibition zones, with reduced or absent zones indicating peptide degradation by trypsin activity.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e\u003ch2\u003eAntibiofilm activity (MBIC, MBEC)\u003c/h2\u003e\u003cp\u003eThe antibiofilm properties were evaluated by determining minimum biofilm inhibition concentration (MBIC) and minimum biofilm eradication concentration (MBEC) against MDRPA (CCARM 2095), following previously described methods\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e,\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. The supplementary information provides a detailed explanation of MBIC and MBEC procedures.\u003c/p\u003e\u003cdiv id=\"Sec25\" class=\"Section3\"\u003e\u003ch2\u003eSalt and serum stability assay\u003c/h2\u003e\u003cp\u003eSalt and serum stability tests were conducted to evaluate efficacy of the peptides under physiological conditions, as previously described \u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. Comprehensive details regarding this method are available in the supplementary materials.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec26\" class=\"Section3\"\u003e\u003ch2\u003eCell viability assay\u003c/h2\u003e\u003cp\u003eCell viability was evaluated using two cell lines: mouse macrophage RAW 264.7 cells and human keratinocyte HaCaT cells. Cells were seeded in 96-well plates and incubated for 24 h at 37\u0026deg;C, in 5% CO₂. After 24 h of peptide treatment, viability was assessed using the WST-8 kit. This assay relies on the reduction of the WST-8 reagent by metabolically active cells, resulting in the formation of an orange formazan product. WST-8 reagent (10 \u0026micro;L/well) was added and incubated for 2 h, and absorbance was measured at 450 nm to evaluate dose-dependent effects across various concentrations and cell types.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec27\" class=\"Section3\"\u003e\u003ch2\u003eMeasurement of pro-inflammatory cytokines\u003c/h2\u003e\u003cp\u003eRAW 264.7 cells were plated in 96-well plates at 3 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells per well and incubated overnight at 37\u0026deg;C. Peptides were added to the cultures, followed by stimulation with 20 ng/mL lipopolysaccharide (LPS) and a 24-hour incubation at 37\u0026deg;C. The LPS-induced activation of the macrophages was expected to trigger the release of inflammatory cytokines, including production of TNF-α and IL-6. TNF-α and IL-6 cytokine was quantified using ELISA kits according to the manufacturer's instructions to evaluate the immunomodulatory effects of the peptides on LPS-stimulated macrophages.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec28\" class=\"Section2\"\u003e\u003ch2\u003eMeasurement of NO production\u003c/h2\u003e\u003cp\u003eNitric oxide (NO) production in LPS-stimulated RAW 264.7 cells was quantified using the Griess assay according to a previously described protocol\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. RAW 264.7 cells (3 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells/well) were cultured in 96-well plates and stimulated with LPS (20 ng/mL) in the presence or absence of peptides for 24 hours. Culture supernatants were transferred to new plates, mixed with equal volumes of 1% Griess reagent (Sigma), and incubated for 15 minutes at room temperature. Nitrite levels were quantified by measuring absorbance at 540 nm using a microplate reader (KLAB, MRX A2000).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec29\" class=\"Section2\"\u003e\u003ch2\u003eLPS binding assay\u003c/h2\u003e\u003cp\u003eThe binding ability of peptides to lipopolysaccharide (LPS) was assessed using a BODIPY-TR cadaverine (BC) displacement assay, as outlined in previous studies\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e,\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. A detailed description of the procedure is provided in the supporting information.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec30\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eAll graphical representations and statistical analyses were performed using one-way analysis of variance (ANOVA) with SPSS 16.0 and SigmaPlot v12.0 software. Data are presented as the means\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD) from three independent experiments conducted in triplicate. Statistical significance was determined using two-sample t-tests, with p-values\u0026thinsp;\u0026lt;\u0026thinsp;0.05 considered statistically significant. Significance levels are indicated as follows: ** for p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, *** for p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, and **** for p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001.\u003c/p\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supported by the National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIT) (No. RS-2023-00210292 and No. 2022R1F1A1068595) and the Global-Learning \u0026amp; Academic Research Institution for Master\u0026apos;s\u0026middot;PhD students, and Postdocs (LAMP) Program of the National Research Foundation of Korea (NRF) grant funded by the Ministry of Education (No. RS-2023-00285353).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eI.J.: Investigation, methodology, data analysis, and writing original draft, S.D.K.: data curation, C.A.: methodology, C.W.L.: writing-review and editing, S.Y.S.: conceptualization, supervision, writing-review and editing, S.Y.: supervision, funding acquisition, writing-review and editing.\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\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analysed during this study are included in this published article and its supplementary information files.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdditional Information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Information\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe online version contains supplementary material available at\u003cstrong\u003e\u003cbr\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eLaxminarayan, R. et al. 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Development of a fluorescent probe displacement method optimized for the rapid identification of lipopolysaccharide-binding agents. \u003cem\u003eComb. Chem. High Throughput Screen.\u003c/em\u003e \u003cb\u003e7\u003c/b\u003e, 239\u0026ndash;249 (2004).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"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":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"antimicrobial peptide, thrombin-derived peptide, D-isomer, antibiofilm, immunomodulation","lastPublishedDoi":"10.21203/rs.3.rs-7306037/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7306037/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe worsening crisis of antibiotic resistance demands innovative therapies to combat multidrug-resistant pathogens. In this study, we report the rational design and optimization of VFR12 (VFRLKKWIQKVI), a thrombin C-terminus-derived 12-mer peptide, via systematic amino acid substitutions and stereochemical modifications. A series of 12-mer analogs were synthesized and assessed for antimicrobial activity, cell selectivity, biofilm disruption, and immunomodulatory effects. Six lead candidates (VFR12-a7, VFR12-a8, VFR12-a7(L), VFR12-a8(L), VFR12-a7(L)-d, VFR12-a8(L)-d) demonstrated potent, broad-spectrum antimicrobial activity against both Gram-positive and Gram-negative bacteria, including multidrug-resistant \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e, while exhibiting minimal hemolytic activity. Notably, D-amino acid variants VFR12-a7(L)-d and VFR12-a8(L)-d showed significantly improved therapeutic indices, complete resistance to proteolysis, and enhanced serum stability compared to their L-isomers. These peptides effectively inhibited biofilm formation and disrupted preformed biofilms while maintaining excellent biocompatibility. Furthermore, anti-inflammatory assessments revealed a significant suppression of LPS-induced production of TNF-α, IL-6, and nitric oxide, along with strong endotoxin neutralization. Among the analogs, VFR12-a8(L)-d emerged as the most promising candidate, combining potent antimicrobial activity with excellent safety and multifaceted therapeutic properties. These findings provide a valuable framework for the development of next-generation host defense peptides with integrated antimicrobial, antibiofilm, and anti-inflammatory properties, offering a multifaceted approach to tackling antibiotic resistance and sepsis.\u003c/p\u003e","manuscriptTitle":"Rational Design of Thrombin-Derived VFR12 Analogs with Enhanced Antimicrobial, Antibiofilm, and Anti-inflammatory Properties","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-19 18:27:30","doi":"10.21203/rs.3.rs-7306037/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-09-03T04:08:56+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-25T14:17:36+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-22T20:33:43+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"163662463287350666569529411182437869778","date":"2025-08-19T21:50:57+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"32682583415401344813465071639231779142","date":"2025-08-13T15:40:25+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"146902420310196035744630401948655004062","date":"2025-08-11T15:37:47+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-08-11T13:27:24+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-08-11T13:25:13+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-08-11T12:13:42+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-08-11T00:53:51+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-08-11T00:50:48+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"29bf0622-9b65-4b08-ac93-9778d1a2139d","owner":[],"postedDate":"August 19th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":53138047,"name":"Biological sciences/Drug discovery"},{"id":53138048,"name":"Biological sciences/Microbiology"}],"tags":[],"updatedAt":"2025-11-24T16:01:15+00:00","versionOfRecord":{"articleIdentity":"rs-7306037","link":"https://doi.org/10.1038/s41598-025-24789-9","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-11-19 15:57:42","publishedOnDateReadable":"November 19th, 2025"},"versionCreatedAt":"2025-08-19 18:27:30","video":"","vorDoi":"10.1038/s41598-025-24789-9","vorDoiUrl":"https://doi.org/10.1038/s41598-025-24789-9","workflowStages":[]},"version":"v1","identity":"rs-7306037","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7306037","identity":"rs-7306037","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}