Sortase A Inhibition: Updated Review of Non-Peptide Ligands for Targeting Staphylococcus Aureus Virulence

Sortase A Inhibition: Updated Review of Non-Peptide Ligands for Targeting Staphylococcus Aureus Virulence | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 29 July 2025 V1 Latest version Share on Sortase A Inhibition: Updated Review of Non-Peptide Ligands for Targeting Staphylococcus Aureus Virulence Authors : Aaheli Basu , Rahul Khanra , and Sarmistha Pal 0000-0002-6142-7118 [email protected] Authors Info & Affiliations https://doi.org/10.22541/au.175376729.98473264/v1 Published Next Research Version of record Peer review timeline 319 views 210 downloads Contents Abstract Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Sortases are a class of bacterial enzymes that are responsible for altering surface proteins and supporting attachment to host cells and enhancing biofilm formation, which poses resistance to the anti-virulence therapy. These enzymes are found in Gram-positive bacteria and can be classified into classes A-F. Sortase A, found in Staphylococcus aureus, is the prototype member of the sortase family and acts as the pioneer to study the working mechanism, applications, and structure of sortases. Sortases attract the attention of researchers as a target for the development of inhibitors used for anti-virulence therapy. Prevention of formation of biofilm is challenging, and hence is targeted using Gram-positive bacteria containing sortase A. In this review, sortase inhibitors are categorized on the basis of specific chemical features such as vinyl sulfone, anthraquinones, isoquinoline derivatives, indoles, pyrrolomycins, thiadiazoles, natural variants, and so on. Further research on sortase in search of inhibitors continues in order to create a new class of anti-infective drugs suitable for humans to prevent the dreadful rise of antimicrobial resistance. INTRODUCTION Antibiotic resistance poses one of the most significant threats to world health in the battle against bacterial infections. Recent research indicate that antimicrobial agents are essential to combat drug-resistant bacteria. This action is necessary to prevent 10 million deaths and $100 trillion in healthcare expenditures by 2050. To confront this escalating dilemma, novel pharmaceuticals must supplant broad-spectrum antibiotics. One approach is to selectively eradicate pathogenic microbes while preserving the indigenous microbiota. Innovative treatments include anti-virulence agents that diminish bacterial growth and survival, alongside bactericidal or bacteriostatic substances for certain infections.(Thappeta et al. , 2020) Bacterial sortases identify and cleave carboxyl-terminal sorting signals from surface proteins. Staphylococcus aureus Sortase A (SrtA) is the first to be comprehended. Sortase A, a membrane-associated cysteine transpeptidase, facilitates the covalent attachment of surface proteins to the cell walls of Gram-positive bacteria. The microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) of S. aureus facilitate bacterial adherence to host tissues, biofilm formation, and evasion of the immune system by inhibiting opsonization and phagocytosis.(Cascioferro et al. , 2014) Numerous bacteria infiltrate surfaces and establish biofilms. Microorganisms within biofilms typically obstruct antibiotic infiltration. Biofilms represent a critical technique employed by bacterial pathogens, regarded as a primary contributor to nosocomial infections, particularly in patients who have recently undergone surgery, possess compromised immune systems, or have indwelling medical devices such as catheters.(Bonaventura et al. , 2020) Before the discovery of antibiotics, severe infections often resulted in fatalities. The rise of antibiotic-resistant pathogens threatens current antibiotic efficacy. Consequently, a critical focus for public health is the creation of innovative antimicrobial agents that possess distinct mechanisms of action, thereby reducing the likelihood of antimicrobial resistance emergence.(Alharthi et al. , 2023) Sortases are a prominent target for anti-virulence therapy; hence, researchers have dedicated the past decade to creating a potent inhibitor. Bacteria employ virulence factors to infiltrate and damage their hosts. Virulence factors comprise surface proteins, toxins, hydrolytic enzymes, and capsules. In the early 1990s, Schneewind and colleagues elucidated the function of the sortase enzyme prior to identifying the corresponding gene. Sortase A (Srt A) from Staphylococcus aureus was the inaugural sortase identified, with several other variants discovered in the past decade. The protein sorting mechanism allocates proteins into the cell wall compartments of Gram-positive bacteria, hence naming this class of enzymes.(Rasko and Sperandio, 2010) Sortase, a membrane-bound cysteine transpeptidase found in Gram-positive bacteria. It was a promising candidate for novel antibiotic classes following its elucidation in 1999. The advantages comprise its extracellular membrane localization, absence of human homologs, and minimal relevance to bacterial development. Sortase A in Staphylococcus aureus serves as the prototype for nearly 1,000 homologs.(Ha et al. , 2020) In order to investigate the suitability of Sortase A inhibitors in preventing the biofilm formation in S. aureus , numerous biological and biophysical experiments must be carried out for confirmation.(Dewan et al. , 2024) Classifying sortase enzymes based on sequence alignment and biological functions is the most straightforward method of characterization, despite the existence of numerous alternative nomenclatures. Given that Srt A is present in nearly all Gram-positive bacteria, the ”housekeeping” sortases classify the majority of cell surface proteins. Only a limited number of bacteria possess Sortase B (Srt B), which selectively sorts a singular substrate (IsdC) under conditions of iron deficiency. Some Gram-positive bacteria produce Sortase C (Srt C) enzymes and pilin proteins to polymerize into pili. Gram-positive bacteria that undergo sporulation selectively co-express sporulation-associated proteins via Sortase D (Srt D). Certain bacteria possess sortase enzyme classes E and F, however their roles remain unidentified. The majority of Gram-positive bacteria possess Srt A and potentially specialized auxiliary sortases. Sortases are present in certain Gram-negative and Archaebacterial species, although their function remains unidentified.(Susmitha et al. , 2021) Table 1. Sortase Classification A LPXTG Surface Protein Anchoring Staphylococcus, Listeria, Streptococcus, Bacillus, Clostridium, Enterobacter, Lactobacillus B (N/S/P)PXTG Haeme Uptake Bacillus, Listeria, Bacillus, C LPXTG Pili Assembly Corynebacterium, Streptococcus, Clostridium, Actinomyces, Enterobacter, Lactobacillus D LPXTA Spore Formation Bacillus E LPXTG Aerial Hyphae Formation, Surface Protein Anchoring, Pilus Attachement Corynebacterium, Streptomyces, Actinomyces F LPXTG Unknown Propionibacterium SORTASE A STRUCTURE The sortase enzyme prototype Strt A of Staphylococcus aureus possesses a well-defined structure. Sortase A (Srt A) is a polypeptide comprising 206 amino acids, with an elongated, unstructured amino-terminal tail rich in non-polar residues for membrane attachment, and a self-folding catalytic domain responsible for transpeptidation.(Suree et al. , 2009a) In this process, the surface protein precusors’ C-terminal sorting signal (LPXTG motif) between threonine (T) and glycine (G) residues is broken.(Ilangovan et al. , 2001) The carboxyl group of the threonine (Thr) residue in the substrate and the thiol group of Cys184 in the enzyme form a covalent thioester bond as a result.(Cascioferro et al. , 2015a) The amino group of Gly in this transient thioester acyl-enzyme intermediate is assailed by a nucleophile, leading to the establishment of an amide bond between the carboxylic group of Thr and the amino group of the cell wall cross-bridge, thereby facilitating the covalent attachment of the substrate protein to the peptidoglycan.(Mazmanian et al. , 1999) The active sites of sortase family enzymes have three conserved residues: His120, Cys184, and Arg197, which are crucial for catalysis. These amino acids diminish sortase activity both in vivo and in vitro when substituted with alanine. These three amino acid residues form a catalytic triad analogous to that seen in amylases, esterases, peptidases, acylases, and lactamases. Every structure comprises an acidic, basic, and nucleophilic residue. The acidic residue, glutamate or aspartate, aligns and polarizes the base, histidine, thereby activating the nucleophile, which may be serine, cysteine, or occasionally threonine. The triad decreases the pKa of the nucleophile residue, enhancing its reactivity for catalysis. The SrtA structure devoid of the substrate (PDB:2KID) reveals isolated and non-interacting His120 and Cys25 side chains. For the extraction of sulfhydryl protons and nucleophilic attack, these side chains must experience a conformational change that promotes substrate-induced activation. The thiol group of Cys 184 in Srt A’s active site nucleophilically attacks the carbonyl group of threonine, resulting in the formation of a thioester acyl enzyme intermediate. The peptidoglycan sequesters this intermediate by an amide bond linking the protein’s C-terminal threonine to the amino group of the pentaglycine cross-bridge. Arg197 stabilizes the tetrahedral oxyanion transition state by deprotonating Cys184 and the Gly5 chain of lipid II prior to forming the acyl-enzyme intermediate. His120 was believed to activate Cys184 by forming an ionic interaction with imidazolium thiolate. Further study indicates that His120 functions as a universal catalytic base. Other Gram-positive bacteria, such as Streptococcus pyogenes, pneumoniae, and Listeria monocytogenes , exhibit significant conservation of the SrtA active site residues Cys184 and His120 found in Staphylococcus aureus . Sortases modify surface proteins, enhance host cell adhesion, and encourage biofilm formation, so conferring resistance to antiviral medication. Gram-positive bacteria synthesize enzymes classified as A through F. Sortase A from Staphylococcus aureus serves as the prototypical sortase, leading investigations into its mechanism, applications, and structure. Researchers are formulating anti-virulence pharmaceuticals aimed at sortases. Sortase A is a bifunctional enzyme including a 206-amino-acid long unstructured amino-terminal tail containing non-polar residues that facilitate protein-membrane linkage, and a self-folding catalytic domain responsible for transpeptidation. Sortases may distinguish between two substrates in each process by creating peptide bonds between two distinct proteins. Sortases have been developed to accommodate a broader range of substrates. Clinics address MRSA infections and septic arthritis with this objective. Inhibiting biofilm formation with antibiotics is challenging; hence, Gram-positive bacteria possessing sortase A are utilized. Chemical features such as sulfone, anthraquinones, isoquinoline derivatives, indoles, pyrrolomycins, thiadiazoles, and natural variants categorize sortase inhibitors. Research on sortase inhibitors persists in producing a novel category of human-compatible anti-infective agents to address antimicrobial resistance. Figure 1. Sortase A structure (PDB ID:1T2W). SORTASE’S ROLE IN VIRULENCE MECHANISM A number of surface polypeptides and polysaccharides, such as streptococcal M protein and staphylococcal protein A,(Navarre and Schneewind, 1999) are connected to the cell wall that encloses Gram-positive bacteria, they play a variety of activities, including attachment to host tissues, evading host defences, and forming biofilms.(Fischetti, 2019) A search for S. aureus mutants incapable of cleaving the protein A discovered that bacteria with a SrtA gene mutation possess surface protein anchoring defects.(Mazmanian et al. , 1999) This enzyme having a membrane-spanning N-terminus and a catalytic C-terminus is encoded by the Srt gene. Srt is required for binding to surface proteins that anchor at their C-terminal end,(Kappel et al. , 2012) and its absence results in this incapacity, and the process of creating an infection is slowed down as a result of the elimination of surface adherence.(Mazmanian et al. , 2000) These enzymes became crucial targets in the hunt for anti-virulence medications because they play a significant role in the attachment of surface proteins to the cell wall. Early research revealed that the cell wall sorting reaction is dependent on an active, chemically sensitive thiol group that it can communicate with.(Ton-That et al. , 1997) Later research showed that the cysteine (Cys) at position 184 of S. aureus’s SrtA is essential for the enzyme’s function.(Ton-That et al. , 1999) Sortase enzymes are encoded by more than 1800 different gene sequences that have been found in about 600 different bacterial species. The majority of them express multiple Srt types, each of which serves to bind different proteins to the cell surface.(Kattke et al. , 2016) Since they are not necessary for bacterial survival, they are becoming targets for anti-virulence medications.(Spirig et al. , 2011) The cell wall sorting signal (CWSS) of the protein is a nucleophilic amino group, and the sortases that have been characterized thus far are members of the cysteine transpeptidase class and catalyzed the reaction that binds a peptide with an LPXTG motif as the sorting signal to the nucleophilic amino group. Due to their sorting signal’s variation, the nucleophilic substrate’s unique characteristics, and also additionally to their function, the Srt family consists of six classes (A-F).(Spirig et al. , 2011) SORTASE A INHIBITORS Numerous methods have been developed for the discovery and analysis of fresh Sortase A inhibitors.(Guo et al. , 2015) Wide diversity in the chemical nature of the sortase A inhibitors (see figure 2) have been found although the development of effective small-molecule SrtA inhibitors is limited due to our incomplete knowledge of the inhibitors’ mode of action in the SrtA binding pocket,.(Yue et al. , 2024)The discovery of β-sitosterol-3- O -glucopyranoside, berberine chloride, bis(indole)-alkaloid, isoaaptamine, kurarinol, curcumin, maltol-3- O -(4- O -cis-p-cumaroyl-6- O -(3-hydroxy-3-methylglu-taroyl)-β-glucopyranoside, (−)-rosmarinic acid was made possible by screening small compound ligands or natural compound libraries .(Nitulescu et al. , 2021) Other approaches of discovery relied on pharmacophore theories, 3D QSAR models and virtual screening.(Maresso and Schneewind, 2008)A number of sortase A inhibitors such as diarylacrilonitriles, aryl(β-amino)ethyl ketones, rhodanines, pyridazinones and pyrazolethiones, morpholinobenzoate derivatives, dihydro-β-carboline, benzo[d]isothiazol-3(2 H )-one-adamantane amine derivatives, and 3,6-disubstituted triazolothiazoles were synthesisedand evaluated.(Nitulescu et al. , 2021) Figure 2 . Wide diversity in the chemical nature of sortase A inhibitors. not-yet-known not-yet-known not-yet-known unknown Figure 3. Role of Sortase A inhibitor in biofilm formation inhibition of Staphylococcus aureus. PLANT EXTRACTS The evaluation of 80 dried extracts and their associated n-hexane, ethyl acetate and water fractions were assessed as Sa-SrtA inhibitors in a screening of therapeutic plant extracts (Figure 4). The range of the measured IC50 was 1.5-39.4g/ml.(Nitulescu et al., 2021) The ethyl acetate fractions of Cocculus orbiculatus (syn. Cocculus trilobus, Fam. Menispermaceae) rhizome, Liriope muscari (syn. Liriope platyphylla, Fam. Asparagaceae) tuber, Fritillaria verticillata (Fam. Liliaceae) tuber, and Toxicodendron vernicifluum (syn. Rhusverniciflua, Fam. Anacardiaceae) bark has shown the best inhibitory effects.(Kim et al., 2002)After one hour of incubation at 37°C, a methanol extract made from dried rhizomes of Curcuma longa (turmeric, family Zingiberaceae) resulted in an 80% suppression of SrtA S. aureus ATCC 6538p activity.(Park et al., 2005) A 44.2% inhibition of the S. mutans SrtA (Sm-SrtA) was obtained by an extract from the dried fruits of Psoralea corylifolia (Fam. Fabaceae) at a concentration of 100 g/mL.(Won et al., 2015) Three different phytoconstituent namely myricetin, palmatine, and esculetin has shown SrtA inhibitory property as evidenced from low IC50 values. Figure 4. Plant extracts (part of the plant) which shows sortase A inhibition. THIOL REACTIVE REAGENTS The broad and unselective reactivity profile of this group’s chemical reagents prevents them from being used in the development of therapeutics.(Marraffini et al., 2006) This group contain several chemical reagents known to interact with biologically active thiol groups that were identified as sortase inhibitors during the process of determining the catalytic mechanism of the enzyme (Figure 5). The first class of sortase inhibitors to emerge was methanethiosulfonates.(Ton-That et al., 1997) By establishing a disulfide link with the cysteine residue, sodium (2-sulphonatoethyl)methylthiosulphonate (MTSES) and (2-(trimethylammonium)ethyl)methylthiosulphonate (MTSET) both prevent the cleavage of sorting signals at the LPXTG motif.(Nitulescu et al., 2021) While alkylating compounds including N-ethylmaleimide, iodoacate and iodoacetamide were found not to be sortase inhibitors, while sodium p -mercurichydroxybenzoate (p HMB) was shown to inhibit several cysteine proteases.(Nitulescu et al., 2021) Dithioreitol (DTT), a sulfhydryl reducing agent, did not affect the Sa-Srt activity but reversed the inhibitory impact of MTSET by regenerating the thiol group.(Ton-That et al., 1999) Figure 5 . Thiol reactive reagents that inhibit sortase A. VINYL SULFONES Based on their electronic propensity to bind to active cysteine residues and create thioether adducts by a 1,4-addition process, several vinyl sulfones were tested against SrtA. The most potent inhibitory action was demonstrated by the chemical 3,3,3 -trifluoro-1-(phenylsulfonyl)-1-propene, which have an IC50 value of 190 µM and an approximately 1.5 times higher MIC value of 317.5 µM. (Nitulescu et al., 2021) Due to its lesser electrophilic nature, the related phenyl vinyl sulfone has a substantially lower inhibiting property, with an IC50 of 736µM. The absence of inhibitory response to phenyl trans -styryl sulfone highlights the significance of molecular size. The establishment of a covalent connection between the phenyl vinyl sulfone and CYS184 of Sa-SrtA was confirmed by the mass spectrometry study. In a fibronectin binding model, phenyl vinyl sulfone at 3000µM greatly reduced the virulence of the S.aureus Newman strain without impairing the bacterial viability (MIC value over 6000µM).(Frankel et al., 2004)The scaffold of several cis-5-phenyl proline methyl esters was given the vinyl sulfone group, although the molecules showed only limited action against SrtA, with IC50 values ranging from 850 to 5000µM.(Kudryavtsev et al., 2009) 3-ARYL ACRYLIC ACID and DERIVATIVES The 3-aryl substituted acrylic acid was identified through examination of the known sortase inhibitors as a common and significant scaffold.(Nitulescu et al., 2021) The formation of phenyl-propanoids, coumarins, lignans, flavonoids, stilbenes, aurones, anthocyanins, spermidines and tannins, all depend on the structural component known as phenylacrylic acid, also known as cinnamic acid.(Guzman, 2014) Cinnamic acid derivatives and related substances, both natural and synthetic, as well as bioisosteric analogues, are included in this group. CINNAMIC ACID DERIVATIVES An extract from the dried flowers of Styphnolobium japonicum (syn. Sophora japonica, Japanese pagoda tree, Fam. Fabaceae) a pair of derivatives of the p-coumaric acid(4-hydroxycinnamic acid), maltol 3-O -(4’-O -trans-p-coumaroyl-6’-O -(3-hydroxy-3-methylglutaroyl))-b -glucopyranoside and its isomer, maltol 3-O -(4’-O -cis-p-coumaroyl-6’-O -(3-hydroxy-3-methylglutaroyl))-b -glucopyranoside was tested for for SrtA inhibitors using the S. mutans strain. With an IC50 value of 58.6µM, the derivative of trans -p-coumaric acid (conformer z) was 1.6 times more potent than the similar derivative of cis -p-coumaric acid (conformer E). With MIC values more than 345 µM, neither substance had any growth-inhibiting effects on S. mutans .(Yang et al., 2015) A Pulsatilla koreana (Korean pasque flower, Fam. Ranunculaceae) extract was fractionated and separated under the direction of bioactivity, producing a number of lignans and cinnamic acid. Caffeic acid, also known as 3,4-dihydroxycinnamic acid, and its two ester derivatives, (-) rosmarinic acid and (+) chicoric acid were found to have the strongest inhibitory effects on Srt A generated from the S. mutans strain (IC50=20.2µM-60.1µM). With an IC50 of more than 100µM, the 3-O -methylated derivate of caffeic acid, ferulic acid, had no inhibitory action on SrtA.(Lee et al., 2014) With a determined IC50 value of 500µM, its isomer,4-O -methylated derivate the isoferulic acid, was tested against Srt A with a minimal effect.(Bi et al., 2016b) Chlorogenic acid (3-O -caffeoylquinic acid), another ester derivative of caffeic acid, was found to be a strong inhibitor of SrtA from S. aureus Newman D2C. With an IC50 of 95.57 µM, it did not impede the growth of bacterial cells (MIC > 1024 µg/mL). Chlorogenic acid dramatically disrupted the pathophysiology of S. aureus in mouse studies and halted the development of renal abscesses.(Wang et al., 2015) SrtADN59 was used as a test subject for two isomers of chlorogenic acid: neochlorogenic acid (5-O -caffeoylquinic acid) and cryptochlorogenic acid (4-O -caffeoylquinic acid) (Figure 8). The IC50 values of both isomers were greater than 500 µM, indicating the significance of the reciprocal locations of the functional Curcumin’s IC50 value of 10.2 µM, which is significantly less than the MIC value of 125 µM, indicated that it was similarly active against S. mutans UA159 SrtA groupings.(Bi et al., 2016b)It was discovered that three main components of the rhizome of Curcuma longa (turmeric, Family Zingiberaceae) inhibited SrtAD24 from S. aureus ATCC 6538p. Curcumin, with an IC50 value of 37.5 µM, had the greatest inhibitory impact, followed by demethoxycurcumin (IC50 = 70.3 µM and 103.5 µM for bisdemethoxycurcumin (Figure 6). All three substances demonstrated no discernible growth inhibition against the Newman strain of S. aureus, with MIC readings more than 200 µg/mL.(Park et al., 2005) A molecule with a weak inhibitory effect on S. mutans SrtA, curcumin can be thought of as a dimer of ferulic acid, demonstrating the intricacy of the structure-activity connections in this chemical class. Because the β-diketo moeity is present, a Michael acceptor group, reacts with cysteine sulfhydryl groups and this is most likely the way it inhibits SrtA.(Magesh et al., 2012) Figure 6. The structures of sortase inhibitors derivatives of cinnamic acid. In red bordered box, the general scaffold of each group. COUMARIN DERIVATIVES The lactone derivative created by intramolecular cyclization of 6-hydroxycaffeic acid is called esculetin (6,7-dihydroxycoumarin also referred to as aesculetin) (Figure 7). Caffeic acid and its methoxy derivative trans-ferulic acid, were found to be inactive while it inhibited Sa-SrtA with an IC50 Value of 37.5µM. When tested against S. aureus ATCC 25923 and S. aureus ATCC 6538, aesculetin showed no discernible antibacterial action. The minimal inhibitory effect induced by umbelliferone(7-hydroxycoumarin) and coumarin on Sa-SrtA served to emphasize the influence of the 6,7-dihydroxy groups present on the chromen-2-one scaffold.(Nitulescu et al., 2017) Psoralen, an umbelliferone furanocoumarin derivative, displayed a negligible inhibitory impact, Sm-SrtA with an IC50 value of about 330µM.(Won et al., 2015) Esculin(esculetin-O -β-glucoside) only slightly inhibited Sa-Srt A, most likely as the result of the 6-hydroxy group being occluded in the etheric bond.(Nitulescu et al., 2021)A dried fruit extract of Poncirus trifoliate, often known as trifoliate orange and belonging to the Rutaceae family contained many prenylated coumarins that were tested for their ability to inhibit S. aureus ATCC6538p SrtA. O -methylponciol B (IC50=26.6µM),7-(6S-hydroperoxy-3,7-dimethyl-2E,7-dienyloxy)-coumarin and 7-O -(7-O -methylponciol B (IC50 = 26.6 M), 7-(6S-hydroperoxy-3,7-dimethyl-2E,7-dienyloxy)-coumarin, and 7-O -(70-peroxygeranyl) coumarin all showed the greatest effects. After 2 hours of treatment with 100 µM, the fibrinogen cell clumping of the S. aureus strain decreased by more than 2-fold, demonstrating the anti-infective potential of these chemicals. Moreover, they lacked S. aureus ATCC6538p growth is significantly inhibited, with MIC values above 300 µM.(Park et al., 2020a) Figure 7. The structures of coumarin derivatives sortase inhibitors. In red bordered box, the general scaffold of each group. AROMATIC FURANONE DERIVATIVES The dark red ascidian Synoicum sp. (Fam. Polyclinidae) found off the coast of southern Korea yielded a number of brominated tris-aromatic 2-furanone derivatives. The 4-(3-bromo-4-hydroxyphenyl)-2-furanone derivative cadmiolide E, which has an IC50 value of 78.8 µM, significantly inhibited Sa-SrtA while simultaneously having potent antibacterial properties. Synoilide B, a similar bis-aromatic diester, had no discernible impact on Sa-SrtA. Cadiolide E and synoilide B have a structural component in common with cinnamic acid.(Won et al., 2012a) Isolated from Synoicum sp., the isocadiolides are a collection of structurally similar compounds that share the bromohydroxyphenyl fragments of the cadiolides but differ in that the central ring no longer contains a furanone and instead has one of the following structures: pyranone, dihydrofuran, or cyclopentene-1,3-dione. Sa-SrtA was moderately inhibited by isocadiolides A and D, however S . aureus ATCC 6538P also experienced bacterial growth inhibition.(Bae et al., 2020) Figure 8. The structures of sortase inhibitors derivatives of cinnamic acid. In red bordered box, the general scaffold of each group. DIACYLACRYLONITRILES The methyl (2E )-2,3-bis(4-methoxyphenyl) acrylate is a trans-p-coumaric acid derivative that is produced synthetically was shown to be a hit when a small-molecule Sa-SrtA inhibitor with an IC50 of 231µM was randomly screened. The equivalent Z-conformer exhibited an inhibitory potency that was nearly 4-fold less, according to the examination of the structure-activity connections. The compound became inactive either by hydrolysis to the associated acid or through hydrogenation of the double bound. The Sa-SrtA inhibition was further enhanced by bioisosterically replacing the ester group with a nitrile. The potency of the diarylacrylonitrile derivatives was greater for the Z -isomers compared to the methyl acrylate derivatives due to the two benzene rings’ trans orientation.(Oh et al., 2004) Numerous structural alterations result in (Z ) DMMA, which has an IC50 of 9.2 µM and reversibly inhibits Sa-SrtA, is 3-(2,5-dimethoxyphenyl)-2-(4-methoxyphenyl) acrylonitrile.(Nitulescu et al., 2021) DMMA has been demonstrated to have no substantial impact on the development of the S. aureus Newman strain, with MIC values exceeding 678 µM, and to inhibit Sa-SrtB activity with an IC50 value of 34.2 µM.(Cho et al., 2022) The substance lowered the mortality rate of Balb/c mice infected with S. aureus Newman in a dose-dependent manner. Intriguingly, the mice treated intraperitoneally with 100 mg/kg/day had a lower survival rate (75%) than mice treated with 20 mg/kg/day (100%); this suggests that DMMA itself may be hazardous. However, even at 4 mg/kg/day, DMMA was still effective (97 percent survival).(Oh et al., 2010) CHALCONES DERIVATIVES Chalcones, which are 1,3-diphenyl-2-propene-1-one derivatives and are classified as cinnamoyl derivatives, have two benzene rings connected by a three-carbon unsaturated chain (Figure 9). The fundamental compound in the chalcone series is called (E )-chalcone.(Li et al., 2016) An IC50 of 5 µM for (E)-chalcone’s activity against Sm-SrtA was established through measurement. By creating a covalent adduct with another substance, the substance caused a gradual and permanent inhibition through a Michael addition process, the Cys205 residue. This mechanism was shown to work by the absence of dihydrochalcone’s saturated analogue’s inhibitory actions.(Wallock-Richards et al., 2015) Chalcone successfully inhibited Sa-SrtA (IC50 = 53.15 µM) while having no discernible effects on the development of S. aureus USA 300 (MIC > 4864 µM). The substance decreased S. aureus’s adherence to fibronectin, the development of biofilms, and bacterial invasion in J774 mouse macrophages model of cells. The S. aureus -infected animals were given chalcone subcutaneous injections of 150 mg/kg every 12 hours had a higher 3-day survival rate than those who weren’t treated.(Mu et al., 2018) Sa-SrtA was inhibited by isoliquiritigenin (20,4,40-trihydroxychalcone), which was obtained from an extract of the vine stem of Spatholobus suberectus (Family: Fabaceae), with an IC50 of 139.7µ M.(Wang et al., 2018a) (E )-Chalcone exhibited a 28.41µ M IC50 against the L. monocytogenes SrtADN70 (Lm-SrtA). Inhibiting SrtA activity decreased the virulence of L. monocytogenes and decreased the mortality of infected mice, with no discernible impact on bacterial growth.(Li et al., 2016) Phloretin, a dihydrochalcone derivative, interestingly had no effect on L. monocytogenes growth (MIC = 933.44 µM) while substantially inhibiting Lm-SrtA (IC50 = 37.24 µM). The phloretin binds, according to models of molecular dynamics the hydroxyl groups of Lm-SrtA in a site close to the active enzymatic centre.(Wang et al., 2017) This explains why there isn’t a double bond, as there is when (E )-chalcone interacts with Sm-SrtA, and why it doesn’t matter. Figure 9 . Aromatic furanone derivatives, diarylacrylonitriles, chalcones derivatives, 3-(2-furyl)acrylic acid, and 3-(thien-2-yl)acrylic acid derivatives. 3-(2-FURYL) ACRYLIC ACID and 3-(THIEN-2-YL) ACRYLIC ACID DERIVATIVES It is possible to think of the 3-(2-furyl) acrylic acid and its thiophene equivalent as cinnamic acid’s bioisosters. Several phenyl-substituted amides are these acids’ derivatives were created and evaluated for their capacity to inhibit SrtAD59 in vitro. The IC50 values ranged from 58 to 571 µM and were stated as exceeding the 600 µM cutoff for several substances. The acrylic double bond was hydrogenated, resulting in inactive compounds, highlighting its significance. The amides with N-methyl substitutions shown to compared to their comparable NH analogues, be less potent inhibitors.(Chenna et al., 2010) In the absence of information detailing how the chemicals in this series affect bacterial growth, it is challenging to evaluate the anti-virulence potential of these substances. FLAVONOIDS As a closed ring derivative of (E )-chalcone, flavone (2-phenyl-4H -chromen-4-one) may be considered (Figure 10). A similar 3-hydroxyflavone scaffold (Uivarosi et al., 2019) is shared by the subclass of flavonoids known as flavonols where SrtA inhibition appears to be dependent on the 3-hydroxyl group.(Nitulescu et al., 2017) The position of the phenyl group on the chromen-4-one scaffold is what differentiates the isoflavones, as the name implies, from the comparable flavones. Flavanones can be categorised as 2,3-dihydroflavones but lack a double bond in the middle of the ring.(Uivarosi et al., 2019) Figure 10. Structures of sortase inhibitors belonging to the flavonoids class. In red box, the general scaffold of each group. Figure 11 . Structures of sortase inhibitors belonging to the flavonoids class. In red box, the general scaffold of each group. FLAVONES The 4’-O -methylated form of apigenin is called acacetin (5,7-dihydroxy-4’-methoxyflavone). The MIC value on S. aureus ATCC25904 was greater than 3800 µM, and the IC50 on S. aureus SrtADN59 was 128.3µ M. 224 µM of acacetin solution decreased to 150 mg/kg/day doses were able to almost completely reduce the number of germs that bind to fibrinogen to greatly prolong the survival of mice against renal abscess brought on by S. aureus. (Bi et al., 2016a) With an IC50 of 67.02µ M, isovitexin, the 6-C substituted glucoside of apigenin, inhibited Sa-SrtADN59 without affecting bacterial growth, the substance prevented S. aureus cell adherence to fibrinogen and the development of biofilms in a dose-dependent manner.(Mu et al., 2018) There is no discernible growth pressure on S. aureus when baicalin, the 7-O -glucuronide of baicalein (5,6,7-trihydroxyflavone), inhibits Sa-SrtB. S. aureus ’ adherence to human alveolar epithelial cells was decreased by baicalin in a dose-dependent manner A549 cell. The results of the molecular dynamics simulations demonstrated the value of both the glucuronide moiety and the chromen-4-one scaffold.(Wang et al., 2018a) Lm-SrtA was inhibited by baicalein in a concentration-dependent manner, and L. monocytogenes entry into Caco-2 cell cultures was reduced.(Lu et al., 2019) By substrate peptide reaction-based fluorescence resonance energy transfer (FRET) screening, identification of oroxylin A glucuronide (OAG )as an efficient inhibitor of Sortase A (SrtA) with an IC50 of 45.61 μg mL−1, was done and achieved efficacy in the treatment of Staphylococcus aureus (S. aureus ) infections. Further demonstration that OAG inhibited the adhesion of the S. aureus to fibrinogen, the surface protein A anchoring and diminished biofilm formation. Notably, OAG exhibits a robust therapeutic effect in a MRSA-induced pneumonia model.(Jiang et al., 2023b) FLAVONOLS With the use of the substrates Dabcyl-QALPETGEE-Edans (for SrtA) and Dabcyl-NPQTN-Edans (for SrtB), a number of natural flavonols were extracted from an ethyl acetate extract of Toxicodendron vernicifluum bark (for SrtB). For example, SrtA, morin (3,5,7,20,40-pentahydroxyflavone) showed the best inhibitory effects. 3,5,7,30,40,50-hexahydroxyflavonol, IC50 = 44.03µ M), myricetin (3,5,7,30,40,50-hexahydroxyflavonol), and IC50 for quercetin is 52.70 µM (3,5,7,30,40-pentahydroxyflavone). IC50 values that were determined were typically less on SrtB than on SrtA, for the most active chemicals. IC50 values for morin (8.54 µM), kaempferol (3,5,7,40-tetrahydroxyflavone, 24.55 µM), and quercetin, whose IC50 value is 33.28 µM. Over 300 µM is the MIC value for S. aureus Newman for every single flavone derivative.(Kang et al., 2006) Both SrtA and SrtB seem to be inhibited differently depending on the amount and locations of the hydroxyl groups on the flavone scaffold. On SrtAD24, several flavonol derivatives were investigated, and a substantial inhibiting by myricetin, with an IC50 value of 4.63 µM, was observed. Quercetin displayed low water solubility and mild inhibitory effects at 10 µM which made testing at higher concentrations difficult. No discernible impact was made by fisetin (3,7,30,40-tetrahydroxyflavone).(Nitulescu et al., 2017) Molecular docking studies and an enzymatic assay revealed that the flavonoid aglycones are more powerful against SrtA than their equivalent glycosides (hyperoside, rutin, and troxerutin), perhaps because there are fewer rotatable bonds.(Nitulescu et al., 2016; Nitulescu et al., 2017) Although the IC50 values were not published, quercetin dramatically reduced Spn-SrtA activity in a dose-dependent manner.(Wang et al., 2018c)With an IC50 value of 72.77 µM, the quercetin glycoside derivative known as quercitrin (syn. quercetin-3-rhamnoside) suppressed the growth of the S. aureus SrtADN59 with no impact on the Newman D2C strain.(Liu et al., 2015) The analysis of morin’s inhibitory action on SrtA from S. mutans yielded a result for the IC50 of 27.2 µM. In addition, S. mutans’ biofilm production was dramatically decreased by 30 µ M morin while bacterial proliferation remained unaffected.(Huang et al., 2014) Quercetin, rutin (quercetin 3-rutinoside), and narcissin (isorhamnetin 3-rutinoside) were obtained from the dried floral extract of Styphnolobium japonicum using bioactivity-guided separation. The substances were evaluated against a S. mutans strain OMZ65-derived recombinant SrtA. Rutin shows an IC50 value of 134.1 µM whereas its 3’-methylated derivative, narcissin, shows an IC50 value of 185.9 µM. Quercetin, the equivalent aglycone, displayed a weaker inhibitory impact with an IC50 of 210.16 µM.(Yang et al., 2016) Apart from these, a flavonol glycoside hibifolin is extracted from Abelmoschus manihot (Linn.) Medicus flower. Studies have been conducted on the biological effects and uses of hibifolin, which include its ability to prevent Alzheimer’s disease, myocardial and cerebral ischemia injury, and anti-inflammatory properties. In this work, we showed that hibifolin, which may be used as adjuvant therapy for S. aureus infections in the future, reduced the pathogenicity of S. aureus by directly targeting SrtA.(Song et al., 2022) ISOFLAVONES With little to no impact on the growth of the S. mutans bacterial species, many flavonoids isolated from Spatholobus suberectus (Fam. Fabaceae) were found to be strong inhibitors of Sm-SrtA. Based on their spectroscopic data, the substances were recognised as two isoflavones: formononetin (7-hydroxy-4’-methoxyisoflavone, IC50 = 41.8 µ), Daidzein (4’,7-dihydroxyisoflavone, IC50 =144.7µM) and one flavone, 7-hydroxy-6-methoxyflavanone which has an IC50 value of 46.1 M.(Park et al., 2017) Neobavaisoflavone (3’-prenyldaidzein, IC50 = 284.1 µM), isolated from the dried fruits of Psoralea corylifolia, inhibited Sm-SrtA by a factor of almost two times less than daidzein.(Won et al., 2015) With an IC50 value of 74.9 µM, formononetin also suppressed Sa-SrtA activity, but its matching 7-O -β-D-glucoside had an impact that was three times less potent.(Cho et al., 2017) FLAVANONES Seven prenylated flavonoids that were extracted from the roots of Sophora flavescens (a shrubby sophora in the family Fabaceae) were tested for their ability to inhibit the activity of SrtA in S. aureus ATCC 6538p. All substances had a moderate inhibitory impact, however only for the 5-methoxyflavanone derivative kurarinol was the IC50 value able to be calculated. Kurarinol has a modest antibacterial activity against S . aureus (MIC=219µM) and an IC50 value of 107 µM.(Oh et al., 2011) Sa-SrtA was inhibited by eriodictyol (3,4,5,7-tetrahydroxyflavanone) with an IC50 value of 7.73 µM in a reversible mechanism, however even at a concentration 50 times higher than IC50, S. aureus growth was unaffected. The substance reduced S. aureus biofilm development and SpA protein anchoring in a dose-dependent manner.(Wang et al., 2021) FLAVANONOLS Without influencing the growth of S. mutans ATCC 25175, astilbin, the 3-O -rhamnoside of taxifolin (syn. dihydroquercetin), inhibits Sm-SrtA with an IC50 value of 16.6 µM. The rhamnoside moiety’s function in binding was represented in the molecular dynamics simulations in the location where an enzyme is active. Astilbin reduced the development of bacterial biofilm by 49% at 142.1 µM, while there was no discernible impact at concentrations of 71 µM or lower. It is thought that due to the amount of enzyme not inhibited, the biofilm inhibitory values are significantly larger than IC50 for SrtA.(Park et al., 2017) This disparity is likely the result of the compound’s poor lipophilicity and cell permeability.(Oh et al., 2011) QUINONE DERIVATIVES Shikonin (IC50 = 0.30 µM) and alkannin (IC50 = 0.36 µM), two naturally occurring 5,8-dihydroxy-1,4-naphthoquinone derivatives (Figure 12), were discovered through the use of a library and the monitoring of SrtAD24 cleavage of the Abz-LPETG-Dnp substrate. Both of the enantiomers of the substances have potent inhibitory effects on the development and survival of S. aureus which limits their effectiveness as anti-virulence drugs significantly.(Hou et al., 2018)Juglone (5-hydroxy-1,4-naphthalenedione) and plumbagin, two related 1,4-naphthoquinone derivatives, showed a strong Sa-SrtA inhibitory action without significantly affecting IC50 values of 1.78 µM and 16.71 µM, respectively without affecting the development of S. epidermidis, E. faecalis, and S. aureus bacteria. The dynamics of inhibition and docking analyses suggested an irreversible mechanism based on the thiol catalytic activity of Cys184 an remainder addition to the naphthoquinone structure’s dual conjugated double system. At 10 µM, lawsone, the 2-hydroxyisomer of juglone, exhibited no discernible impact on Sa-SrtA because it doesn’t react much with nucleophilic substances.(Nitulescu et al., 2019) While juglone’s chemical cousin Rhein showed promising inhibitory effects at 10 µM, the experiment for calculating the IC50 was hampered by Rhein’s low water solubility.(Nitulescu et al., 2017) Two emodin (6-methyl-1,3,8-trihydroxyanthraquinone) moieties joined by a single C-C bond yield ed the bisanthraquinone compound skyrin, which demonstrated Sa-SrtA inhibitory actions with IC50 values in the range of 24-31 µM. (Reddy Venkata Thappeta et al., n.d.) Unfortunately, the robust growth reduces the anti-virulence potential on S. aureus Newman strain inhibitory activity (MIC values about 10 µM). At 100 µM, Emodin had little impact on Sa-SrtA.(Nitulescu et al., 2017) By attaching to the nucleophilic thiol group of Cys184 in a Michael addition process, a pair of pyranoquinone derivatives that are structurally similar to the 1,4-naphthoquinones were shown to irreversibly block Sa-SrtA with IC50 values of 6.2 µM and 19.4 µM. Derivatives are diastereomers with just one carbon’s stereochemistry different from one another. The isomer R has a stronger affinity for Sa-SrtA. Both substances contribute a lot in reduced development of biofilm at 80 µM but had no effect on the growth of S. aureus though exhibiting a good anti-virulence profile at 150 µM. (Hou et al., 2018) Figure 12. Structures of sortase inhibitors belonging to the class of quinones. In red box, the general scaffold of each group. INDOLES DERIVATIVES Several 5-hydroxyindole derivatives (Figure 13) were identified during the investigation of an extract from tropical marine sponges of the Hyrtios species (fam. Thorectidae)(Lee et al., 2009) and evaluated for impact on Sa-SrtA.(Lee et al., 2010)Although serotonin had no discernible impact, its N-closed ring analogue derivative,1-carboxy-6-hydroxy-3,4-dihydro-β-carboline, showed a moderately inhibitory function against Sa-SrtA (IC50=290µM).Considering its format, a string of 3-indoleglyoxylic acid derivatives and they were assessed as Sa-SrtA inhibitory agent once the matching N -phenyl amide was produced. With IC50 values in the range of, the compounds generated from indole and 2-methylindole had strong inhibitory activities. With IC50 values in the range of 61 to 174 µM, the compounds generated from indole and 2-methylindole had strong inhibitory activities, nevertheless, those made from 5-hydroxy and 5-methoxy indole had no discernible impact. Nevertheless, those made from 5-hydroxy and 5-methoxy indole had no discernible impact. The presence of both carbonyl groups in the related them inactive towards SrtA without them. The finding of (2-amino-6-chloro-1H -indol -3yl)morpholino methanone with the associated IC50 value of 10µM came from the screening of a number of indole derivatives and related heterocyclic compounds on S . Pyogenes SrtAΔN81(Sp-SrtA). The substance resembles the derivatives of 3-indoleglyoxylic acid structurally, but it had no discernible impact on Sa-SrtAΔN59. The relevance of the morpholino moiety for the inhibition of Sp-SrtA was shown by docking experiments and by the lack of impact of the equivalent piperidine derivative.(Lee et al., 2010) On a brilliant yellow sponge Spongosorites sp. (Fam. Halichondriidae) extract, the Sa-SrtA inhibition assay-guided separation produced a number of indole derivatives, which are compounds with two indole moieties joined by a heterocyclic scaffold. The discovered inhibitors can be divided into topsentins (bis(indoylyl)imidazole derivatives) and hamacanthins(bis(indolyl)pyrazinone derivatives) depending on how heterocycles are joined. Deoxytopsentin (IC50=48µM), bromodeoxytopsentin (IC50=48µM), bromotopsentin (IC50=39.7µM) from the first group produced the best outcomes, but it also significantly impacted S.aureus Newman bacterial growth. The potential therapeutic benefits of the hamacanthin derivatives as anti-virulence agents are constrained by their robust antibacterial properties and significantly reduced inhibitory potency compared to the related topsentins.(Oh et al., 2005) The core 2-methoxy-1-imidazole-5-one linking ring of the spongosoritins B and C, two structurally similar compounds, inhibited Sa-SrtA with IC50 values of 62.7 and 43.9 µM respectively.(Park et al., 2021) As topsentin analogues, a number of derivatives of bis(indolyl)-1,2,4-oxadizole were created, and their impact on S. aureus biofilm development was assessed. Three substances were found to be effective biofilm inhibitors, and they all showed a significant decrease in Sa- SrtA’sAs impact.(Parrino et al., 2021) Multiple chromatographic techniques were used to extract and separate six β-carboline alkaloids included to the eudistomin Y group from the marine ascidian Synoicum sp . (Fam. Polyclinidae).(Won et al., 2012b) The indole and pyridine moiety are joined to form the β-carboline scaffold.(Szabó et al., 2021) Additionally, a tunicate from the Eudistoma genus had the chemicals.(Wang et al., 2008) The only compounds that shown inhibitory effect on were eudistomin and eudistomin y5.The only compounds that shown inhibitory effect on Sa-SrtA were eudistomin Y4 (1-(3-bromo-4-hydroxybenzoyl)-6-bromo-β-carboline) with IC50 163.2 µM and its isomer eudistomin y5 with IC50 146.4 µM.Additionally, the compounds displayed potent antibacterial properties against S.aureus ATCC 6538p, with MIC values approximately in the range of 7µM and 14 µM respectively. The inhibitory capacity of Sa-SrtA was significantly lowered by hydrogenation of the carbonyl group to a hydroxyl group.(Won et al., 2012b) One type of 1-phenyl-dihydro-b-carboline and 1-phenyl-tetrahydro-b-carboline derivatives were created, and several of them demonstrated effective inhibitory activity against SrtA with IC50 values between 25 and 115 M. Most of these chemicals had no effect on the survival of 629215the S. aureus bacterium.(Lee et al., 2010) Figure 13. Structures of indole-containing sortase inhibitors. PYRROLOMYCINS & ANALOGUES Halogenated antibiotics known as pyrrolomycins were discovered in the fermentation broth of Actinosporangium sp . and Streptomyces sp. (Cascioferro et al., 2015b) With IC50 values of 300 µM and 250 µM, respectively, the natural pyrrolomycins C and F2a moderately inhibited Sa-SrtA (Figure 14). Both substances exhibit strong antibacterial effects on S. aureus, as evidenced by their MIC values. (Cascioferro et al., 2015b) When evaluated on Sa-SrtA, four synthesised variants demonstrated IC50 values in the 130–160 µM range and significantly disrupted staphylococcal biofilm formation.(Nitulescu et al., 2021) The chemical compound (4,5-dichloro-1H -pyrrol-2-yl)-[2,4- dihydroxy-3-(4-methyl-pentyl)-phenyl]-methanone caused Sa-SrtA inhibition (IC50 = 47 µM) and a decline in biofilm development, but potency in anti-staphylococcal activity.(Reddy Venkata Thappeta et al., n.d.) ISOQUINOLINE DERIVATIVES One of the active ingredients that inhibits Sa-SrtA in Coptis chinensis rhizome extract (Chinese Goldthread, Family Ranunculaceae) is berberine chloride (IC50 =16.7µg/mL)(Kim et al., 2004) (Figure 14). As Sa-SrtA inhibitors, palmatine, the dimethoxy analogue of berberine, and hydrastine, a connected phthalide-isoquinoline derivative, were also studied. Berberine chloride had an IC50 value of 23.4 µM and MIC values of 269 µM for S. aureus, and over 1000 µM for S.epidermidis . The MIC value for palmatine chloride was greater than 1000 µM on both S.aureus and S.epidermis .(Kim et al., 2004) Other Sa-SrtA inhibition experiments frequently include berberine chloride as a positive control, and depending on the precise techniques employed, IC50 values communicated as - 85.9µM,(Park et al., 2020b) 99.8 µM(Cho et al., 2017) or 120µM(Raimondi et al., 2019). Another experiment corroborated palmatine’s Sa-SrtA inhibitory action (IC50 = 52.8 µM), which correlated with a low toxicity in the Daphnia magna test.(Nitulescu et al., 2017) The dioxolane counterpart of berberine, coptisine chloride, reduced the activity of Sa-SrtB (IC50 = 24.6 µM) without evident antibacterial effect on S.aureus 29213.(Wang et al., 2018b) Sa-SrtA inhibitors were found in three aaptamine alkaloids that were isolated from the marine sponge Aaptos aaptos (Family Suberitidae). The N -methyl group present in isoaaptamine, which showed the strongest inhibitory impact (IC50=16.22 µM), is likely to blame. The IC50 value for its isomer, aaptamine, was 102.9 µM, while that of its N-demethyl analogue, demethylaaptamine the IC50 for was 85.9 µM. Using isoaaptamine, a mild growth inhibition on S. aureus strain Newman with a MIC value of 219 µM was reported.(Jang et al., 2007) Figure 14 . Structures of Pyrrolomycins analogues and Isoquinoline derivatives, sortase inhibitors. ARYL β-AMINOETHYL KETONES Using the fluorescence resonance energy transfer substrate approach, a library of 135,625 small compounds was screened, and a series of SrtA inhibitors were discovered. Several of the 6154 compounds that showed over 20% inhibitions were chosen based on reactivity, genotoxic potential, and drug-like characteristics, resulting in 407 substances. To get rid of nonselective agents, the chemicals in this group were evaluated against papain, a eukaryotic protease having an active site thiol. The 3-(dimethylamino)-1-(2-thienyl)-1-propanone (AAEK1) and 1-(3,4- dichlorophenyl)-3-(dimethylamino)propan-1-one (AAEK2) aryl b- aminoethyl ketones were chosen for further study. For Sa-SrtA of S. aureus, the drugs’ IC50 values were 47 µM and 15 µM, respectively. The SrtA homologue from B. anthracis was more effectively inhibited by AAEK1 and AAEK2, with IC50 values of 4.8 µM and 5.6 µM, respectively. (Maresso et al., 2007) The interaction between AAEK1 and SrtA was studied using mass spectrometry and X-ray crystallography, and it was discovered that the inhibitory mechanism is based on the removal of the dimethylamino group and production of the thiol in the enzyme Cys’s thienyl vinyl ketone that forms a covalent bond with it.(Maresso et al., 2007) Figure 15. Structures of sortase inhibitors containing aryl β-aminoethyl ketones. Figure 16. Structures of sortase inhibitors containing heteroaromatic cycles. PYRAZOLETHIONES and PYRIDAZINONES On Sa-SrtADN59, a high-throughput screening test revealed a number of promising compounds to be powerful SrtA inhibitors. The study’s scope was expanded to include freshly synthesised or structurally related substances that belonged to the pyrazolethione and pyridazinone classes and were readily available commercially. The IC50 for the pyrazolethione derivatives ranged from 0.30 µM to 115 µM. The decrease in potency following its substitution with a ketone group and the docking investigations both served to highlight the significance of the thione group. The most effective compounds (had an IC50 of 0.30 µM for 5-methyl-2-phenyl-4-[(2,4,6-tribromoanilino)methylene]pyrazole-3-thione and 0.76 µM for 5-methyl-2-phenyl-4-[(2-pyridylamino)methylene]pyrazole-3-thione) either of it having little impact on growth of S. aureus at the measured 500 µM concentration.(Suree et al., 2009b) IC50 values for several of the examined pyridazinone derivatives ranged from 0.20 µM to 219 µM, however the bulk of them were stated as being above the 50µM cutoff. The pyridazinone ring’s substituents’ nature had a significant impact on the inhibitory activity (Figure 15). The most successful outcomes were recorded for 4-ethoxy-2-phenyl-5-mercaptopyridazin-3-one with IC50= 0.20 µM, whereas that of its 4-ethylsulfanyl analogue is 1.4 µM and also has an IC50 value of 1.0 µM for 4-chloro-5-ethoxy-2-phenyl-pyridazin-3-one. At 500 µM, 4-ethylsulfanyl-2-phenyl-5-mercaptopyridazin-3-one had little effect on the growth of S. aureus, whereas the other two chemicals virtually completely prevented bacterial growth at the similar concentration.(Suree et al., 2009b) Docking investigations, mass spectrometry, and NMR spectroscopy demonstrated that the Sa-SrtA is covalently modified by 5-mercaptopyridazin-3-one derivatives by the formation of a disulfide associate with Cys184. Potent new pyridazinone compounds have been discovered and marked as inhibitor of Sa-SrtA, 2-(3-fluorophenyl)-4-(3-hydroxypropoxy)-5-mercaptopyridazin-3-one being the most effective inhibitor with an IC50 of 0.45 µM and 0.021 µM for Sa-SrtA and Ba-SrtA respectively.(Chan et al., 2017) BENZISOTHIAZOLINONES On Sa-SrtADN59, a high-throughput screening test revealed a number of promising compounds to be powerful SrtA inhibitors. N -(adamantan-1-yl)-2-(3-oxobenzo[d]isothiazol-2(3H )-yl) acetamide (Figure 16) was shown to be a potential lead compound that permanently inhibited SrtA by creating a disulphide bond with the residue Cys184 (IC50 = 6.11 µM). Considering its framework, a number of connected derivatives were created and examined. The substances reported IC50 values ranging from 3.39 µM to 7.06 µM and measured MIC values ranged from 20.02 µM to 163.91 µM against S. aureus . Almost all of the derivatives had substantial cytotoxic effects on the NIH 3T3 mouse embryo fibroblast cell line, restricting their therapeutic advancement.(Zhulenkovs et al., 2014) DERIVATIVES of 2-PHENYL-BENZOXAZOLE and 2-PHENYL-BENZOFURAN A number of 2-phenyl-benzo[d ]oxazole-7-carboxamide derivatives were created to replicate the form and bonding characteristics of the distinctive LPXTG motif seen in SrtA (Figure 16). On Sa-SrtADN24, the compounds had IC50 values that ranged from 19.8 µM to 184.2 µM. The type of substitution on the phenyl fragment’s para position has a significant impact on the inhibitory function, with hydroxyl groups esterified using different phenolic acids showing the best outcomes. The scaffold of N-alkylbenzoxazole-7-carboxamide serves as a structural analogue of the L-leucyl-L-prolyl fragment found in the substrates of SrtA, and exhibited low inhibitory effects of the compounds with no substitute (IC50 > 200 µM) at the seventh place.(Zhang et al., 2016) A number of N -alkyl-2-phenyl-benzofuran-3-carboxamide derivatives were produced and evaluated on Sa-SrtA, yielding IC50 values ranging from 30.8 µM to over 200 µM. These derivatives were comparable to the associated benzoxazole compounds, the strongest inhibitory effects were seen for the N -i butyl-carboxamides and those with a dihydroxybenzoate moiety.(He et al., 2017) There are no data on how these benzoxazole and benzofuran affect bacterial growth to assess their potential for advancement as anti-virulence treatments. THIADIAZOLES and TRIAZOLOTHIADIAZOLES DERIVATIVES Similar to the 2-phenyl-benzoxazole-7-carboxamide derivatives and their analogues, 2-phenyl-benzofuran-3-carboxamide derivatives, these molecules all have a core 1,3,4-thiadiazole scaffold in common (Figure 16). These resemblances are not reflected in a typical SrtA suppression mechanism as necessary. Using a library of over 28,500 chemicals, 5-[(7-nitro-2,1,3-benzoxadiazol-4-yl)sulfanyl]-1,3,4-thiadiazol-2-amine was shown to be a powerful SrtA inhibitor with an IC50 value of 6.2 µM. The removal of the oxadiazole scaffold was done because the chance of cytotoxicity brought on by its presence and a number of 5-amino-1,3,4-thiadiazole-2-thiol was created and put to the test. The substituted benzyl sulphide compounds with the greatest inhibitory action were N -(5-((4-nitrobenzyl)thio)-1,3,4- thiadiazol-2-yl)nicotinamide (IC50 = 3.8 µM) is the best substance for this series. The results of the experiments indicate that this chemical is reduced by the enzyme and converted to nicotinamide-thiadiazole thiol fragment and then forms a covalent disulphide bond with the residue Cys184.(Wehrli et al., 2019) Using a molecular docking analysis on drug-like structures and an experimental validation assay on Sa-SrtADN24, topsentin derivatives based on the bis(indolyl)imidazole scaffold were found as 6-(2-chlorophenyl)-3- (4-pyridyl) -[1,2,4] triazolo[3,4-b ] [1,3,4] thiadiazole (IC50 = 37.7 µM) as a strong inhibitor. The creation and examination of many analogues provided the optimal 3-(4-pyridinyl) -6-(2-sodiumsulfonate phenyl) [1,2,4] triazolo [3,4- b ][1,3,4]thiadiazole, whose IC50 for reversibly inhibiting SrtA was 9.3 µM. On S. aureus, the substance had a MIC value of more than 40,000 µM in method for dilution of microtiter broth. Given dosages of 40 mg/Kg spaced out by 12 hours for a Newman enhanced the 20-day S. aureus infection in BALB/c mice over a period of 5 days. 53.3 percent of the animals survived, compared to 0 percent of the untreated animals.(Wehrli et al., 2019) 1,2,4-THIADIAZOLIDINE-3,5-DIONE DERIVATIVES Tideglusib (IC50 = 0.6 M), an irreversible non- ATP-competitive glycogen synthase kinase 3β (GSK-3β) inhibitor, was found after a screening for Sa-SrtA inhibitors on a collection of around 2400 clinical medicines and candidates.(Yang et al., 2020) Surprisingly, the the potential GSK-3 inhibition mechanism includes, even if it has not been clearly shown, it might be analogous in the case of SrtA to the Cys199 residue of the enzyme.(Domínguez et al., 2012) Tideglusib slowed the S. aureus Newman strain’s growth only little. Many thiadiazolidinedione derivatives were created and tested to find relevant structural activity, connection building and enhancing the anti-virulence profile. Swapping out the 2-naphtyl group with an ethyl, benzyl, or phenethyl reduced the Sa-SrtA inhibitory action but significantly increased the antimicrobial effects. The 3,5-dimethylisoxazole ring when replaced with the naphtyl maintained the SrtA inhibition along with improved water solubility. BALB/c mice were treated with tideglusib at a dose of 40 mg/kg/day. S. aureus USA300 infection increased the 10-day survival rate to 40% from just 10% in the uninfected group. not-treated animals.(Yang et al., 2020) 2-(2-PHENYLHYDRAZINYLIDENE) ALKANOIC ACIDS and DERIVATIVES A number of 1,3-dicarbonyl-2-phenylhydrazinylidene compounds were created and tested against the of S. aureus ’s SrtADN59-6His enzyme. The 3-oxo-2-(2-(3,4-dichlorophenyl)hydrazinylidene)butanoic acid showed the best inhibitory impact and IC50 has a value of 50 µM. The substance resulted in a 39 percent suppression of the biofilm at 100 µM. S. aureus 29213 experienced a 71 percent decrease and S. aureus 25923 formation of biofilm. The substances with MICs greater than 270 µM had no discernible impact on the growth of S. aureus. (Maggio et al., 2016) The chemicals in this class share structural similarities with the derivatives of pyrazolethione pointing to a possible shared mechanism for SrtA suppression. Fritillaria verticillata ’s bulbs were used to produce -Sitosterol-3-O -glucopyranoside, a Sa-SrtA inhibitor (IC50 = 31.7 M), although it had no effect on Sa-SrtB. OTHER NON-CLASSIFIED SrtA INHIBITORS Many Srt inhibitors have distinctive chemical structures, making it challenging to categorise them into a single class. Here are several pertinent chemicals with various structures. Fritillaria verticillata’s bulbs were used to produce β-Sitosterol-3-O -glucopyranoside, a Sa-SrtA inhibitor (IC50 = 31.7 M), although it had no effect on Sa-SrtB.(Kim et al., 2003; Kang et al., 2006) With MIC values of 693 µM, the substance showed diminished antibacterial efficacy against S. aureus . Sitosterol was discovered to have no effect on SrtA or bacterial cell proliferation, highlighting how important the glucoside moiety is.(Kim et al., 2003) Without having a noticeable effect on S.aureus ATCC6538p, Aspermytin A, a polyketide isolated from the culture of a marine-derivedfungus Aspergillus sp. prevented Sa-SrtA .(Park et al., 2020b)Natural dibenzyl compound erianin inhibited Sa-SrtA (IC50 = 65.7 µM) without having any antibacterial effects on S. aureus (MIC 1600 µM). Mice that had been infected with S . aureus and were given 50 mg/Kg erianin three times per day for three days had a 9-day survival rate of 30% against 0% in the animals that weren’t given any treatment.(Ouyang et al., 2018) Halisulfate 1 was isolated from the sponge Coscinoderma sp . (Family Spongiidae) and showed substantial antibacterial properties on S . aureus as well as potent inhibitory effects on Sa-SrtA (IC50 = 36 µM).(Bae et al., 2011) Figure 17. Structures of several sortase inhibitors containing steroid nucleus, sulfate group etc. Echinacoside (ECH), a naturally occurring polyphenol, was discovered to be a possible SrtA inhibitor in vitro with an IC50 of 38.42 μM using fluorescence resonance energy transfer (FRET). It was shown that SrtA-mediated fibrinogen binding, surface protein A anchoring, and biofilm formation in S. aureus were inhibited by ECH. By calculating the KA-binding constant of 3.09 × 105 L/mol and determining the binding mode of ECH to SrtA, the fluorescence quenching assay demonstrated a direct interaction between the two molecules. Crucially, ECH and vancomycin together provided defense against MRSA-induced pneumonia in mice models. Consequently, ECH, either by itself or in conjunction with vancomycin, may function as a possible antivirulence agent against S. aureus infections.(Jiang et al., 2023a) not-yet-known not-yet-known not-yet-known unknown SUMMARY AND CONCLUSION Instead of creating chemicals with bactericidal properties in response to the rise of resistant bacteria, a lot of attention has been focused on targeting bacterial pathogenicity factors. Should antivirulence if the appropriate measures are taken, the pathogen without killing them, they can no longer pose a threat, so resulting in a low selective pressure to create resistance to the anti-virulent treatment. SrtA, one of the virulence components is a target that shows great promise. In Gram-positive bacteria, sortase enzymes are widely distributed and are responsible for building pili and attaching proteins to the cell wall. They make for attractive therapeutic targets because of their significant function in exhibiting virulence factors. Additionally, it is expected that sortases will soon be utilised as pharmacological targets and the treatment of a variety of ailments, some of which include maybe in the process of conception. Amazing development has been made in the last ten years in learning about the many types of sortases from gram-positive bacteria, A, B, C, and D. SrtA from S. aureus, which has major industrial value and serves as an essential therapeutic target, has been the focus of a sizable percentage of the work done on sortases thus far. Sortases play a critical role in biofilm formation and attachment to host tissues, signalling to the host, and evading the host immune response even though they are not necessary for bacterial cell survival. Finally, despite the fact that numerous small molecule sortase inhibitors have been discovered, no medication using them to treat bacterial infections has yet been created. Given the incidence of germs that are resistant to antibiotics and increasing effort to create sortase A inhibitors that are effective as medicines, which will surely result in the identification and characterization of more potent and selected antivirulent agent. FUNDING INFORMATION The study is supported by the seed funding by Research Funding & Management Office (RFMO), JIS Group of Institutions (RFMO/SF/JISU/24-25/002). CONFLICTS OF INTEREST There is no conflict of interest to declare. ACKNOWLEDGEMENTS We acknowledge JIS University. 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Information & Authors Information Version history V1 Version 1 29 July 2025 Peer review timeline Published Next Research Version of Record 1 Apr 2026 Published Copyright This work is licensed under a Non Exclusive No Reuse License. Keywords staphylococcus aureus anti-microbial resistance anti-virulence biofilm sortase a Authors Affiliations Aaheli Basu JIS University View all articles by this author Rahul Khanra JIS University View all articles by this author Sarmistha Pal 0000-0002-6142-7118 [email protected] JIS University View all articles by this author Metrics & Citations Metrics Article Usage 319 views 210 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Aaheli Basu, Rahul Khanra, Sarmistha Pal. Sortase A Inhibition: Updated Review of Non-Peptide Ligands for Targeting Staphylococcus Aureus Virulence. Authorea . 29 July 2025. 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