Targeting Bacterial Sortases in Search of Anti-virulence Therapies with Low Risk of Resistance Development

Increasingly ineffective antibiotics and rapid spread of multi- and pan-resistant bacteria represent a global health threat; hence, the need of developing new antimicrobial medicines. A first step in this direction is identifying new molecular targets, such as virulence factors. Sortase A represents a virulence factor essential for the pathogenesis of Gram-positive pathogens, some of which have a high risk for human health. We present here an exhaustive collection of sortases inhibitors grouped by relevant chemical features: vinyl sulfones, 3-aryl acrylic acids and derivatives, flavonoids, naphtoquinones, anthraquinones, indoles, pyrrolomycins, isoquinoline derivatives, aryl β-aminoethyl ketones, pyrazolethiones, pyridazinones, benzisothiazolinones, 2-phenyl-benzoxazole and 2-phenyl-benzofuran derivatives, thiadiazoles, triazolothiadiazoles, 2-(2-phenylhydrazinylidene)alkanoic acids, and 1,2,4-thiadiazolidine-3,5-dione. This review focuses on highlighting their structure–activity relationships, using the half maximal inhibitory concentration (IC50), when available, as an indicator of each compound effect on a specific sortase. The information herein is useful for acquiring knowledge on diverse natural and synthetic sortases inhibitors scaffolds and for understanding the way their structural variations impact IC50. It will hopefully be the inspiration for designing novel effective and safe sortase inhibitors in order to create new anti-infective compounds and to help overcoming the current worldwide antibiotic shortage.


Introduction
The worldwide increasing resistance of bacterial pathogens to antibiotics imposes alternative strategies to treat infectious diseases [1,2]. Drug resistant bacteria pose a great threat and more and more infections fail to respond to available treatments [3]. Methicillinresistant Staphylococcus aureus, vancomycin-resistant Enterococcus, and carbapenem-resistant Enterobacteriaceae are just some examples of major resistant bacteria that cause serious diseases sometimes resulting in the death of the patients [4]. The identification and analysis of the bacterial mechanisms of pathogenicity emerged as a promising strategy for drug development. The virulence factors are used by pathogens to colonize, invade, and persist within a susceptible host, surviving its immune system [5]. Bacteria use virulence factors to disable the host immune system and to invade its tissues. Drugs blocking these factors without killing the bacteria create less evolutionary pressure and diminish the chances of resistant genes to emerge. Various strategies are studied for implementation into future therapies, like the inhibition of the quench pathogen quorum sensing (QS) systems, disrupting the biosynthesis of the functional membrane microdomains, the inhibition of the biofilm formation, and toxins' neutralization [6].
The center of attention for this paper is one of the most promising anti-virulence strategies targeting prominent Gram-positive pathogens: the inhibition of bacterial sortases (Srt)

Sortase A Inhibitors
There are several works that present known Srt inhibitors, but most of them chose to classify these substances as natural and synthesis compounds [29,30]. We presented here an exhaustive collection of SrtA inhibitors grouped by relevant chemical features highlighting their structure-activity relationships, rather than using their source as classification criteria. The half maximal inhibitory concentration (IC 50 ) presented here should be considered as a relative, and not an absolute, indicator of each compound's effect on a specific Srt target. The differences in testing protocols can be important and should not be ignored. When available, the minimum inhibitory concentration (MIC) values are presented and represent the lowest tested concentration of the compound at which no bacteria growth was observed. Low Srt's IC 50 values coupled with high MIC values are an indicator of the compound's potential to disrupt the pathogenesis of the bacteria without affecting its viability, placing no pressure on bacteria to develop drug-resistant mechanisms [31].

Plant Extracts
A screening of medicinal plant extracts as Sa-SrtA inhibitors reported the evaluation of 80 dried extracts and their corresponding n-hexane, ethyl acetate, and water fractions. The measured IC 50 values were in the range of 1.5-39.4 µg/mL The best inhibitory effects were registered for 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. Rhus verniciflua, Fam. Anacardiaceae) bark [32]. The evaluation of the anti-virulence potential of these extracts is limited, because this study offers no information on extracts' impact on the bacterial growth.
A methanol extract obtained from Curcuma longa (turmeric, Fam. Zingiberaceae) dried rhizomes (80 µg/mL) produced a 80% inhibition of SrtA S. aureus ATCC 6538p activity after 1 h of incubation at 37 • C [33]. An extract from the dried fruits of Psoralea corylifolia (Fam. Fabaceae) produced a 44.2% inhibition of the S. mutans SrtA (Sm-SrtA) at 100 µg/mL [34]. A series of compounds were isolated from these extracts and are described in this study based on their structures.

Thiol Reactive Reagents
This group contains several chemical reagents know to interact with biologically active thiol groups that were identified as sortases inhibitors in the process of discovering the catalytic mechanism of the enzyme, but their broad and unselective reactivity profile renders them unfit for therapeutic development [35]. Methanethiosulfonates emerged as the first class of sortase inhibitors. Both (2-(trimethylammonium)ethyl)methanethiosulfonate (MTSET) and sodium (2-sulfonatoethyl)methanethiosulfonate (MTSES) interfere with the cleavage of sorting signals at the LPXTG motif [12] by forming a disulfide bond with the cysteine residue ( Figure 1) [13]. Several cysteine proteases inhibitors were tested, identifying sodium p-hydroxymercuribenzoate (pHMB) as a sortase inhibitor, whereas alkylating reagents such as N-ethylmaleimide, iodoacetate, and iodoacetamide were not [12]. Sulfhydryl reducing agents, like dithiothreitol (DTT), had no effect on Sa-SrtA activity, but they reversed the inhibitory effect of MTSET by regenerating the thiol group [13].
A methanol extract obtained from Curcuma longa (turmeric, Fam. Zingiberaceae) dried rhizomes (80 μg/mL) produced a 80% inhibition of SrtA S. aureus ATCC 6538p activity after 1 h of incubation at 37 °C [33]. An extract from the dried fruits of Psoralea corylifolia (Fam. Fabaceae) produced a 44.2% inhibition of the S. mutans SrtA (Sm-SrtA) at 100 μg/mL [34]. A series of compounds were isolated from these extracts and are described in this study based on their structures.

Thiol Reactive Reagents
This group contains several chemical reagents know to interact with biologically active thiol groups that were identified as sortases inhibitors in the process of discovering the catalytic mechanism of the enzyme, but their broad and unselective reactivity profile renders them unfit for therapeutic development [35]. Methanethiosulfonates emerged as the first class of sortase inhibitors. Both (2-(trimethylammonium)ethyl)methanethiosulfonate (MTSET) and sodium (2-sulfonatoethyl)methanethiosulfonate (MTSES) interfere with the cleavage of sorting signals at the LPXTG motif [12] by forming a disulfide bond with the cysteine residue ( Figure 1) [13]. Several cysteine proteases inhibitors were tested, identifying sodium p-hydroxymercuribenzoate (pHMB) as a sortase inhibitor, whereas alkylating reagents such as N-ethylmaleimide, iodoacetate, and iodoacetamide were not [12]. Sulfhydryl reducing agents, like dithiothreitol (DTT), had no effect on Sa-SrtA activity, but they reversed the inhibitory effect of MTSET by regenerating the thiol group [13].

Vinyl Sulfones
Several vinyl sulfones derivatives were evaluated against SrtAΔ24, based on their electrophilic potential to block active cysteine residues by forming thioether adducts through 1,4-addition reaction. The compound 3,3,3-trifluoro-1-(phenylsulfonyl)-1-propene exhibited the highest inhibitory effect, with an IC50 value of 190 μM and approximatively 1.5 folds higher MIC value (317.5 μM). The related phenyl vinyl sulfone has a significantly reduced inhibitory potency, with an IC50 of 736 μM, probably because of the lower electrophilic character ( Figure 1). Phenyl trans-styryl sulfone had no inhibitory effect, indicating the importance of the molecule size. Mass spectrometry analysis confirmed the formation of a covalent bond between the phenyl vinyl sulfone and Sa-SrtA's Cys184. Phenyl vinyl sulfone at 3000 μM reduced significantly the virulence of S. aureus Newman strain in a fibronectin binding model, without effecting the bacterial viability (MIC value over 6000 μM) [36].
The vinyl sulfone group was added to a scaffold of various cis-5-phenyl proline methyl esters, but the compounds exhibited only a modest activity towards SrtAΔ24, with IC50 values ranging from 850 μM to over 5000 μM [37].

3-Aryl Acrylic Acids and Derivatives
Our analysis of the known sortases inhibitors revealed the 3-aryl substituted acrylic acid as a common and important scaffold, and therefore, we chose it as criteria for clustering them together in this group. The phenylacrylic acid, better known as cinnamic acid, is the structural core of various natural aromatic carboxylic acids with important roles in the biosynthesis of phenyl-propanoids, coumarins, lignans, flavonoids, stilbenes, aurones,

Vinyl Sulfones
Several vinyl sulfones derivatives were evaluated against SrtA∆24, based on their electrophilic potential to block active cysteine residues by forming thioether adducts through 1,4-addition reaction. The compound 3,3,3-trifluoro-1-(phenylsulfonyl)-1-propene exhibited the highest inhibitory effect, with an IC 50 value of 190 µM and approximatively 1.5 folds higher MIC value (317.5 µM). The related phenyl vinyl sulfone has a significantly reduced inhibitory potency, with an IC 50 of 736 µM, probably because of the lower electrophilic character (Figure 1). Phenyl trans-styryl sulfone had no inhibitory effect, indicating the importance of the molecule size. Mass spectrometry analysis confirmed the formation of a covalent bond between the phenyl vinyl sulfone and Sa-SrtA's Cys184. Phenyl vinyl sulfone at 3000 µM reduced significantly the virulence of S. aureus Newman strain in a fibronectin binding model, without effecting the bacterial viability (MIC value over 6000 µM) [36].
The vinyl sulfone group was added to a scaffold of various cis-5-phenyl proline methyl esters, but the compounds exhibited only a modest activity towards SrtA∆24, with IC 50 values ranging from 850 µM to over 5000 µM [37].

3-Aryl Acrylic Acids and Derivatives
Our analysis of the known sortases inhibitors revealed the 3-aryl substituted acrylic acid as a common and important scaffold, and therefore, we chose it as criteria for clustering them together in this group. The phenylacrylic acid, better known as cinnamic acid, is the structural core of various natural aromatic carboxylic acids with important roles in the biosynthesis of phenyl-propanoids, coumarins, lignans, flavonoids, stilbenes, aurones, anthocyanins, spermidines, and tannins [38]. This group contains natural and synthetic cinnamic acid derivatives and related compounds, as well as bioisosteric analogues.
Another ester derivative of caffeic acid, the chlorogenic acid (3-O-caffeoylquinic acid), was identified as a potent inhibitor against SrtA from S. aureus Newman D2C. It exhibited an IC 50 of 95.57 µM, without inhibition of bacterial cell growth (MIC greater than 1024 µg/mL). In mice studies, chlorogenic acid significantly interfered in the pathogenesis of S. aureus and prevented renal abscess formation [19]. Two isomers of the chlorogenic acid, cryptochlorogenic acid (4-O-caffeoylquinic acid) and neochlorogenic acid (5-O-caffeoylquinic acid), were tested against SrtA∆N59. Both isomers exhibited an IC 50 over 500 µM, highlighting the importance of the functional groups' reciprocal positions [41].
Three major constituents of the Curcuma longa (turmeric, Fam. Zingiberaceae) rhizome were found to inhibit SrtA∆24 from S. aureus ATCC 6538p. The highest inhibitory effect was produced by curcumin with an IC 50 value of 37.5 µM, followed by demethoxycurcumin (IC 50 = 70.3 µM) and bisdemethoxycurcumin (IC 50 = 103.5 µM). All three compounds showed no significant growth inhibitory activity against S. aureus strain Newman, with MIC values over 200 µg/mL [33]. Curcumin was found to be active also against S. mutans UA159 SrtA, with an IC 50 value of 10.2 µM, a value considerably lower than the registered MIC value of 125 µM [21]. Curcumin can be considered a dimer of ferulic acid, compound with low inhibitory effect of S. mutans SrtA, indicating the complexity of the structureactivity relationships in this chemical class. Due to the presence of the β-diketo moeity, a Michael acceptor group, curcumin is known to react with cysteine sulfhydryl groups [42] and is most likely its mechanism for SrtA inhibition.
The chemical structures of the cinnamic acid derivatives identified as Srt inhibitors are presented in Figure 2.

Aromatic Furanone Derivatives
A series of brominated tris-aromatic 2-furanones derivatives were isolated from the dark red ascidian Synoicum sp. (Fam. Polyclinidae) collected from the coast of southern Korea. Cadiolide E, a 4-(3-bromo-4-hydroxyphenyl)-2-furanone derivative (Figure 4), exhibited a significant inhibition on Sa-SrtA with an IC 50 value of 78.8 µM, but also strong antibacterial effects. The related bis-aromatic diester, synoilide B, showed no significant effect on Sa-SrtA [44]. Structurally, both cadiolide E and synoilide B share a common fragment with the cinnamic acid.

Coumarin Derivatives
Esculetin (6,7-dihydroxycoumarin, also known as aesculetin) is the lactone derivative formed by intramolecular cyclization of the 6-hydroxycaffeic acid. It inhibited Sa-SrtA with an IC50 value of 37.5 μM, while caffeic acid and its methoxy-derivative, the transferulic acid, proved to be inactive. Esculetin exhibited no significant antimicrobial activity against S. aureus ATCC 25923 and S. aureus ATCC 6538. The impact of the 6,7-dihydroxy  also had no significant inhibitory effect on S. aureus ATCC6538p growth, the MIC values being over 300 μM [43].

Aromatic Furanone Derivatives
A series of brominated tris-aromatic 2-furanones derivatives were isolated from the dark red ascidian Synoicum sp. (Fam. Polyclinidae) collected from the coast of southern Korea. Cadiolide E, a 4-(3-bromo-4-hydroxyphenyl)-2-furanone derivative (Figure 4), exhibited a significant inhibition on Sa-SrtA with an IC50 value of 78.8 μM, but also strong antibacterial effects. The related bis-aromatic diester, synoilide B, showed no significant effect on Sa-SrtA [44]. Structurally, both cadiolide E and synoilide B share a common fragment with the cinnamic acid.  A group of structurally related compounds named isocadiolides isolated from Synoicum sp. share the bromohydroxyphenyl fragments with the cadiolides, but differ in the nature of the central ring which is no longer a furanone, but one of the following structures: cyclopentene-1,3-dione, dihydrofuran, or pyranone. Isocadiolides A−D moderately inhibited Sa-SrtA, but also the bacterial development of S. aureus ATCC 6538P [45].  Figure 4. Aromatic furanone derivatives, diarylacrylonitriles, chalcones derivatives, 3-(2-furyl)acrylic acid, and 3-(thien-2yl)acrylic acid derivatives. A group of structurally related compounds named isocadiolides isolated from Synoicum sp. share the bromohydroxyphenyl fragments with the cadiolides, but differ in the nature of the central ring which is no longer a furanone, but one of the following structures: cyclopentene-1,3-dione, dihydrofuran, or pyranone. Isocadiolides A−D moderately inhibited Sa-SrtA, but also the bacterial development of S. aureus ATCC 6538P [45].

Diarylacrylonitriles
A synthetic derivative of the trans-p-coumaric acid, the methyl (2E)-2,3-bis(4-methoxyphenyl)acrylate ( Figure 4) was identified as hit in a random screening for a small-molecule Sa-SrtA inhibitors with an IC 50 of 231 µM. The analysis of the structure-activity relationships revealed that the corresponding Z-conformer had close to 4-fold less inhibitory potency. The hydrolysis to the corresponding acid or the hydrogenation of the double bound both rendered the compound inactive. The bioisosteric replacement of the ester group with a nitrile significantly improved the Sa-SrtA inhibition. Contrary to the methyl acrylate derivatives, the potency of the diarylacrylonitrile derivatives was higher for the Z-isomers, the two benzene rings being in trans orientation [18].

Chalcones Derivatives
Chalcones are derivatives of 1,3-diphenyl-2-propene-1-one that consist of two benzene rings linked by a three carbon unsaturated chain and can be considered cinnamoyl derivatives. (E)-chalcone is the basic member of the chalcone series [23]. The activity of (E)-chalcone against Sm-SrtA was measured and an IC 50 of 5 µM was determined. The compound produced a slow and irreversible inhibition by forming a covalent adduct with the Cys205 residue through a Michael addition mechanism. This mechanism was demonstrated by the lack of inhibitory effects of the saturated analogue, dihydrochalcone [20]. Chalcone effectively inhibited Sa-SrtA (IC 50 = 53.15 µM) without visibly altering S. aureus USA 300 growth (MIC > 4864 µM). The compound reduced the adhesion of S. aureus to fibronectin, biofilm formation, and bacterial invasion in a J774 mouse macrophages cells model. The S. aureus infected mice treated with subcutaneous injections of chalcone of 150 mg/kg at 12-h intervals had a higher 3-days survival rate compared to those untreated [48]. Isoliquiritigenin (2 ,4,4 -trihydroxychalcone) isolated from an extract of the vine stem of Spatholobus suberectus (Fam. Fabaceae) inhibited Sa-SrtA with an IC 50 of 139.7 µM [49].
(E)-Chalcone ( Figure 4) inhibited also the L. monocytogenes SrtA∆N70 (Lm-SrtA) with an IC 50 of 28.41 µM. The inhibition of SrtA activity reduced L. monocytogenes virulence and decreases the mortality of infected mice, with no visible influence on bacterial growth [23]. Interestingly, the dihydrochalcone derivative phloretin (Figure 4) effectively inhibited Lm-SrtA (IC 50 = 37.24 µM) and produced no significant impact on L. monocytogenes growth (MIC = 933.44 µM). Molecular dynamics simulations indicated that the phloretin binds to Lm-SrtA through its hydroxyl groups in a region adjacent to the active enzymatic center [50]. This explains why the lack of the double bond is not important as in the case of the interaction of (E)-chalcone and Sm-SrtA.

3-(2-Furyl)Acrylic Acid and 3-(Thien-2-yl)Acrylic Acid Derivatives
The 3-(2-furyl)acrylic acid and its thiophene analogue can be considered bioisosters of the cinnamic acid. A series of phenyl-substituted amides derivatives of these acids ( Figure 4) were synthesized and tested for their in vitro inhibitory effect against SrtA∆59. The IC 50 values ranged from 58 to 571 µM and for some compounds were described as over the 600 µM threshold. The hydrogenation of the acrylic double bond led to inactive compounds, thus underlining its importance. The N-methyl substituted amides proved to be weaker inhibitors than their corresponding NH analogues [51]. It is difficult to assess the anti-virulence potential of the compounds in this series in the absence of data regarding their impact on bacterial growth.

Flavonoids
Flavone (2-phenyl-4H-chromen-4-one) may be regarded as a closed ring derivative of (E)-chalcone. The flavonols are a subclass of flavonoids sharing a common 3hydroxyflavone scaffold [52], where the 3-hydroxyl group seems to be essential for SrtA inhibition [28]. As the name suggests, the isoflavones are isomers of their corresponding flavones, differing in the position of the phenyl group on the chromen-4-one scaffold. Flavanones lack the double bond in the central ring and can be considered 2,3dihydroflavones [52]. The chemical structures of Srt inhibitors belonging to this group are illustrated in Figure 5.   Acacetin (5,7-dihydroxy-4 -methoxyflavone) represents the 4 -O-methylated derivative of apigenin. It exhibited an IC 50 of 128.3 µM on S. aureus SrtA∆N59 and a MIC value on S. aureus ATCC25904 greater than 3800 µM. A solution of 224 µM acacetin reduced almost to half the bacteria binding to fibrinogen, and in doses of 150 mg/kg/day, was able to protect mice from renal abscess induced by S. aureus, significantly increasing survival rates [53].
Isovitexin, the 6-C substituited glucoside of apigenin, inhibited Sa-SrtA∆N59 with an IC 50 of 67.02 µM. The compound inhibited in a dose-dependent manner S. aureus cell adhesion to fibrinogen and biofilm formation without influencing the bacterial growth [54].
Baicalin, the 7-O-glucuronide of baicalein (5,6,7-trihydroxyflavone), inhibits Sa-SrtB with an IC 50 value of 57.9 µM with no significant growth pressure on S. aureus. Baicalin reduced in a dose-dependent manner the adhesion of S. aureus to human alveolar epithelial A549 cells. The molecular dynamics simulations indicated the importance of both the chromen-4-one scaffold and the glucuronide moiety [55]. Baicalein inhibited in a concentration-dependent manner Lm-SrtA and attenuated L. monocytogenes entrance into Caco-2 cells cultures [56].

Flavonols
A series of natural flavonols were isolated from an ethyl acetate extract of Toxicodendron vernicifluum bark and tested on SrtA and SrtB of S. aureus ATCC6538p using as substrate Dabcyl-QALPETGEE-Edans (for SrtA) and Dabcyl-NPQTN-Edans (for SrtB). In the case of SrtA, the best inhibition effects were registered for morin ( A number of flavonol derivatives were tested on SrtA∆24 and a significant inhibitory effect were recorded only for myricetin with an IC 50 value of 4.63 µM. Quercetin presented weak inhibitory effects at 10 µM, and its low water solubility hindered the testing at higher concentrations. Fisetin (3,7,3 ,4 -tetrahydroxyflavone) had no significant effect [28]. The enzymatic assay coupled with molecular docking studies indicated that the flavonoid aglycones are more potent towards SrtA than their corresponding glycosides (hyperoside, rutin, and troxerutin), probably due to the lower number of rotatable bonds [28,58]. Quercetin significantly inhibited Spn-SrtA activity in a dose-dependent manner, but the IC 50 values was not reported [22]. Quercitrin (syn. quercetin-3-rhamnoside), a quercetin glycoside derivative, inhibited the S. aureus SrtA∆N59 with an IC 50 value of 72.77 µM, with little effect on Newman D2C strain growth [59].

Flavanones
A series of seven prenylated flavonoids isolated from the roots of Sophora flavescens (shrubby sophora, Fam. Fabaceae) were evaluated for inhibitory activity against SrtA from S. aureus ATCC 6538p. All compounds produced a medium inhibitory effect, but the IC 50 value could be calculated only for kurarinol, a derivative of 5-methoxyflavanone. Kurarinol has an IC 50 value of 107 µM and a medium antibacterial activity against S. aureus (MIC = 219 µM) [63]. Eriodictyol (3,4,5,7-tetrahydroxyflavanone) inhibited Sa-SrtA with an IC 50 value of 7.73 µM in a reversible mechanism and did not block S. aureus development even at a concentration 50-fold higher than IC 50. The compound decreased in a dose-dependent manner the biofilm formation and the anchoring of the SpA protein in S. aureus [64].

Flavanonols
Astilbin, the 3-O-rhamnoside of taxifolin (syn. dihydroquercetin) inhibits Sm-SrtA with an IC 50 value of 16.6 µM without affecting the proliferation of S. mutans ATCC 25175. The molecular dynamics simulations reflected the role of the rhamnoside moiety in binding the enzyme active site. Astilbin produced a 49% reduction in bacterial biofilm formation at 142.1 µM, but no significant effect at 71 µM or lower concentrations. The authors consider that the biofilm inhibitory levels are much higher than IC 50 for SrtA, because of the amount of not inhibited enzyme [65]. In our opinion, the compound's low lipophilicity and cell permeability [66] is probably the cause of this difference.

Quinone Derivatives
A screening for SrtA inhibitors using a clinical drug and a natural product-based library and the monitoring of SrtA∆24 cleavage of Abz-LPETG-Dnp substrate, identified two natural 5,8-dihydroxy-1,4-naphthoquinone derivatives, shikonin (IC 50 = 0.30 µM) and alkannin (IC 50 = 0.36 µM). The compounds are enantiomers and they both strongly inhibit the growth and viability of S. aureus [67] limiting considerably their use as anti-virulence agents. Similar 1,4-naphthoquinone derivatives, juglone (5-hydroxy-1,4-naphthalenedione) and its 2-methyl analogue, plumbagin, demonstrated a potent Sa-SrtA inhibitory effect with IC 50 values of 1.78 µM and 16.71 µM respectively, without significantly influencing the bacterial growth of S. aureus, E. faecalis, and S. epidermidis. The inhibition kinetics and docking studies indicated an irreversible mechanism based on the Cys184's thiol catalytic residue addition to the dual conjugated double system of the naphthoquinone structure. Lawsone, the 2-hydroxy-isomer of juglone, had no significant effect on Sa-SrtA at 10 µM because of its low reactivity towards nucleophilic agents [68]. The chemical structures of these naphthoquinone derivatives are presented in Figure 6.

Indoles Derivatives
In the course of investigating an extract from tropical marine sponges from Hyrtios species (Fam. Thorectidae), a series of derivatives of 5-hydroxyindole were isolated [70] and tested for their effect on Sa-SrtA [71]. Serotonin (5-hydroxytryptamine) had no significant effect, but its N-closed ring analogue derivative, 1-carboxy-6-hydroxy-3,4dihydro-β-carboline, demonstrated moderated inhibitory action against Sa-SrtA (IC50 = 290 μM). Based on its structure, a series of 3-indoleglyoxylic acid derivatives and their corresponding N-phenyl-amide were synthesized and evaluated as Sa-SrtA inhibitors. The compounds derived from indole and 2-methylindole, exhibited potent inhibitory properties with IC50 values in the range of 61 to 174 μM, while those derived from 5hydroxy-and 5-methoxy-indole showed no significant effect. The related 3-glyceroindole and 3-indoleacetic acid were not active towards SrtA, indicating the importance of both carbonyl groups in the structure [71].
The screening of a series of indole derivatives and related heterocyclic compounds on S. pyogenes SrtA∆N81 (Sp-SrtA) led to the identification of (2-amino-6-chloro-1H-indol-3-yl)-morpholino-methanone with the corresponding IC50 value of 10 μM. The compound is structurally similar to the 3-indoleglyoxylic acid derivatives, but it had no significant effect on Sa-SrtA∆N59. The importance of the morpholino moiety for the inhibition of Sp-SrtA was demonstrated by docking studies and by the lack of effect of the corresponding piperidine derivative [72].
The Sa-SrtA inhibition assay-guided separation on a bright yellow sponge Spongosorites sp. (Fam. Halichondriidae) extract yielded several indole derivatives consisting of two indole moieties connected by a heterocyclic scaffold. Based on the nature of the joining heterocyle, the identified SrtA inhibitors can be classified as topsentins (bis(indolyl)imidazole derivatives) and hamacanthins (bis(indolyl)pyrazinone derivatives). From the first group, the best results were obtained for deoxytopsentin (IC50 = 48 μM), bromodeoxytopsentin (IC50 = 48 μM), bromotopsentin (IC50 = 39.7 μM), but they also had a major impact on S. aureus Newman bacterial growth. The hamacanthin derivatives had slightly less inhibitory potency than the related topsentins and strong antibacterial effects that limits their usefulness as anti-virulence potential therapies [73]. Two structurally related compounds, the spongosoritins B and C, sharing a central 2methoxy-1-imidazole-5-one connecting ring, inhibited Sa-SrtA with IC50 values of 62.7 and 43.9 μM, respectively [74]. A series of bis(indolyl)-1,2,4-oxadizole derivatives were synthesized as topsentin analogues and their effects on S. aureus biofilm formation were evaluated. Three compounds were identified as potent biofilm inhibitors and all demonstrated a significant reduction in Sa-SrtA's effect [75]. A pair of pyranoquinone derivatives structurally related to the 1,4-naphthoquinones were identified to irreversibly inhibit Sa-SrtA with IC 50 values of 6.2 µM and 19.4 µM by bonding to the nucleophilic thiol group of Cys184 in a Michael addition reaction. The two derivatives ( Figure 6) are diastereomers differing in the stereochemistry of just one carbon atom, the isomer R being more active towards Sa-SrtA. Both compounds significantly attenuated the biofilm production at 80 µM and had no impact on the growth of S. aureus at 150 µM, demonstrating a good anti-virulence profile [67].

Indoles Derivatives
In the course of investigating an extract from tropical marine sponges from Hyrtios species (Fam. Thorectidae), a series of derivatives of 5-hydroxyindole were isolated [70] and tested for their effect on Sa-SrtA [71]. Serotonin (5-hydroxytryptamine) had no significant effect, but its N-closed ring analogue derivative, 1-carboxy-6-hydroxy-3,4-dihydroβ-carboline, demonstrated moderated inhibitory action against Sa-SrtA (IC 50 = 290 µM). Based on its structure, a series of 3-indoleglyoxylic acid derivatives and their corresponding N-phenyl-amide were synthesized and evaluated as Sa-SrtA inhibitors. The compounds derived from indole and 2-methylindole, exhibited potent inhibitory properties with IC 50 values in the range of 61 to 174 µM, while those derived from 5-hydroxy-and 5-methoxyindole showed no significant effect. The related 3-glyceroindole and 3-indoleacetic acid were not active towards SrtA, indicating the importance of both carbonyl groups in the structure [71].
The screening of a series of indole derivatives and related heterocyclic compounds on S. pyogenes SrtA∆N81 (Sp-SrtA) led to the identification of (2-amino-6-chloro-1H-indol-3yl)-morpholino-methanone with the corresponding IC 50 value of 10 µM. The compound is structurally similar to the 3-indoleglyoxylic acid derivatives, but it had no significant effect on Sa-SrtA∆N59. The importance of the morpholino moiety for the inhibition of Sp-SrtA was demonstrated by docking studies and by the lack of effect of the corresponding piperidine derivative [72].
The Sa-SrtA inhibition assay-guided separation on a bright yellow sponge Spongosorites sp. (Fam. Halichondriidae) extract yielded several indole derivatives consisting of two indole moieties connected by a heterocyclic scaffold. Based on the nature of the joining heterocyle, the identified SrtA inhibitors can be classified as topsentins (bis(indolyl)imidazole derivatives) and hamacanthins (bis(indolyl)pyrazinone derivatives). From the first group, the best results were obtained for deoxytopsentin (IC 50 = 48 µM), bromodeoxytopsentin (IC 50 = 48 µM), bromotopsentin (IC 50 = 39.7 µM), but they also had a major impact on S. aureus Newman bacterial growth. The hamacanthin derivatives had slightly less inhibitory potency than the related topsentins and strong antibacterial effects that limits their usefulness as anti-virulence potential therapies [73]. Two structurally related compounds, the spongosoritins B and C, sharing a central 2-methoxy-1-imidazole-5-one connecting ring, inhibited Sa-SrtA with IC 50 values of 62.7 and 43.9 µM, respectively [74]. A series of bis(indolyl)-1,2,4-oxadizole derivatives were synthesized as topsentin analogues and their effects on S. aureus biofilm formation were evaluated. Three compounds were identified as potent biofilm inhibitors and all demonstrated a significant reduction in Sa-SrtA's effect [75].
A number of six β-carboline alkaloids belonging to the eudistomin Y class were extracted and separated by multiple chromatographic methods from the marine ascidian Synoicum sp. (Fam. Polyclinidae) [76]. The β-carboline scaffold is based on an indole ring fused with a pyridine moiety [77]. The compounds were also isolated from a tunicate of the Eudistoma genus [78]. Only eudistomin Y4 (1-(3-bromo-4-hydroxybenzoyl)-6-bromoβ-carboline) and its isomer eudistomin Y5 presented inhibitory activity on Sa-SrtA with IC 50 163.2 µM and 146.4 µM, respectively. The compounds demonstrated also strong antibacterial effects on S. aureus ATCC 6538p with MIC values close to 7 µM and 14 µM, respectively. The hydrogenation of the carbonyl group to a hydroxyl group reduced considerably the Sa-SrtA inhibitory capacity [76].
A series of 1-phenyl-dihydro-β-carboline and 1-phenyl-tetrahydro-β-carboline derivatives were synthesized and some of them showed good inhibitory activity against SrtA, with IC 50 values in the range of 25 to 115 µM. The majority of these compounds had no impact on S. aureus bacterial viability [71].
The structures of the indole derivatives identified as Srt inhibitors are presented in Figure 7. A number of six β-carboline alkaloids belonging to the eudistomin Y class were extracted and separated by multiple chromatographic methods from the marine ascidian Synoicum sp. (Fam. Polyclinidae) [76]. The β-carboline scaffold is based on an indole ring fused with a pyridine moiety [77]. The compounds were also isolated from a tunicate of the Eudistoma genus [78]. Only eudistomin Y4 (1-(3-bromo-4-hydroxybenzoyl)-6-bromoβ-carboline) and its isomer eudistomin Y5 presented inhibitory activity on Sa-SrtA with IC50 163.2 μM and 146.4 μM, respectively. The compounds demonstrated also strong antibacterial effects on S. aureus ATCC 6538p with MIC values close to 7 μM and 14 μM, respectively. The hydrogenation of the carbonyl group to a hydroxyl group reduced considerably the Sa-SrtA inhibitory capacity [76].
A series of 1-phenyl-dihydro-β-carboline and 1-phenyl-tetrahydro-β-carboline derivatives were synthesized and some of them showed good inhibitory activity against SrtA, with IC50 values in the range of 25 to 115 μM. The majority of these compounds had no impact on S. aureus bacterial viability [71].
The structures of the indole derivatives identified as Srt inhibitors are presented in Figure 7.
Three aaptamines alkaloids (Figure 8) isolated from the marine sponge Aaptos aaptos (Fam. Suberitidae) were identified as Sa-SrtA inhibitors. Isoaaptamine had the highest inhibitory effect (IC50 = 16.22 μM), probably due to the presence of the N-methyl group. Its isomer, aaptamine had an IC50 value of 102.9 μM, while its N-demethyl analog, demethylaaptamine, had an IC50 value of 85.9 μM. Isoaaptamine showed a moderate growth inhibition on S. aureus strain Newman with a MIC value of 219 μM [84].

Aryl β-Aminoethyl Ketones
A series of SrtA inhibitors emerged after a screening of a 135,625 small molecules library using the fluorescence resonance energy transfer substrate method. A number of 6154 compounds displayed over 20% percentage inhibitions and were subjected to selection based on reactivity, genotoxic potential, and drug-like properties leading to 407 compounds. The compounds in this set were tested against papain, a eukaryotic protease with an active site thiol, in order to remove nonselective agents. Two aryl β-aminoethyl
Three aaptamines alkaloids (Figure 8) isolated from the marine sponge Aaptos aaptos (Fam. Suberitidae) were identified as Sa-SrtA inhibitors. Isoaaptamine had the highest inhibitory effect (IC 50 = 16.22 µM), probably due to the presence of the N-methyl group. Its isomer, aaptamine had an IC 50 value of 102.9 µM, while its N-demethyl analog, demethylaaptamine, had an IC 50 value of 85.9 µM. Isoaaptamine showed a moderate growth inhibition on S. aureus strain Newman with a MIC value of 219 µM [84].

Aryl β-Aminoethyl Ketones
A series of SrtA inhibitors emerged after a screening of a 135,625 small molecules library using the fluorescence resonance energy transfer substrate method. A number of 6154 compounds displayed over 20% percentage inhibitions and were subjected to selection based on reactivity, genotoxic potential, and drug-like properties leading to 407 compounds. The compounds in this set were tested against papain, a eukaryotic protease with an active site thiol, in order to remove nonselective agents. Two aryl βaminoethyl ketones, 3-(dimethylamino)-1-(2-thienyl)-1-propanone (AAEK1) and 1-(3,4dichlorophenyl)-3-(dimethylamino)propan-1-one (AAEK2), were selected for further inves-tigation ( Figure 9). The compounds had IC 50 values of 47 µM and 15 µM, respectively, for Sa-SrtA of S. aureus. AAEK1 and AAEK2 showed better inhibition of the SrtA homologue from B. anthracis with IC 50 values of 4.8 µM, respectively 5.6 µM [85]. Mass spectrometry and X-ray crystallography studies of AAEK1 and SrtA interaction revealed that the inhibition mechanism is based on the elimination of the dimethylamino group and formation of the thienyl vinyl ketone that covalently binds to the enzyme Cys's thiol [85].
Pharmaceuticals 2021, 14, x FOR PEER REVIEW 16 of 24 dichlorophenyl)-3-(dimethylamino)propan-1-one (AAEK2), were selected for further investigation (Figure 9). The compounds had IC50 values of 47 μM and 15 μM, respectively, for Sa-SrtA of S. aureus. AAEK1 and AAEK2 showed better inhibition of the SrtA homologue from B. anthracis with IC50 values of 4.8 μM, respectively 5.6 μM [85]. Mass spectrometry and X-ray crystallography studies of AAEK1 and SrtA interaction revealed that the inhibition mechanism is based on the elimination of the dimethylamino group and formation of the thienyl vinyl ketone that covalently binds to the enzyme Cys's thiol [85].

Pyrazolethiones and Pyridazinones
A high-throughput screening assay on Sa-SrtA∆N59 identified several lead compounds as potent SrtA inhibitors. The research was extended on commercially available structurally similar compounds or newly synthesized ones belonging to the class of pyrazolethiones and the class of pyridazinones. The pyrazolethione derivatives presented IC 50 in the range of 0.30 µM to 115 µM. The importance of the thione group was demonstrated by the decrease of the potency after its replacement with a ketone group and by the docking studies. The most active compounds ( Figure 9) were 5-methyl-2-phenyl-4-[(2,4,6tribromoanilino)methylene]pyrazole-3-thione (IC 50 = 0.30 µM) and 5-methyl-2-phenyl-4-[(2-pyridylamino)methylene]pyrazole-3-thione (IC 50 = 0.76 µM), both with a low effect on S. aureus growth at the tested concentration of 500 µM [86].

Benzisothiazolinones
A high-throughput screening assay on Sa-SrtA∆N59 identified several lead compounds as potent SrtA inhibitors. N-(adamantan-1-yl)-2-(3-oxobenzo[d]isothiazol-2(3H)yl)acetamide ( Figure 9) was identified as a lead molecule that irreversibly inhibited SrtA (IC 50 = 6.11 µM) by forming a disulphide bond with the Cys184 residue. Based on its structure, a series of related derivatives were synthesized and tested. The compounds presented IC 50 values in the range of 3.39 µM to 7.06 µM and MIC values measured against S. aureus ranging from 20.02 µM to 163.91 µM. The majority of the derivatives had significant cytotoxic effects on mouse embryo fibroblast cell line NIH 3T3, limiting their therapeutically development [88].

Derivatives of 2-Phenyl-Benzoxazole and 2-Phenyl-Benzofuran
A series of 2-phenyl-benzo[d]oxazole-7-carboxamide derivatives were designed to mimic the shape and bonding properties of the characteristic SrtA's motif LPXTG. The compounds presented IC 50 values on Sa-SrtA∆N24 in the range of 19.8 µM to 184.2 µM. The nature of the substitution on the para position of the phenyl fragment has a great influence on the inhibitory activity, the best results being observed for a hydroxyl group esterified with various phenolic acids. The N-alkyl-benzoxazole-7-carboxamide scaffold functions as a structural analog of the L-leucyl-L-prolyl fragment of SrtA's substrates and its importance was demonstrated by the low inhibitory effects of the compounds with no substituent in 7-position (IC 50 > 200 µM) [89].
Based on the structure of the 2-phenyl-benzoxazole-7-carboxamide derivatives, a series of N-alkyl-2-phenyl-benzofuran-3-carboxamide derivatives (Figure 9) were synthesized and tested on Sa-SrtA registering IC 50 values from 30.8 µM up to over 200 µM. Similar to the related benzoxazole derivatives, the best inhibitory effects were observed for the N-ibutyl-carboxamide derivatives and for those containing a dihydroxybenzoate moiety [90]. There are no data on the bacteria growth impact of these benzoxazole and benzofuran derivatives in order to evaluate their potential development as anti-virulence solutions.

Thiadiazoles Derivatives. Triazolothiadiazoles Derivatives
The compounds of this class share a common 1,3,4-thiadiazole central scaffold and an "L" geometry that is similar to the 2-phenyl-benzoxazole-7-carboxamide derivatives and their analogues 2-phenyl-benzofuran-3-carboxamide derivatives. These similarities are not necessary reflected in a common mechanism of SrtA inhibition.

1,2,4-Thiadiazolidine-3,5-Dione Derivatives
A screening for Sa-SrtA inhibitors on a collection of close to 2400 clinical drugs and candidates led to the identification of tideglusib (IC 50 = 0.6 µM), an irreversible non-ATP-competitive glycogen synthase kinase 3β (GSK-3β) inhibitor [92]. Interestingly, the proposed GSK-3β inhibition mechanism, even if not unequivocally demonstrated, involves the enzyme's Cys199 residue [93] and it could be similar in the SrtA case. Tideglusib minimally inhibited the growth of the S. aureus Newman strain. A series of thiadiazolidinedione derivatives were synthetized and tested to identify relevant structure activity relationships and to improve the anti-virulence profile. The replacement of the 2-naphtyl group with an ethyl, or benzyl, or phenethyl decreased the inhibitory activity on Sa-SrtA and considerably enhanced the antibacterial effects. The substitution of the naphtyl with a 3,5-dimethylisoxazole ring retained the SrtA inhibition, coupled with a better water solubility. The treatment with tideglusib in a dose of 40 mg/kg/day of BALB/c mice infected with S. aureus USA300 improved the 10-days survival rate to 40%, as to 10% in non-treated animals [92].

Various Structures
A number of Srt inhibitors have unique chemical scaffolds and are difficult to enclose to a certain class. Here are presented some relevant compounds with diverse structures (Figure 10). [25,57]. The compound demonstrated reduced antimicrobial activity against S. aureus with MIC values of 693 μM. Sitosterol was found to be inactive on SrtA and also on bacterial cell growth, indicating the importance of the glucoside moiety [25]. Aspermytin A, a polyketide isolated from the culture of the marine-derived fungus Aspergillus sp. F452 inhibited Sa-SrtA (IC50 = 146 μM) without a significant activity against S. aureus ATCC6538p [81]. Erianin, a natural dibenzyl compound, inhibited Sa-SrtA (IC50 = 65.7 μM) lacking any antibacterial effect on S. aureus (MIC ~ 1600 μM). The S. aureus-inoculated mice treated with 50 mg/Kg erianin administered three times a day for a period of 3 days had a 9-days survival rate of 30%, compared with 0% in the non-treated animals [95]. Halisulfate 1 was isolated from the sponge Coscinoderma sp. (Fam. Spongiidae) and demonstrated potent inhibitory effects on Sa-SrtA (IC50 = 36 μM), but also strong antibacterial effects on S. aureus [96].

Conclusions
A large diversity of Srt inhibitors is known. Generally, they are classified depending on their source of origin: natural or synthetic. The focus of this review was to identify and classify Srt inhibitors based on their structural features, significant for enzymatic inhibition. The IC50 values are presented as an indicator of each compound's antivirulence potential coupled, when available, with MIC values as a measure of the resistance risks. We classified the inhibitors based on their mechanistic relevant scaffolds: β-Sitosterol-3-O-glucopyranoside has been isolated from the bulbs of Fritillaria verticillata as Sa-SrtA inhibitor (IC 50 = 31.7 µM), but with little activity against Sa-SrtB [25,57]. The compound demonstrated reduced antimicrobial activity against S. aureus with MIC values of 693 µM. Sitosterol was found to be inactive on SrtA and also on bacterial cell growth, indicating the importance of the glucoside moiety [25]. Aspermytin A, a polyketide isolated from the culture of the marine-derived fungus Aspergillus sp. F452 inhibited Sa-SrtA (IC 50 = 146 µM) without a significant activity against S. aureus ATCC6538p [81]. Erianin, a natural dibenzyl compound, inhibited Sa-SrtA (IC 50 = 65.7 µM) lacking any antibacterial effect on S. aureus (MIC~1600 µM). The S. aureus-inoculated mice treated with 50 mg/Kg erianin administered three times a day for a period of 3 days had a 9-days survival rate of 30%, compared with 0% in the non-treated animals [95]. Halisulfate 1 was isolated from the sponge Coscinoderma sp. (Fam. Spongiidae) and demonstrated potent inhibitory effects on Sa-SrtA (IC 50 = 36 µM), but also strong antibacterial effects on S. aureus [96].

Conclusions
A large diversity of Srt inhibitors is known. Generally, they are classified depending on their source of origin: natural or synthetic. The focus of this review was to identify and classify Srt inhibitors based on their structural features, significant for enzymatic inhibition. The IC 50 values are presented as an indicator of each compound's anti-virulence potential coupled, when available, with MIC values as a measure of the resistance risks. We classified the inhibitors based on their mechanistic relevant scaffolds: vinyl sulfones, 3-aryl acrylic acids and derivatives, flavonoids, naphtoquinones, anthraquinones, indoles, pyrrolomycins, isoquinoline derivatives, aryl β-aminoethyl ketones, pyrazolethiones, pyridazinones, benzisothiazolinones, 2-phenyl-benzoxazole and 2-phenyl-benzofuran derivatives, thiadiazoles, triazolothiadiazoles, 2-(2-phenylhydrazinylidene)alkanoic acids, and 1,2,4-thiadiazolidine-3,5-dione. We highlighted the structure-activity relationships of the inhibitors using IC 50 , when possible, for assessing the effect of each compound on a specific SrtA.
In a time of emerging bacterial resistance, sortases represent promising molecular targets for developing new antibiotics: a Srt inhibitor possesses a clear potential for being used as an alternative or complementary treatment of Gram-positive bacterial infections. Even if no candidates were promoted to clinical testing, this strategy could lead to the extension of the existing clinical antibacterial pipeline. We consider that an essential first step consists in having extensive knowledge on the already existing classes of inhibitors and understanding how certain structural features impact their efficacy as enzymatic inhibitors-efficacy expressed through IC 50 's value. The purpose of this review was to offer such information, in a concise and accurate manner, as a useful tool for developing selective and effective Srt inhibitors.

Data Availability Statement:
No new data were created or analyzed in this study. Data sharing is not applicable to this article.

Conflicts of Interest:
The authors declare no conflict of interest.