The Mechanism of Inhibition of Mycobacterial (p)ppGpp Synthetases by a Synthetic Analog of Erogorgiaene.

The synthesis of (p)ppGpp alarmones plays a vital role in the regulation of metabolism suppression, growth rate control, virulence, bacterial persistence, and biofilm formation. The (p)ppGpp alarmones are synthesized by proteins of the RelA/SpoT homolog (RSH) superfamily, including long bifunctional RSH proteins and small alarmone synthetases. Here, we investigated enzyme kinetics and dose-dependent enzyme inhibition to elucidate the mechanism of 4-(4,7-dimethyl-1,2,3,4-tetrahydronaphthalen-1-yl)pentanoic acid (DMNP) action on the (p)ppGpp synthetases RelMsm and RelZ from Mycolicibacterium smegmatis and RelMtb from Mycobacterium tuberculosis. DMNP was found to inhibit the activity of RelMtb. According to the enzyme kinetics analysis, DMNP acts as a noncompetitive inhibitor of RelMsm and RelZ. Based on the results of molecular docking, the DMNP-binding site is located in the proximity of the synthetase domain active site. This study might help in the development of alarmone synthetase inhibitors, which includes relacin and its derivatives, as well as DMNP - a synthetic analog of the marine coral metabolite erogorgiaene. Unlike conventional antibiotics, alarmone synthetase inhibitors target metabolic pathways linked to the bacterial stringent response. Although these pathways are not essential for bacteria, they regulate the development of adaptation mechanisms. Combining conventional antibiotics that target actively growing cells with compounds that impede bacterial adaptation may address challenges associated with antimicrobial resistance and bacterial persistence.

A Small-Molecule Inhibitor of the Anthranilyl-CoA Synthetase PqsA for the Treatment of Multidrug-Resistant Pseudomonas aeruginosa.

One of the challenges associated with the treatment of Pseudomonas aeruginosa infections is the high prevalence of multidrug resistance (MDR). Since conventional antibiotics are ineffective at treating such bacterial infections, innovative antibiotics acting upon novel targets or via mechanisms are urgently required. In this study, we identified a quorum sensing inhibitor (QSI), norharmane, that uniquely shows weak antibacterial activity but strongly inhibits pyocyanin production and biofilm formation of MDR P. aeruginosa. Biophysical experiments and molecular docking studies showed that norharmane competes with anthraniloyl-AMP for anthranilyl-CoA synthetase PqsA of P. aeruginosa at the ligand-binding pocket, which is not exploited by current inhibitors, thereby altering transcription regulatory activity. Moreover, norharmane exhibits synergy with polymyxin B. This synergism exhibits a high killing rate, low probability of resistance selection, and minimal cytotoxicity. Notably, norharmane can effectively boost polymyxin B activity against MDR P. aeruginosa-associated infections in animal models. Together, our findings provide novel insight critical to the design of improved PqsA inhibitors, and an effective combination strategy to overcome multiantibiotic bacterial resistance using conventional antibiotics and QSIs. IMPORTANCE Pseudomonas aeruginosa is a dominant hospital-acquired bacterial pathogen typically found in immunocompromised individuals. It is particularly dangerous for patients with chronic lung diseases and was identified as a serious threat for patients in the 2019 Antimicrobial Resistance Threats report (https://www.cdc.gov/drugresistance/biggest-threats.html). In this study, we used activity-based high-throughput screening to identify norharmane, a potent and selective inhibitor of P. aeruginosa PqsA, which is a well-conserved master quorum sensing (QS) regulator in multidrug resistant (MDR) P. aeruginosa. This compound competitively binds anthranilyl-CoA synthetase PqsA at the anthraniloyl-AMP binding domain, which has not been exploited by known inhibitors. Remarkably, norharmane can significantly block the production of the virulence factor, pyocyanin (87%), and biofilm formation (80%) in MDR P. aeruginosa. Furthermore, norharmane is capable of augmenting polymyxin B activity against MDR P. aeruginosa in cell cultures and animal models. Taken together, these results suggest that norharmane may be an effective adjuvant for combating multiantibiotic bacterial resistance.

A synthetic diterpene analogue inhibits mycobacterial persistence and biofilm formation by targeting (p)ppGpp synthetases.

Bacterial persistence coupled with biofilm formation is directly associated with failure of antibiotic treatment of tuberculosis. We have now identified 4-(4,7-DiMethyl-1,2,3,4-tetrahydroNaphthalene-1-yl)Pentanoic acid (DMNP), a synthetic diterpene analogue, as a lead compound that was capable of suppressing persistence and eradicating biofilms in Mycobacterium smegmatis. By using two reciprocal experimental approaches - ΔrelMsm and ΔrelZ gene knockout mutations versus relMsm and relZ overexpression technique - we showed that both RelMsm and RelZ (p)ppGpp synthetases are plausible candidates for serving as targets for DMNP. In vitro, DMNP inhibited (p)ppGpp-synthesizing activity of purified RelMsm in a concentration-dependent manner. These findings, supplemented by molecular docking simulation, suggest that DMNP targets the structural sites shared by RelMsm, RelZ, and presumably by a few others as yet unidentified (p)ppGpp producers, thereby inhibiting persister cell formation and eradicating biofilms. Therefore, DMNP may serve as a promising lead for development of antimycobacterial drugs.

Design, synthesis, and evaluation of transition-state analogs as inhibitors of the bacterial quorum sensing autoinducer synthase CepI.

Quorum sensing is a bacterial signaling system that involves the synthesis, secretion and detection of signal molecules called autoinducers. The main autoinducer in Gram-negative bacteria are acylated homoserine lactones, produced by the LuxI family of autoinducer synthases and detected by the LuxR family of autoinducer receptors. Quorum sensing allows for changes in gene expression and bacterial behaviors in a coordinated, cell density dependent manner. Quorum sensing controls the expression of virulence factors in some human pathogens, making quorum sensing an antibacterial drug target. Here we describe the design and synthesis of transition-state analogs of the autoinducer synthase enzymatic reaction and the evaluation of these compounds as inhibitors of the synthase CepI. One such compound potently inhibits CepI and constitutes a new type of inhibitor against this underdeveloped antibacterial target.

Inhibiting CBX4 efficiently protects hepatocellular carcinoma cells against sorafenib resistance.

BACKGROUND: This study aimed to investigate the possible role of inhibiting chromobox protein homologue 4 (CBX4) to deregulate of cancer stem cells (CSCs) and to evaluate the contribution of these molecules to sorafenib resistance in advanced hepatocellular carcinoma (HCC). METHODS: HCC cell lines and a xenograft mouse model with resistance to sorafenib were employed to analyse the effects of miR424 on CSC characteristics. RNA expression was analysed by RT-PCR and next-generation sequencing in a cohort of HCC cancer patients and sorafenib-resistant (SR) cell lines, respectively, to validate the key microRNAs and targets in the network. RESULTS: MicroRNA and mRNA profiles of SR cell lines identified miR424 and its direct target CBX4 as significantly associated with stem-cell-like properties, poor survival, and clinical characteristics. Functional experiments demonstrated that miR424 suppressed CBX4 and CBX4 induced nuclear translocation of YAP1 protein but was not associated with protein production. When YAP1 and CBX4 were modulated with CA3 and UNC3866, tumorigenicity and stem-like properties were extremely inhibited, thus indicating that these compounds exerted a strong anti-tumour effect in vivo against SR HCC cells. CONCLUSIONS: Our results revealed that blocking CBX4 expression is critical in response to sorafenib resistance with advanced HCC.

Structural characterization, antifungal and cytotoxic profiles of quaternized heteropolysaccharide from Anadenanthera colubrina.

In the present work, we investigated the minimal inhibitory concentration (MIC) against fungal strains (Fonsecaea pedrosoi, Microsporum canis, Candida albicans, Cryptococcus neoformans), and cytotoxicity to normal cell lines for modified red angico gum (AG) with eterifying agent N-chloride (3-chloro-2-hydroxypropyl) trimethylammonium (CHPTAC). Quaternized ammonium groups were linked to AG backbone using N-(3-chloro-2-hydroxypropyl) trimethylammonium chloride. The chemical features of the quaternized gum derivatives (QAG) were analyzed by: FTIR, elemental analysis, Zeta potential and gel permeation chromatography. The angico quaternizated gum presented a degree of substitution (DS) of 0.22 and Zeta potential of +36.43. For the antifungal test, it was observed that unmodified gum did not inhibit fungal growth. While, QAG inhibited the growth of most fungi used in this study. By AFM technique QAG interacted with the fungal surface, altering wall roughness significantly. The probable affinity of fragments of the QAG structure for the fungal enzyme 5I33 (Adenylosuccinate synthetase) has been shown by molecular docking. Low cytotoxicity was observed for polymers (unmodified gum and QAG). The results demonstrate that the quaternized polymer of AG presented in this study is a quite promising biomaterial for biotechnological applications.

Sulfonated inhibitors of the RNA editing ligases validate the essential role of the MRP1/2 proteins in kinetoplastid RNA editing.

The RNA editing core complex (RECC) catalyzes mitochondrial U-insertion/deletion mRNA editing in trypanosomatid flagellates. Some naphthalene-based sulfonated compounds, such as C35 and MrB, competitively inhibit the auto-adenylylation activity of an essential RECC enzyme, kinetoplastid RNA editing ligase 1 (KREL1), required for the final step in editing. Previous studies revealed the ability of these compounds to interfere with the interaction between the editosome and its RNA substrates, consequently affecting all catalytic activities that comprise RNA editing. This observation implicates a critical function for the affected RNA binding proteins in RNA editing. In this study, using the inhibitory compounds, we analyzed the composition and editing activities of functional editosomes and identified the mitochondrial RNA binding proteins 1 and 2 (MRP1/2) as their preferred targets. While the MRP1/2 heterotetramer complex is known to bind guide RNA and promote annealing to its cognate pre-edited mRNA, its role in RNA editing remained enigmatic. We show that the compounds affect the association between the RECC and MRP1/2 heterotetramer. Furthermore, RECC purified post-treatment with these compounds exhibit compromised in vitro RNA editing activity that, remarkably, recovers upon the addition of recombinant MRP1/2 proteins. This work provides experimental evidence that the MRP1/2 heterotetramer is required for in vitro RNA editing activity and substantiates the hypothesized role of these proteins in presenting the RNA duplex to the catalytic complex in the initial steps of RNA editing.

Targeting the CK1α/CBX4 axis for metastasis in osteosarcoma.

Osteosarcoma, an aggressive malignant cancer, has a high lung metastasis rate and lacks therapeutic target. Here, we reported that chromobox homolog 4 (CBX4) was overexpressed in osteosarcoma cell lines and tissues. CBX4 promoted metastasis by transcriptionally up-regulating Runx2 via the recruitment of GCN5 to the Runx2 promoter. The phosphorylation of CBX4 at T437 by casein kinase 1α (CK1α) facilitated its ubiquitination at both K178 and K280 and subsequent degradation by CHIP, and this phosphorylation of CBX4 could be reduced by TNFα. Consistently, CK1α suppressed cell migration and invasion through inhibition of CBX4. There was a reverse correlation between CK1α and CBX4 in osteosarcoma tissues, and CK1α was a valuable marker to predict clinical outcomes in osteosarcoma patients with metastasis. Pyrvinium pamoate (PP) as a selective activator of CK1α could inhibit osteosarcoma metastasis via the CK1α/CBX4 axis. Our findings indicate that targeting the CK1α/CBX4 axis may benefit osteosarcoma patients with metastasis.

THE CONCISE GUIDE TO PHARMACOLOGY 2019/20: Enzymes.

The Concise Guide to PHARMACOLOGY 2019/20 is the fourth in this series of biennial publications. The Concise Guide provides concise overviews of the key properties of nearly 1800 human drug targets with an emphasis on selective pharmacology (where available), plus links to the open access knowledgebase source of drug targets and their ligands (www.guidetopharmacology.org), which provides more detailed views of target and ligand properties. Although the Concise Guide represents approximately 400 pages, the material presented is substantially reduced compared to information and links presented on the website. It provides a permanent, citable, point-in-time record that will survive database updates. The full contents of this section can be found at http://onlinelibrary.wiley.com/doi/10.1111/bph.14752. Enzymes are one of the six major pharmacological targets into which the Guide is divided, with the others being: G protein-coupled receptors, ion channels, nuclear hormone receptors, catalytic receptors and transporters. These are presented with nomenclature guidance and summary information on the best available pharmacological tools, alongside key references and suggestions for further reading. The landscape format of the Concise Guide is designed to facilitate comparison of related targets from material contemporary to mid-2019, and supersedes data presented in the 2017/18, 2015/16 and 2013/14 Concise Guides and previous Guides to Receptors and Channels. It is produced in close conjunction with the International Union of Basic and Clinical Pharmacology Committee on Receptor Nomenclature and Drug Classification (NC-IUPHAR), therefore, providing official IUPHAR classification and nomenclature for human drug targets, where appropriate.

Chirality-Driven Mode of Binding of α-Aminophosphonic Acid-Based Allosteric Inhibitors of the Human Farnesyl Pyrophosphate Synthase (hFPPS).

Thienopyrimidine-based allosteric inhibitors of the human farnesyl pyrophosphate synthase (hFPPS), characterized by a chiral α-aminophosphonic acid moiety, were synthesized as enantiomerically enriched pairs, and their binding mode was investigated by X-ray crystallography. A general consensus in the binding orientation of all (R)- and (S)-enantiomers was revealed. This finding is a prerequisite for establishing a reliable structure-activity relationship (SAR) model.

N-Acyl Homoserine Lactone Analog Modulators of the Pseudomonas aeruginosa Rhll Quorum Sensing Signal Synthase.

Virulence in the Gram-negative pathogen Pseudomonas aeruginosa relies in part on the efficient functioning of two LuxI/R dependent quorum sensing (QS) cascades, namely, the LasI/R and RhlI/R systems that generate and respond to N-(3-oxo)-dodecanoyl-l-homoserine lactone and N-butyryl-l-homoserine lactone, respectively. The two acyl homoserine lactone (AHL) synthases, LasI and RhlI, use 3-oxododecanoyl-ACP and butyryl-ACP, respectively, as the acyl-substrates to generate the corresponding autoinducer signals for the bacterium. Although AHL synthases represent excellent targets for developing QS modulators in P. aeruginosa, and in other related bacteria, the identification of potent and signal synthase specific inhibitors has represented a significant technical challenge. In the current study, we sought to test the utility of AHL analogs as potential modulators of an AHL synthase and selected RhlI in P. aeruginosa as an initial target. We systematically varied the chemical functionalities of the AHL headgroup, acyl chain tail, and head-to-tail linkage to construct a small library of signal analogs and evaluated them for RhlI modulatory activity. Although the native N-butyryl-l-homoserine lactone did not inhibit RhlI, we discovered that several of our long-chain, unsubstituted acyl-d-homoserine lactones and acyl-d-homocysteine thiolactones inhibited while a few of the 3-oxoacyl-chain counterparts activated the enzyme. Additional mechanistic investigations with acyl-substrate analogs and docking experiments with AHL analogs revealed two distinct inhibitor and activator binding pockets in the enzyme. This study provides the first evidence of the yet untapped potential of AHL analogs as signal synthase modulators of QS pathways.

Stringent response protein as a potential target to intervene persistent bacterial infection.

More than half of the world's population is infected with persistent bacterial infections, consequently, persisters are gradually becoming a major public health concern. During the persistent phase, bacterial pathogens deploy many regulatory strategies to compensate unfavorable host environmental conditions. The stringent response is one of such gene regulatory mechanisms which is stimulated by nutrient starvation. It is regulated by the synthesis of highly phosphorylated signaling nucleotides, (p)ppGpp or alarmone. (p)ppGpp is synthesized by ppGpp synthetases, and these proteins are classified as RelA/SpoT homolog (RSH) proteins. Subsequently, (p)ppGpp modulate several molecular and biochemical processes ranging from transcription to metabolism. Imperativeness of (p)ppGpp synthetases has been investigated by numerous approaches including microbiology and animal studies, thereby establishing that Rel enzyme deleted strains of pathogenic bacteria were unable to transform in persister form. In this review, we summarize recent findings to corroborate the rationality to consider (p)ppGpp synthetase as a potential target in discovering a novel class of antimicrobial agents to combat persistent infections. Moreover, inhibition studies on Mycobacterium tuberculosis (p)ppGpp synthetase shows that these inhibitors prevent dormant state transition and biofilm formation. Also, we have highlighted the structural biology of (p)ppGpp synthetases, which may provide significant information that could be used in structure-based inhibitor design.

Evaluation of the published kinase inhibitor set to identify multiple inhibitors of bacterial ATP-dependent mur ligases.

The Mur ligases form a series of consecutive enzymes that participate in the intracellular steps of bacterial peptidoglycan biosynthesis. They therefore represent interesting targets for antibacterial drug discovery. MurC, D, E and F are all ATP-dependent ligases. Accordingly, with the aim being to find multiple inhibitors of these enzymes, we screened a collection of ATP-competitive kinase inhibitors, on Escherichia coli MurC, D and F, and identified five promising scaffolds that inhibited at least two of these ligases. Compounds 1, 2, 4 and 5 are multiple inhibitors of the whole MurC to MurF cascade that act in the micromolar range (IC50, 32-368 µM). NMR-assisted binding studies and steady-state kinetics studies performed on aza-stilbene derivative 1 showed, surprisingly, that it acts as a competitive inhibitor of MurD activity towards D-glutamic acid, and additionally, that its binding to the D-glutamic acid binding site is independent of the enzyme closure promoted by ATP.

Targeting adenylate-forming enzymes with designed sulfonyladenosine inhibitors.

Adenylate-forming enzymes are a mechanistic superfamily that are involved in diverse biochemical pathways. They catalyze ATP-dependent activation of carboxylic acid substrates as reactive acyl adenylate (acyl-AMP) intermediates and subsequent coupling to various nucleophiles to generate ester, thioester, and amide products. Inspired by natural products, acyl sulfonyladenosines (acyl-AMS) that mimic the tightly bound acyl-AMP reaction intermediates have been developed as potent inhibitors of adenylate-forming enzymes. This simple yet powerful inhibitor design platform has provided a wide range of biological probes as well as several therapeutic lead compounds. Herein, we provide an overview of the nine structural classes of adenylate-forming enzymes and examples of acyl-AMS inhibitors that have been developed for each.

Isatins Inhibit N5-CAIR Synthetase by a Substrate Depletion Mechanism.

The continued rise of antibiotic-resistant infections coupled with the limited pipeline of new antimicrobials highlights the pressing need for the development of new antibacterial agents. One potential pathway for new agents is de novo purine biosynthesis as studies have shown that bacteria and lower eukaryotes synthesize purines differently than humans. Microorganisms utilize two enzymes, N5-CAIR synthetase and N5-CAIR mutase, to convert 5-aminoimidazole ribonucleotide (AIR) into 4-carboxy-5-aminoimidazole ribonucleotide (CAIR) through the intermediate N5-carboxy-5-aminoimidazole ribonucleotide (N5-CAIR). In contrast, vertebrates directly convert AIR to CAIR via the enzyme AIR carboxylase. A high-throughput screen against N5-CAIR synthetase identified a group of compounds with a 2,3-indolinedione (isatin) core that inhibited the enzyme. While initial studies suggested that isatins inhibited the enzyme by a noncompetitive mechanism, here we show that isatins inhibit N5-CAIR synthetase by a substrate depletion mechanism. Unexpectedly, we found that isatin reacts rapidly and reversibly with the substrate AIR. The rate of the reaction is dependent upon the substituents on the phenyl moiety of isatin, with 5- and 7-bromoisatin being faster than 4-bromoisatin. These studies suggest that care should be taken when exploring isatin compounds because the biological activity could be a result of their reactivity.

Kinetic Analyses of the Siderophore Biosynthesis Inhibitor Salicyl-AMS and Analogues as MbtA Inhibitors and Antimycobacterial Agents.

There is a paramount need for expanding the drug armamentarium to counter the growing problem of drug-resistant tuberculosis. Salicyl-AMS, an inhibitor of salicylic acid adenylation enzymes, is a first-in-class antibacterial lead compound for the development of tuberculosis drugs targeting the biosynthesis of salicylic-acid-derived siderophores. In this study, we determined the Ki of salicyl-AMS for inhibition of the salicylic acid adenylation enzyme MbtA from Mycobacterium tuberculosis (MbtAtb), designed and synthesized two new salicyl-AMS analogues to probe structure-activity relationships (SAR), and characterized these two analogues alongside salicyl-AMS and six previously reported analogues in biochemical and cell-based studies. The biochemical studies included determination of kinetic parameters ( Kiapp, konapp, koff, and tR) and analysis of the mechanism of inhibition. For these studies, we optimized production and purification of recombinant MbtAtb, for which Km and kcat values were determined, and used the enzyme in conjunction with an MbtAtb-optimized, continuous, spectrophotometric assay for MbtA activity and inhibition. The cell-based studies provided an assessment of the antimycobacterial activity and postantibiotic effect of the nine MbtAtb inhibitors. The antimycobacterial properties were evaluated using a strain of nonpathogenic, fast-growing Mycobacterium smegmatis that was genetically engineered for MbtAtb-dependent susceptibility to MbtA inhibitors. This convenient model system greatly facilitated the cell-based studies by bypassing the methodological complexities associated with the use of pathogenic, slow-growing M. tuberculosis. Collectively, these studies provide new information on the mechanism of inhibition of MbtAtb by salicyl-AMS and eight analogues, afford new SAR insights for these inhibitors, and highlight several suitable candidates for future preclinical evaluation.

Insights into ß-ketoacyl-chain recognition for ß-ketoacyl-ACP utilizing AHL synthases.

Beta-ketoacyl-ACP utilizing enzymes in fatty acid, polyketide and acyl-homoserine lactone biosynthetic pathways are important targets for developing antimicrobial, anticancer and antiparasitic compounds. Published reports on successful isolation of beta-ketoacyl-ACPs in a laboratory remain scarce to date and thus most beta-ketoacyl-ACP utilizing enzymes are routinely characterized using small molecule substrates in lieu of the bonafide 3-oxoacyl-ACPs. We report the systematic investigation into the electronic, geometric and spatial aspects of beta-ketoacyl-chain recognition to develop 3-oxoacyl-ACP substrate mimics for two beta-ketoacyl-ACP utilizing quorum signal synthases.

Selective protein unfolding: a universal mechanism of action for the development of irreversible inhibitors.

High-throughput differential scanning fluorimetry of GFP-tagged proteins (HT-DSF-GTP) was applied for the identification of novel enzyme inhibitors acting by a mechanism termed: selective protein unfolding (SPU). Four different protein targets were interrogated with the same library to identify target-selective hits. Several hits selectively destabilized bacterial biotin protein ligase. Structure-activity relationship data confirmed a structure-dependent mechanism of protein unfolding. Simvastatin and altenusin were confirmed to irreversibly inactivate biotin protein ligase. The principle of SPU combined with HT-DSF-GTP affords an invaluable and innovative workflow for the identification of new inhibitors with potential applications as antimicrobials and other biocides.

Combination Therapy Strategy of Quorum Quenching Enzyme and Quorum Sensing Inhibitor in Suppressing Multiple Quorum Sensing Pathways of P. aeruginosa.

The threat of antibiotic resistant bacteria has called for alternative antimicrobial strategies that would mitigate the increase of classical resistance mechanism. Many bacteria employ quorum sensing (QS) to govern the production of virulence factors and formation of drug-resistant biofilms. Targeting the mechanism of QS has proven to be a functional alternative to conventional antibiotic control of infections. However, the presence of multiple QS systems in individual bacterial species poses a challenge to this approach. Quorum sensing inhibitors (QSI) and quorum quenching enzymes (QQE) have been both investigated for their QS interfering capabilities. Here, we first simulated the combination effect of QQE and QSI in blocking bacterial QS. The effect was next validated by experiments using AiiA as QQE and G1 as QSI on Pseudomonas aeruginosa LasR/I and RhlR/I QS circuits. Combination of QQE and QSI almost completely blocked the P. aeruginosa las and rhl QS systems. Our findings provide a potential chemical biology application strategy for bacterial QS disruption.

Reconstitution of Staphylococcus aureus Lipoteichoic Acid Synthase Activity Identifies Congo Red as a Selective Inhibitor.

Lipoteichoic acid (LTA) is an anionic surface polymer that is essential for normal growth of Staphylococcus aureus, making the LTA polymerase, LTA synthase (LtaS), a proposed drug target for combating Staphylococcal infections. LtaS is a polytopic membrane protein with five membrane-spanning helices and an extracellular domain, and it uses phosphatidylglycerol to assemble a glycerol phosphate chain on a glycosylated diacylglycerol membrane anchor. We report here the first reconstitution of LtaS polymerization activity and show that the azo dye Congo red inhibits this enzyme both in vitro and in cells. Related azo dyes and the previously reported LtaS inhibitor 1771 have weak or no in vitro inhibitory activity. Synthetic lethality with mutant strains known to be nonviable in the absence of LTA confirms selective inhibition by Congo red. As the only validated LtaS inhibitor, Congo red can serve as a probe to understand how inhibiting lipoteichoic acid biosynthesis affects cell physiology and may also guide the discovery of more potent inhibitors for use in treating S. aureus infections.