Target For Antimicrobial Drug Development Research Articles

A novel antibiotic class targeting the lipopolysaccharide transporter

Carbapenem-resistant Acinetobacter baumannii (CRAB) has emerged as a major global pathogen with limited treatment options1. No new antibiotic chemical class with activity against A. baumannii has reached patients in over 50 years1. Here we report the identification and optimization of tethered macrocyclic peptide (MCP) antibiotics with potent antibacterial activity against CRAB. The mechanism of action of this molecule class involves blocking the transport of bacterial lipopolysaccharide from the inner membrane to its destination on the outer membrane, through inhibition of the LptB2FGC complex. A clinical candidate derived from the MCP class, zosurabalpin (RG6006), effectively treats highly drug-resistant contemporary isolates of CRAB both in vitro and in mouse models of infection, overcoming existing antibiotic resistance mechanisms. This chemical class represents a promising treatment paradigm for patients with invasive infections due to CRAB, for whom current treatment options are inadequate, and additionally identifies LptB2FGC as a tractable target for antimicrobial drug development.

Development of Gallium(III) as an Antimicrobial Drug Targeting Pathophysiologic Iron Metabolism of Human Pathogens.

The treatment of infections is becoming more difficult due to emerging resistance of pathogens to existing drugs. As such, alternative druggable targets, particularly those that are essential for microbe viability and thus make it harder to develop resistance, are desperately needed. In turn, once identified, safe and effective agents that disrupt these targets must be developed. Microbial acquisition and use of iron is a promising novel target for antimicrobial drug development. In this Review we look at the various facets of iron metabolism critical to human infection with pathogenic microbes and the various ways in which it can be targeted, altered, disrupted, and taken advantage of to halt or eliminate microbial infections. Although a variety of agents will be touched upon, the primary focus will be on the potential use of one or more gallium complexes as a new class of antimicrobial agents. In vitro and in vivo data on the activity of gallium complexes against a variety of pathogens including ESKAPE pathogens, mycobacteria, emerging viruses, and fungi will be discussed in detail, as well as pharmacokinetics, novel formulations and delivery approaches, and early human clinical results.

Computational Modelling, Functional Characterization and Molecular Docking to Lead Compounds of Bordetella pertussis Diaminopimelate Epimerase.

Bordetella pertussis, the causative agent of whooping cough, is an opportunistic virulent bacterial pathogen that is resistant to a wide range of antibiotics due to a variety of resistance mechanisms. Looking at the increasing number of infections caused by B. pertussis and its resistance to diverse antibiotics, it is essential to develop alternative strategies to fight against B. pertussis. Diaminopimelate epimerase (DapF) is an important enzyme of the lysine biosynthesis pathway in B. pertussis that catalyzes the formation of meso-2, 6-diaminoheptanedioate (meso-DAP), which is an important step in lysine metabolism. Therefore, Bordetella pertussis diaminopimelate epimerase (DapF) becomes an ideal target for antimicrobial drug development. In the present study, computational modelling, functional characterization, binding studies, and docking studies of BpDapF with lead compounds were carried out using different in silico tools. In silico prediction results in the secondary structure, 3-D structure analysis, and protein-protein interaction analysis of BpDapF. Docking studies further showed the respective amino acid residues for ligands in the phosphate‑binding loop of BpDapF play a vital role in the formation of H‑bonds with these ligands. The site where the ligand was bound is a deep groove, which is regarded as the binding cavity of the protein. Biochemical studies indicated that Limonin (binding energy - 8.8kcal/mol), Ajmalicine (binding energy - 8.7kcal/mol), Clinafloxacin (binding energy - 8.3kcal/mol), Dexamethasone (binding energy - 8.2kcal/mol), and Tetracycline (binding energy - 8.1kcal/mol) exhibited promising binding towards the drug target DapF of B. pertussis in comparison with the binding between other drugs and act as the potential inhibitors of BpDapF that eventually can reduce the catalytic activity of BpDapF.

2nd Workshop in Microbial Molecular Biology

2nd Workshop in Microbial Molecular Biology

Screening for small molecule inhibitors of SAH nucleosidase using an SAH riboswitch

Due to the emergence of multidrug resistant pathogens, it is imperative to identify new targets for antibiotic drug discovery. The S-adenosylhomocysteine (SAH) nucleosidase enzyme is a promising target for antimicrobial drug development due to its critical functions in multiple bacterial processes including recycling of toxic byproducts of S-adenosylmethionine (SAM)-mediated reactions and producing the precursor of the universal quorum sensing signal, autoinducer-2 (AI-2). Riboswitches are structured RNA elements typically used by bacteria to precisely monitor and respond to changes in essential bacterial processes, including metabolism. Natural riboswitches fused to a reporter gene can be exploited to detect changes in metabolism or in physiological signaling. We performed a high-throughput screen (HTS) using an SAH-riboswitch controlled β-galactosidase reporter gene in Escherichia coli to discover small molecules that inhibit SAH recycling. We demonstrate that the assay strategy using SAH riboswitches to detect the effects of SAH nucleosidase inhibitors can quickly identify compounds that penetrate the barriers of Gram-negative bacterial cells and perturb pathways involving SAH.

The Anti-Fungal Activity of Nitropropenyl Benzodioxole (NPBD), a Redox-Thiol Oxidant and Tyrosine Phosphatase Inhibitor.

Phylogenetically diverse fungal species are an increasing cause of severe disease and mortality. Identification of new targets and development of new fungicidal drugs are required to augment the effectiveness of current chemotherapy and counter increasing resistance in pathogens. Nitroalkenyl benzene derivatives are thiol oxidants and inhibitors of cysteine-based molecules, which show broad biological activity against microorganisms. Nitropropenyl benzodioxole (NPBD), one of the most active antimicrobial derivatives, shows high activity in MIC assays for phylogenetically diverse saprophytic, commensal and parasitic fungi. NPBD was fungicidal to all species except the dermatophytic fungi, with an activity profile comparable to that of Amphotericin B and Miconazole. NPBD showed differing patterns of dynamic kill rates under different growth conditions for Candida albicans and Aspergillus fumigatus and was rapidly fungicidal for non-replicating vegetative forms and microconidia. It did not induce resistant or drug tolerant strains in major pathogens on long term exposure. A literature review highlights the complexity and interactivity of fungal tyrosine phosphate and redox signaling pathways, their differing metabolic effects in fungal species and identifies some targets for inhibition. A comparison of the metabolic activities of Amphotericin B, Miconazole and NPBD highlights the multiple cellular functions of these agents and the complementarity of many mechanisms. The activity profile of NPBD illustrates the functional diversity of fungal tyrosine phosphatases and thiol-based redox active molecules and contributes to the validation of tyrosine phosphatases and redox thiol molecules as related and complementary selective targets for antimicrobial drug development. NPBD is a selective antifungal agent with low oral toxicity which would be suitable for local treatment of skin and mucosal infections.

Structural characterization of aspartate-semialdehyde dehydrogenase from Pseudomonas aeruginosa and Neisseria gonorrhoeae

Gonorrhoea infection rates and the risk of infection from opportunistic pathogens including P. aeruginosa have both risen globally, in part due to increasing broad-spectrum antibiotic resistance. Development of new antimicrobial drugs is necessary and urgent to counter infections from drug resistant bacteria. Aspartate-semialdehyde dehydrogenase (ASADH) is a key enzyme in the aspartate biosynthetic pathway, which is critical for amino acid and metabolite biosynthesis in most microorganisms including important human pathogens. Here we present the first structures of two ASADH proteins from N. gonorrhoeae and P. aeruginosa solved by X-ray crystallography. These high-resolution structures present an ideal platform for in silico drug design, offering potential targets for antimicrobial drug development as emerging multidrug resistant strains of bacteria become more prevalent.

Pantothenate and CoA biosynthesis in Apicomplexa and their promise as antiparasitic drug targets.

The Apicomplexa phylum comprises thousands of distinct intracellular parasite species, including coccidians, haemosporidians, piroplasms, and cryptosporidia. These parasites are characterized by complex and divergent life cycles occupying a variety of host niches. Consequently, they exhibit distinct adaptations to the differences in nutritional availabilities, either relying on biosynthetic pathways or by salvaging metabolites from their host. Pantothenate (Pan, vitamin B5) is the precursor for the synthesis of an essential cofactor, coenzyme A (CoA), but among the apicomplexans, only the coccidian subgroup has the ability to synthesize Pan. While the pathway to synthesize CoA from Pan is largely conserved across all branches of life, there are differences in the redundancy of enzymes and possible alternative pathways to generate CoA from Pan. Impeding the scavenge of Pan and synthesis of Pan and CoA have been long recognized as potential targets for antimicrobial drug development, but in order to fully exploit these critical pathways, it is important to understand such differences. Recently, a potent class of pantothenamides (PanAms), Pan analogs, which target CoA-utilizing enzymes, has entered antimalarial preclinical development. The potential of PanAms to target multiple downstream pathways make them a promising compound class as broad antiparasitic drugs against other apicomplexans. In this review, we summarize the recent advances in understanding the Pan and CoA biosynthesis pathways, and the suitability of these pathways as drug targets in Apicomplexa, with a particular focus on the cyst-forming coccidian, Toxoplasma gondii, and the haemosporidian, Plasmodium falciparum.

Pharmacophore-Based Virtual Screening and Molecular Dynamics Simulation for Identification of a Novel DNA Gyrase B Inhibitor with Benzoxazine Acetamide Scaffold.

DNA gyrase B is one of the enzyme targets for antimicrobial drug development, and its absence in mammals makes it a suitable target for the creation of safe antibacterial drugs. We identified six novel hits as DNA gyrase B inhibitors in the present study by employing 3D-pharmacophore structure-based virtual screening. The lead compounds complied with drug-likeness rules and lacked toxicity. Compound 4 (ZINC32858011) showed the highest inhibitory activity with an IC50 value of 6.3 ± 0.1 μM against the DNA gyrase enzyme. In contrast, the positive controls ciprofloxacin and novobiocin used in enzyme inhibition assay had IC50 values of 14.4 ± 0.2 and 12.4 ± 0.2 μM, respectively. The molecular docking of the six hits demonstrated that compounds 1, 2, 4, and 6 had suitable fitting modes inside the binding pocket. Molecular dynamics simulations were carried out for the six hits and the rmsd, rmsf, radius of gyration, and solvent accessible surface area parameters obtained from 100 ns molecular dynamics simulations for the six compounds complexed with a DNA gyrase B protein indicated that compound 4 (ZINC32858011) formed the most stable complex with DNA gyrase B. The binding free energy calculation with the MM-PBSA method suggested that the van der Waals interaction, followed by electrostatic force, played a significant role in the binding. Per-residue free binding energy decomposition showed that Ile78 contributed the most for the binding energy followed by Asn46, Asp49, Glu50, Asp73, Ile78, Pro79, Ala86, Ile90, Val120, Thr165, and Val167.

Antibacterial Profile of a Microbicidal Agent Targeting Tyrosine Phosphatases and Redox Thiols, Novel Drug Targets.

The activity profile of a protein tyrosine phosphatase (PTP) inhibitor and redox thiol oxidant, nitropropenyl benzodioxole (NPBD), was investigated across a broad range of bacterial species. In vitro assays assessed inhibitory and lethal activity patterns, the induction of drug variants on long term exposure, the inhibitory interactions of NPBD with antibiotics, and the effect of plasma proteins and redox thiols on activity. A literature review indicates the complexity of PTP and redox signaling and suggests likely metabolic targets. NPBD was broadly bactericidal to pathogens of the skin, respiratory, urogenital and intestinal tracts. It was effective against antibiotic resistant strains and slowly replicating and dormant cells. NPBD did not induce resistant or drug-tolerant phenotypes and showed low cross reactivity with antibiotics in synergy assays. Binding to plasma proteins indicated lowered in-vitro bioavailability and reduction of bactericidal activity in the presence of thiols confirmed the contribution of thiol oxidation and oxidative stress to lethality. This report presents a broad evaluation of the antibacterial effect of PTP inhibition and redox thiol oxidation, illustrates the functional diversity of bacterial PTPs and redox thiols, and supports their consideration as novel targets for antimicrobial drug development. NPBD is a dual mechanism agent with an activity profile which supports consideration of tyrosine phosphatases and bacterial antioxidant systems as promising targets for drug development.

Dual Transcriptomic Analyses Unveil Host-Pathogen Interactions Between Salmonella enterica Serovar Enteritidis and Laying Ducks (Anas platyrhynchos).

Salmonella enteritidis (SE) is a pathogen that can readily infect ovarian tissues and colonize the granulosa cell layer such that it can be transmitted via eggs from infected poultry to humans in whom it can cause food poisoning. Ducks are an important egg-laying species that are susceptible to SE infection, yet the host–pathogen interactions between SE and ducks have not been thoroughly studied to date. Herein, we performed dual RNA-sequencing analyses of these two organisms in a time-resolved infection model of duck granulosa cells (dGCs) by SE. In total, 10,510 genes were significantly differentially expressed in host dGCs, and 265 genes were differentially expressed in SE over the course of infection. These differentially expressed genes (DEGs) of dGCs were enriched in the cytokine–cytokine receptor interaction pathway via KEGG analyses, and the DEGs in SE were enriched in the two-component system, bacterial secretion system, and metabolism of pathogen factors pathways as determined. A subsequent weighted gene co-expression network analysis revealed that the cytokine–cytokine receptor interaction pathway is mostly enriched at 6 h post-infection (hpi). Moreover, a number of pathogenic factors identified in the pathogen–host interaction database (PHI-base) are upregulated in SE, including genes encoding the pathogenicity island/component, type III secretion, and regulators of systemic infection. Furthermore, an intracellular network associated with the regulation of SE infection in ducks was constructed, and 16 cytokine response-related dGCs DEGs (including IL15, CD40, and CCR7) and 17 pathogenesis-related factors (including sseL, ompR, and fliC) were identified, respectively. Overall, these results not only offer new insights into the mechanisms underlying host–pathogen interactions between SE and ducks, but they may also aid in the selection of potential targets for antimicrobial drug development.

DESIGN, INSILICO SCREENING, MOLECULAR DOCKING, SYNTHESIS AND BIOLOGICAL EVALUATION OF BENZO-FUSED FIVE MEMBERED NITROGEN CONTAINING HETEROCYCLE AGAINST DNA GYRASE SUBUNIT B AS POTENTIAL ANTIMICROBIAL AGENT

Because of its function in DNA replication, DNA gyrase subunit B (1KZN) is a promising target for antimicrobial drug development. There is an urgent requirement for the designing and improvement of novel antimicrobial drugs due to the rapid development of antimicrobial drug resistance. The aim of this study is to use molecular docking to design, synthesise, and identify benzo-fused five-membered nitrogen containing heterocycle against DNA gyrase subunit B (1KZN). Using an effective procedure, 2-(1H-1,2,3-Benzotriazol-1-yl)-N-substituted acetamide was synthesised based on the literature review. The antimicrobial activity of all synthesised compounds was tested against four different organisms: E. coli, P. aeruginosa, S. aureus, and Candida albicans. The compound binds to the active site of DNA gyrase subunit B (1KZN) in a docking study, indicating that it may have antimicrobial activity. The compounds BT4 and BT6 have the antimicrobial capacity, according to the findings of this report. BT3 has the ability to be an antibacterial agent for Staphylococcus aureus.

(p)ppGpp/GTP and Malonyl-CoA Modulate Staphylococcus aureus Adaptation to FASII Antibiotics and Provide a Basis for Synergistic Bi-Therapy.

Fatty acid biosynthesis (FASII) enzymes are considered valid targets for antimicrobial drug development against the human pathogen Staphylococcus aureus However, incorporation of host fatty acids confers FASII antibiotic adaptation that compromises prospective treatments. S. aureus adapts to FASII inhibitors by first entering a nonreplicative latency period, followed by outgrowth. Here, we used transcriptional fusions and direct metabolite measurements to investigate the factors that dictate the duration of latency prior to outgrowth. We show that stringent response induction leads to repression of FASII and phospholipid synthesis genes. (p)ppGpp induction inhibits synthesis of malonyl-CoA, a molecule that derepresses FapR, a key regulator of FASII and phospholipid synthesis. Anti-FASII treatment also triggers transient expression of (p)ppGpp-regulated genes during the anti-FASII latency phase, with concomitant repression of FapR regulon expression. These effects are reversed upon outgrowth. GTP depletion, a known consequence of the stringent response, also occurs during FASII latency, and is proposed as the common signal linking these responses. We next showed that anti-FASII treatment shifts malonyl-CoA distribution between its interactants FapR and FabD, toward FapR, increasing expression of the phospholipid synthesis genes plsX and plsC during outgrowth. We conclude that components of the stringent response dictate malonyl-CoA availability in S. aureus FASII regulation, and contribute to latency prior to anti-FASII-adapted outgrowth. A combinatory approach, coupling a (p)ppGpp inducer and an anti-FASII, blocks S. aureus outgrowth, opening perspectives for bi-therapy treatment.IMPORTANCEStaphylococcus aureus is a major human bacterial pathogen for which new inhibitors are urgently needed. Antibiotic development has centered on the fatty acid synthesis (FASII) pathway, which provides the building blocks for bacterial membrane phospholipids. However, S. aureus overcomes FASII inhibition and adapts to anti-FASII by using exogenous fatty acids that are abundant in host environments. This adaptation mechanism comprises a transient latency period followed by bacterial outgrowth. Here, we use metabolite sensors and promoter reporters to show that responses to stringent conditions and to FASII inhibition intersect, in that both involve GTP and malonyl-CoA. These two signaling molecules contribute to modulating the duration of latency prior to S. aureus adaptation outgrowth. We exploit these novel findings to propose a bi-therapy treatment against staphylococcal infections.

New Spins on Old Drugs: Enhancing Activity of Antifungals

In this issue of Cell Chemical Biology, Caplan etal. (2020) describe a series of studies in the human fungal pathogen Candida albicans to identify a new target for antimicrobial drug development. Beginning with an unbiased compound screen, they identify new mechanisms to address rising resistance to currently used anti-infective agents.

Binding of metronidazole to Enterococcus faecalis homoserine kinase: Binding studies, docking studies, and molecular dynamics simulation studies

Background: Enterococcus faecalis (Ef) is an opportunistic virulent bacterial pathogen resistant to a diverse class of antibiotics by possession of a wide range of resistance mechanisms. Aim and Objective: Looking at the increasing number of infections caused by Ef, it is essential to develop alternative strategies to fight against Ef. In this regard, homoserine kinase (HSK) is an important enzyme of threonine biosynthesis pathway in Ef. Threonine is an essential amino acid, and HSK catalyzes the formation of O-phospho-L-homoserine production, which is an important step in threonine metabolism. Therefore, Enterococcus faecalis homoserine kinase (EfHSK) becomes an ideal target for antimicrobial drug development. Methodology: We report binding studies, docking studies, and molecular dynamics (MD) simulation studies of EfHSK. Fluorescence spectroscopy studies indicated the binding of metronidazole with EfHSK. Result: Docking studies further showed that amino acid residues such as Asn124 and Gly83 in the phosphate-binding loop of EfHSK play a vital role in the formation of H-bonds with the ligand metronidazole. The site where ligand was bound is a deep groove, which is regarded as the binding cavity of the protein. Docking studies were further confirmed by MD simulation studies. Conclusion: Metronidazole might be considered as a potential inhibitor of EfHSK, as this occupies the binding pocket and eventually can reduce the kinase activity of this enzyme.

Identification of potential antivirulence agents by substitution-oriented screening for inhibitors of Streptococcus pyogenes sortase A

Antimicrobial resistance resulting in ineffective treatment of infectious diseases is an increasing global problem, particularly in infections with pathogenic bacteria. In some bacteria, such as Streptococcus pyogenes, the pathogenicity is strongly linked to the attachment of virulence factors. Their attachment to the cellular membrane is a transpeptidation reaction, catalyzed by sortase enzymes. As such, sortases pose an interesting target for the development of new antivirulence strategies that could yield novel antimicrobial drugs. Using the substitution-oriented fragment screening (SOS) approach, we discovered a potent and specific inhibitor (C10) of sortase A from S. pyogenes. The inhibitor C10 showed high specificity towards S. pyogenes sortase A, with an IC50 value of 10 μM and a Kd of 60 μM. We envision that this inhibitor could be employed as a starting point for further exploration of sortase's potential as therapeutic target for antimicrobial drug development.

Lipoteichoic acid deficiency permits normal growth but impairs virulence of Streptococcus pneumoniae

Teichoic acid (TA), a crucial cell wall constituent of the pathobiont Streptococcus pneumoniae, is bound to peptidoglycan (wall teichoic acid, WTA) or to membrane glycolipids (lipoteichoic acid, LTA). Both TA polymers share a common precursor synthesis pathway, but differ in the final transfer of the TA chain to either peptidoglycan or a glycolipid. Here, we show that LTA exhibits a different linkage conformation compared to WTA, and identify TacL (previously known as RafX) as a putative lipoteichoic acid ligase required for LTA assembly. Pneumococcal mutants deficient in TacL lack LTA and show attenuated virulence in mouse models of acute pneumonia and systemic infections, although they grow normally in culture. Hence, LTA is important for S. pneumoniae to establish systemic infections, and TacL represents a potential target for antimicrobial drug development.

Alanine racemase is essential for the growth and interspecies competitiveness of Streptococcus mutans.

D-alanine (D-Ala) is an essential amino acid that has a key role in bacterial cell wall synthesis. Alanine racemase (Alr) is a unique enzyme that interconverts L-alanine and D-alanine in most bacteria, making this enzyme a potential target for antimicrobial drug development. Streptococcus mutans is a major causative factor of dental caries. The factors involved in the survival, virulence and interspecies interactions of S. mutans could be exploited as potential targets for caries control. The current study aimed to investigate the physiological role of Alr in S. mutans. We constructed alr mutant strain of S. mutans and evaluated its phenotypic traits and interspecies competitiveness compared with the wild-type strain. We found that alr deletion was lethal to S. mutans. A minimal supplement of D-Ala (150 μg·mL−1) was required for the optimal growth of the alr mutant. The depletion of D-alanine in the growth medium resulted in cell wall perforation and cell lysis in the alr mutant strain. We also determined the compromised competitiveness of the alr mutant strain relative to the wild-type S. mutans against other oral streptococci (S. sanguinis or S. gordonii), demonstrated using either conditioned medium assays or dual-species fluorescent in situ hybridization analysis. Given the importance and necessity of alr to the growth and competitiveness of S. mutans, Alr may represent a promising target to modulate the cariogenicity of oral biofilms and to benefit the management of dental caries.

β-Hydroxyacyl-acyl Carrier Protein Dehydratase (FabZ) from Francisella tularensis and Yersinia pestis: Structure Determination, Enzymatic Characterization, and Cross-Inhibition Studies.

The bacterial system for fatty acid biosynthesis (FAS) contains several enzymes whose sequence and structure are highly conserved across a vast array of pathogens. This, coupled with their low homology and difference in organization compared to the equivalent system in humans, makes the FAS pathway an excellent target for antimicrobial drug development. To this end, we have cloned, expressed, and purified the β-hydroxyacyl-acyl carrier protein dehydratase (FabZ) from both Francisella tularensis (FtFabZ) and Yersinia pestis (YpFabZ). We also solved the crystal structures and performed an enzymatic characterization of both enzymes and several mutant forms of YpFabZ. Additionally, we have discovered two novel inhibitors of FabZ, mangostin and stictic acid, which show similar potencies against both YpFabZ and FtFabZ. Lastly, we selected several compounds from the literature that have been shown to be active against single homologues of FabZ and tested them against both YpFabZ and FtFabZ. These results have revealed clues as to which scaffolds are likely to lead to broad-spectrum antimicrobials targeted against FabZ as well as modifications to existing FabZ inhibitors that may improve potency.

Crystallographic and kinetic study on riboflavin synthase from Brucella abortus

Riboflavin synthase (RS) catalyzes the last step of riboflavin biosynthesis in microorganisms and plants, which corresponds to the dismutation of two molecules of 6,7-dimethyl-8-ribityllumazine to yield one molecule of riboflavin and one molecule of 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione [1]. Due to the absence of this enzyme in animals and the fact that most pathogenic bacteria show a strict dependence on riboflavin biosynthesis, RS has been proposed as a potential target for antimicrobial drug development. Eubacterial, fungal and plant RSs assemble as homotrimers lacking C3-symmetry [2]. Every monomer can bind two substrate molecules, yet there is only one active site for the whole enzyme, which is located at the interface between two neighboring chains. This work reports the crystallographic structure of RS from the pathogenic bacterium Brucella abortus (the etiological agent of the disease brucellosis) in its apo form, in complex with riboflavin, and in complex with two different product analogues, being the first time in which an intact RS trimer is solved with bound ligands. These crystal models support the hypothesis for an enhanced flexibility in the particle and also highlight the role of the ligands in assembling the unique active site. Kinetic and binding studies were also performed to complement these findings. The structural and biochemical information generated may be useful for the rational design of novel RS inhibitors with antimicrobial activity.