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N-(5-(5-nitro-2-oxo-1,2-dihydro-3H-indol-3-ylidene)4-oxo-2-thioxo-1,3-thiazolidin-3-yl)nicotinamide, a 2-oxoindolinylidene derivative with novel structure scaffold, was evaluated for inhibition potency against the MurD enzyme from Escherichia coli using an enzyme steady-state kinetics study. The compound exerted competitive inhibition with respect to UMA, a MurD substrate, and affected bacterial growth. Furthermore, we isolated and purified (13)C selectively labeled MurD enzyme from E. coli and evaluated the binding interactions of the new compound using the (1)H/(13)C-HSQC 2D NMR method. Molecular dynamics calculations showed stable structure for the MurD-inhibitor complex. The binding mode of novel inhibitor was determined and compared to naphthalene-N-sulfonamide-d-Glu derivatives, transition state mimicking inhibitors, UMA and AMP-PCP, an ATP analog. It binds to the UDP/MurNAc binding region. In contrast to transition state mimicking inhibitors, it does not interact with the enzyme's C-terminal domain, which can be beneficial for ligand binding. A pharmacophore pattern was established for the design of novel drugs having a propensity to inhibit a broad spectrum of Mur enzymes., (Copyright © 2014 Elsevier Masson SAS. All rights reserved.)

In the present study, we used crystal structure of mycobacterial pantothenate synthetase (PS) bound with 2-(2-(benzofuran-2-ylsulfonylcarbamoyl)-5-methoxy-1H-indol-1-yl) acetic acid inhibitor for virtual screening of antitubercular compound database to identify new scaffolds. One of the identified lead was modified synthetically to obtain thirty novel analogues. These synthesized compounds were evaluated for Mycobacterium tuberculosis (MTB) PS inhibition study, in vitro antimycobacterial activities and cytotoxicity against RAW 264.7 cell line. Among the compounds tested, N'-(1-naphthoyl)-2-methylimidazo[1,2-a]pyridine-3-carbohydrazide (5b) was found to be the most active compound with IC₅₀ of 1.90 ± 0.12 μM against MTB PS, MIC of 4.53 μM against MTB with no cytotoxicity at 50 μM. The binding affinity of the most potent inhibitor 5b was further confirmed biophysically through differential scanning fluorimetry., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Enzymes catalyzing the biosynthesis of bacterial peptidoglycan represent traditionally a collection of highly selective targets for novel antibacterial drug design. Four members of the bacterial Mur ligase family-MurC, MurD, MurE and MurF-are involved in the intracellular steps of peptidoglycan biosynthesis, catalyzing the synthesis of the peptide moiety of the Park's nucleotide. In our previous virtual screening campaign, a chemical class of benzene-1,3-dicarboxylic acid 2,5-dimethylpyrrole derivatives exhibiting dual MurD/MurE inhibition properties was discovered. In the present study we further investigated this class of compounds by performing inhibition assays on all four Mur ligases (MurC-MurF). Furthermore, molecular dynamics (MD) simulation studies of one of the initially discovered compound 1 were performed to explore its geometry as well as its energetic behavior based on the Linear Interaction Energy (LIE) method. Further in silico virtual screening (VS) experiments based on the parent active compound 1 were conducted to optimize the discovered series. Selected hits were assayed against all Escherichia coli MurC-MurF enzymes in biochemical inhibition assays and molecules 10-14 containing benzene-1,3-dicarboxylic acid 2,5-dimethylpyrrole coupled with five member-ring rhodanine moiety were found to be multiple inhibitors of the whole MurC-MurF cascade of bacterial enzymes in the micromolar range. Steady-state kinetics studies suggested this class to act as competitive inhibitors of the MurD enzyme towards d-Glu. These compounds represent novel valuable starting point in the development of novel antibacterial agents., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Increasing bacterial resistance to available antibiotics stimulated the discovery of novel efficacious antibacterial agents. The biosynthesis of the bacterial peptidoglycan, where the MurD enzyme is involved in the intracellular phase of the UDP-MurNAc-pentapeptide formation, represents a collection of highly selective targets for novel antibacterial drug design. In our previous computational studies, the C-terminal domain motion of the MurD ligase was investigated using Targeted Molecular Dynamic (TMD) simulation and the Off-Path Simulation (OPS) technique. In this study, we present a drug design strategy using multiple protein structures for the identification of novel MurD ligase inhibitors. Our main focus was the ATP-binding site of the MurD enzyme. In the first stage, three MurD protein conformations were selected based on the obtained OPS/TMD data as the initial criterion. Subsequently, a two-stage virtual screening approach was utilized combining derived structure-based pharmacophores with molecular docking calculations. Selected compounds were then assayed in the established enzyme binding assays, and compound 3 from the aminothiazole class was discovered to act as a dual MurC/MurD inhibitor in the micomolar range. A steady-state kinetic study was performed on the MurD enzyme to provide further information about the mechanistic aspects of its inhibition. In the final stage, all used conformations of the MurD enzyme with compound 3 were simulated in classical molecular dynamics (MD) simulations providing atomistic insights of the experimental results. Overall, the study depicts several challenges that need to be addressed when trying to hit a flexible moving target such as the presently studied bacterial MurD enzyme and show the possibilities of how computational tools can be proficiently used at all stages of the drug discovery process.

A chemical database of 30 representative imidazo-azines was built and screened against important tropical disease targets by computational docking. After three rounds of screening, an interaction profile was generated and analyzed. On the basis of binding energy and ligand efficiency, it was concluded that in general, imidazo-azine scaffold has a potential of being selective and simultaneous inhibitor against the five receptors Pf-dihydrofolate reductase, Pf-enoyl acyl carrier protein reductase, Pf-protein kinase 7, Mt-pantothenate synthetase and Mt-thymidine monophosphate kinase. Interestingly, two compounds 2-(4-chlorophenyl)-N-cyclohexyl-6-methylH-imidazo[1,2-a]pyridine-3-amine (MCL011) and N-cyclohexyl-2-(4-methoxyphenyl)-6-methylH-imidazo[1,2-a]pyridine-3-amine (MCL017) showed highest binding energy against four targets namely Pf-dihydrofolate reductase, Pf-enoyl acyl carrier protein reductase, Pf-protein kinase 7 and Mt-pantothenate synthetase. Eventually, in order to improve the decision making and success rate in actual efficacy evaluations other criteria such as lead-likeness were envisaged., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Twenty six 2,6-disubstituted 4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carboxamide derivatives were designed by molecular hybridization approach using and synthesized from piperidin-4-one by five step synthesis. Compounds were evaluated for Mycobacterium tuberculosis (MTB) pantothenate synthetase (PS) inhibition study, in vitro activities against MTB, cytotoxicity against RAW 264.7 cell line. Among the compounds, 6-(4-nitrophenylsulfonyl)-2-(5-nitrothiophene-2-carboxamido)-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carboxamide (11) was found to be the most active compound with IC50 of 5.87 ± 0.12 μM against MTB PS, inhibited MTB with MIC of 9.28 μM and it was non-cytotoxic at 50 μM. The binding affinity of the most potent inhibitor 11 was further confirmed biophysically through differential scanning fluorimetry., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The biosynthesis of pantothenate, the core of coenzyme A (CoA), has been considered an attractive target for the development of antimicrobial agents since this pathway is essential in prokaryotes, but absent in mammals. Pantothenate synthetase, encoded by the gene panC, catalyzes the final condensation of pantoic acid with β-alanine to afford pantothenate via an intermediate pantoyl adenylate. We describe the synthesis and biochemical characterization of five PanC inhibitors that mimic the intermediate pantoyl adenylate. These inhibitors are competitive inhibitors with respect to pantoic acid and possess submicromolar to micromolar inhibition constants. The observed SAR is rationalized through molecular docking studies based on the reported co-crystal structure of 1a with PanC. Finally, whole cell activity is assessed against wild-type Mtb as well as a PanC knockdown strain where PanC is depleted to less than 5% of wild-type levels., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

MurF ligase is a crucial enzyme that catalyses the ultimate intracellular step of bacterial peptidoglycan biosynthesis, and thus represents an attractive target for antibacterial drug discovery. We designed, synthesized and evaluated a new series of cyanothiophene-based inhibitors of MurF enzymes from Streptococcus pneumoniae and Escherichia coli. The target compounds had increased polarity compared to the first generation of inhibitors, with demonstrated enzyme inhibitory potencies in the low micromolar range. Furthermore, the best inhibitors displayed promising antibacterial activities against selected Gram-positive and Gram-negative strains. These results represent an important step towards the development of new antibacterial agents targeting peptidoglycan biosynthesis., (Copyright © 2013 Elsevier Masson SAS. All rights reserved.)

Recently, the search for new drugs against tuberculosis (TB) has been a hot topic and the search for new inhibitors against validated drug targets and pathways other than those currently targeted by known drugs is suggested to be the most promising way forward. Mycobacterium tuberculosis pantothenate synthetase (MTBPS) happens to be one of such targets. In a quest to carry out virtual screening for active inhibitors against MTBPS and to get ideas for the design of new inhibitors against this target, we have docked a set of pyrazole-based inhibitors to the active site of this enzyme. The docking solutions were post processed using the MM-PB(GB)SA method and molecular dynamic simulations in order to analyze and validate the two previously proposed binding modes. The results show that both the MM-PBSA and MM-GBSA were able to discriminate between active and inactive compounds. Moreover, the pharmacophore-based scoring method proved efficient in discriminating the active compounds from inactives. From this work a protocol for screening of potential inhibitors of the enzyme from commercially available databases has been devised.

Aminoimidazole carboxamide ribotide (AICAR) is a purine biosynthetic intermediate and a by-product of histidine biosynthesis. In bacteria, yeast, and humans, accumulation of AICAR has been shown to affect an array of cellular processes by both direct and indirect mechanisms. In purine biosynthesis, AICAR is the substrate of the bifunctional protein phosphoribosylaminoimidazolecarboxamide formyltransferase/IMP cyclohydrolase (PurH, EC 2.1.2.3/3.5.4.10). Strains lacking PurH accumulate AICAR and have a defect in the synthesis of the 4-amino-5-hydroxymethyl-2-methylpyrimidine (HMP) moiety of thiamine. The formation of HMP is also compromised in vivo when coenzyme A (CoA) levels are reduced. Our results show that the in vivo accumulation of AICAR decreased total CoA pools and, further, that AICAR inhibited the activity of pantoate β-alanine ligase in vitro (PanC, EC 6.3.2.1). These results demonstrated a mechanism of AICAR action and provide new insights into the metabolic consequences of disrupting purine metabolism.

Tuberculosis (TB) is an infectious disease caused by many strains of mycobacteria, but commonly Mycobacterium tuberculosis. As a possible method of reducing the drug resistance of M. tuberculosis, this research investigates the inhibition of Folylpolyglutamate synthetase, a protein transcript from the resistance association gene folC. After molecular docking to screen the traditional Chinese medicine (TCM) database, the candidate TCM compounds, with Folylpolyglutamate synthetase, were selected by molecular dynamics. The 10,000 ps simulation in association with RMSD analysis and total energy and structural variation defined the protein-ligand interaction. The selected TCM compounds Saussureamine C, methyl 3-O-feruloylquinate, and Labiatic acid have been found to inhibit the activity of bacteria and viruses and to regulate immunity. We also suggest the possible pathway in protein for each ligand. Compared with the control, similar interactions and structural variations indicate that these compounds might have an effect on Folylpolyglutamate synthetase. Finally, we suggest Saussureamine C is the best candidate compound as the complex has a high score, maintains its structural composition, and has a larger variation value than the control, thus inhibiting the drug resistance ability of Mycobacterium tuberculosis.

In light of the low success rate of target-based genomics and HTS (High Throughput Screening) approaches in anti-infective drug discovery, in silico structure-based drug design (SBDD) is becoming increasingly prominent at the forefront of drug discovery. In silico SBDD can be used to identify novel enzyme inhibitors rapidly, where the strength of this approach lies with its ability to model and predict the outcome of protein-ligand binding. Over the past 10 years, our group have applied this approach to a diverse number of anti-infective drug targets ranging from bacterial D-ala-D-ala ligase to Plasmodium falciparum DHODH. Our search for new inhibitors has produced lead compounds with both enzyme and whole-cell activity with established on-target mode of action. This has been achieved with greater speed and efficiency compared with the more traditional HTS initiatives and at significantly reduced cost and manpower.

The synthesis of the peptide stem of bacterial peptidoglycan involves four enzymes, the Mur ligases (MurC, D, E and F). Among them, MurD is responsible for the ATP-dependent addition of d-glutamic acid to UDP-MurNAc-l-Ala, a reaction which involves acyl-phosphate and tetrahedral intermediates. Like most enzymes of peptidoglycan biosynthesis, MurD constitutes an attractive target for the design and synthesis of new antibacterial agents. Escherichia coli MurD has been the first Mur ligase for which the tridimensional (3D) structure was solved. Thereafter, several co-crystal structures with different ligands or inhibitors were released. In the present review, we will deal with work performed on substrate specificity, reaction mechanism and 3D structure of E. coli MurD. Then, a part of the review will be devoted to recent work on MurD orthologs from species other than E. coli and to cellular organization of Mur ligases and in vivo regulation of the MurD activity. Finally, we will review the different classes of MurD inhibitors that have been designed and assayed to date with the hope of obtaining new antibacterial compounds.

The enzyme pantothenate synthetase, PanC, is an attractive drug target in Mycobacterium tuberculosis. It is essential for the in vitro growth of M. tuberculosis and for survival of the bacteria in the mouse model of infection. PanC is absent from mammals. We developed an enzyme-based assay to identify inhibitors of PanC, optimized it for high-throughput screening, and tested a large and diverse library of compounds for activity. Two compounds belonging to the same chemical class of 3-biphenyl-4- cyanopyrrole-2-carboxylic acids had activity against the purified recombinant protein, and also inhibited growth of live M. tuberculosis in manner consistent with PanC inhibition. Thus we have identified a new class of PanC inhibitors with whole cell activity that can be further developed.

Forty 3-phenyl-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine derivatives were synthesized from piperidin-4-one by five step synthesis and evaluated for Mycobacterium tuberculosis (MTB) pantothenate synthetase (PS) inhibition study, in vitro activities against MTB, cytotoxicity against RAW 264.7 cell line. Among the compounds, 1-benzoyl-N-(4-nitrophenyl)-3-phenyl-6,7-dihydro-1H-pyrazolo[4,3-c]pyridine-5(4H)-carboxamide (6ac) was found to be the most active compound with IC₅₀ of 21.8 ± 0.8 μM against MTB PS, inhibited MTB with MIC of 26.7 μM and it was non-cytotoxic at 50 μM., (Copyright © 2013 Elsevier Masson SAS. All rights reserved.)

d-Cycloserine is a second-line drug approved for use in the treatment of patients infected with Mycobacterium tuberculosis, the etiologic agent of tuberculosis. The unique mechanism of action of d-cycloserine, compared with those of other clinically employed antimycobacterial agents, represents an untapped and exploitable resource for future rational drug design programs. Here, we show that d-cycloserine is a slow-onset inhibitor of MtDdl and that this behavior is specific to the M. tuberculosis enzyme orthologue. Furthermore, evidence is presented that indicates d-cycloserine binds exclusively to the C-terminal d-alanine binding site, even in the absence of bound d-alanine at the N-terminal binding site. Together, these results led us to propose a new model of d-alanine:d-alanine ligase inhibition by d-cycloserine and suggest new opportunities for rational drug design against an essential, clinically validated mycobacterial target.

In fragment-based drug discovery, the weak affinities exhibited by fragments pose significant challenges for screening. Biophysical techniques are used to address this challenge, but there is no clear consensus on which cascade of methods is best suited to identify fragment hits that ultimately translate into bound X-ray structures and provide bona fide starting points for synthesis. We have benchmarked an integrated biophysical approach for fragment screening and validation against Mycobacterium tuberculosis pantothenate synthetase. A primary screen of 1,250 fragments library was performed by thermal shift, followed by secondary screen using one-dimensional NMR spectroscopy (water ligand observed gradient spectroscopy and saturation transfer difference binding experiments) and ultimate hit validation by isothermal titration calorimetry and X-ray crystallography. Our multibiophysical approach identified three distinct binding sites for fragments and laid a solid foundation for successful structure-based elaboration into potent inhibitors.

Peptidoglycan is an essential component of the bacterial cell wall, and enzymes involved in its biosynthesis represent validated targets for antibacterial drug discovery. MurF catalyzes the final intracellular peptidoglycan biosynthesis step: the addition of D-Ala-D-Ala to the nucleotide precursor UDP-MurNAc-L-Ala-γ-D-Glu-meso-DAP (or L-Lys). As MurF has no human counterpart, it represents an attractive target for the development of new antibacterial drugs. Using recently published cyanothiophene inhibitors of MurF from Streptococcus pneumoniae as a starting point, we designed and synthesized a series of structurally related derivatives and investigated their inhibition of MurF enzymes from different bacterial species. Systematic structural modifications of the parent compounds resulted in a series of nanomolar inhibitors of MurF from S. pneumoniae and micromolar inhibitors of MurF from Escherichia coli and Staphylococcus aureus. Some of the inhibitors also show antibacterial activity against S. pneumoniae R6. These findings, together with two new co-crystal structures, represent an excellent starting point for further optimization toward effective novel antibacterials., (Copyright © 2013 Elsevier Masson SAS. All rights reserved.)

Tubulin is subject to a reversible post-translational modification involving polyglutamylation and deglutamylation of glutamate residues in its C-terminal tail. This process plays key roles in regulating the function of microtubule associated proteins, neuronal development, and metastatic progression. This study describes the synthesis and testing of three phosphinic acid-based inhibitors that have been designed to inhibit both the glutamylating and deglutamylating enzymes. The compounds were tested against the polyglutamylase TTLL7 using tail peptides as substrates (100 μM) and the most potent inhibitor displayed an IC₅₀ value of 150 μM. The incorporation of these compounds into tubulin C-terminal tail peptides may lead to more potent TTLL inhibitors., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The biosynthetic pathway of the bacterial peptidoglycan, where MurD is an enzyme involved at the intracellular stage of its construction, represents a collection of highly selective macromolecular targets for novel antibacterial drug design. In this study as part of our investigation of the MurD bacterial target two recently discovered classes of the MurD ligase inhibitors were investigated resulting from the lead optimization phases of the N-sulfonamide D-Glu MurD inhibitors. Molecular dynamics simulations, based on novel structural data, in conjunction with the linear interaction energy (LIE) method suggested the transferability of our previously obtained LIE coefficients to further D-Glu based classes of MurD inhibitors. Analysis of the observed dynamical behavior of these compounds in the MurD active site was supported by static drug design techniques. These results complement the current knowledge of the MurD inhibitory mechanism and provide valuable support for the D-Glu paradigm of the inhibitor design.

Assuming that bicyclic β-lactams endowed with high conformational adaptability should more easily form acyl-enzyme complexes with PBP2a than the traditional antibiotics, we have prepared a series of bis-2-oxo-azetidinyl macrocycles as potential inhibitors. The compounds are formally "head-head" (HH) cyclodimers of 1-(ω-alkenoyl)-3-(S)-(ω'-alkenoylamino)-2-azetidinones, with various lengths of the alkene chains, obtained by two successive metathesis reactions using the Grubbs catalyst. All compounds behave as acylating inhibitors of PBP2a and one β-lactam (5c), embedded into the largest ring (32 atoms), features an activity close to that of Ceftobiprole. Conformational analyses, theoretical reactivity models and docking experiments in PBP2a cavity allow to propose a novel pharmacophore, i.e. the 3-(S)-acylamino-1-acyl-2-azetidinone ring, with the syn-conformation of the imide function, associated to a flexible macrocycle favoring the opening of the active site., (Copyright © 2013 Elsevier Masson SAS. All rights reserved.)

The use of the MM2QM tool in a combined docking + molecular dynamics (MD) + molecular mechanics (MM) + quantum mechanical (QM) binding affinity prediction study is presented, and the tool itself is discussed. The system of interest is Mycobacterium tuberculosis (MTB) pantothenate synthetase in complexes with three highly similar sulfonamide inhibitors, for which crystal structures are available. Starting from the structure of MTB pantothenate synthetase in the "open" conformation and following the combined docking + MD + MM + QM procedure, we were able to capture the closing of the enzyme binding pocket and to reproduce the position of the ligands with an average root mean square deviation of 1.6 Å. Protein-ligand interaction energies were reproduced with an average error lower than 10%. The discussion on the MD part and a protein flexibility importance is carried out. The presented approach may be useful especially for finding analog inhibitors or improving drug candidates., (Copyright © 2012 Wiley Periodicals, Inc.)

D-cycloserine (DCS) is an antibiotic that is currently used in second-line treatment of tuberculosis. DCS is a structural analogue of D-alanine, and targets two enzymes involved in the cytosolic stages of peptidoglycan synthesis: alanine racemase (Alr) and D-alanine:D-alanine ligase (Ddl). The mechanisms of inhibition of DCS have been well-assessed using Alr and Ddl enzymes from various bacterial species, but little is known regarding the interactions of DCS with the mycobacterial orthologues of these enzymes. We have over-expressed and purified recombinant Mycobacterium tuberculosis Ddl (MtDdl; Rv2981c), and report a kinetic examination of the enzyme with both its native substrate and DCS. MtDdl is activated by K(+), follows an ordered ter ter mechanism and displays distinct affinities for D-Ala at each D-Ala binding site (K(m,D-Ala1) = 0.075 mm, K(m,D-Ala2) = 3.6 mm). ATP is the first substrate to bind and is necessary for subsequent binding of D-alanine or DCS. The pH dependence of MtDdl kinetic parameters indicate that general base chemistry is involved in the catalytic step. DCS was found to competitively inhibit D-Ala binding at both MtDdl D-Ala sites with equal affinity (K(i,DCS1) = 14 μm, K(i,DCS2) = 25 μm); however, each enzyme active site can only accommodate a single DCS molecule at a given time. The pH dependence of K(i,DCS2) revealed a loss of DCS binding affinity at high pH (pK(a) = 7.5), suggesting that DCS binds optimally in the zwitterionic form. The results of this study may assist in the design and development of novel Ddl-specific inhibitors for use as anti-mycobacterial agents., (© 2013 The Authors Journal compilation © 2013 FEBS.)

D-Glutamic acid-adding enzyme (MurD ligase) catalyses the addition of D-glutamic acid to UDP-N-acetylmuramoyl-L-alanine, an essential cytoplasmic step in the pathway for bacterial cell-wall peptidoglycan synthesis. As such, it represents an important antibacterial drug-discovery target enzyme. Recently, several series of compounds have been synthesised and found to inhibit MurD from Escherichia coli, the best one having an IC(50) value of 8 μM. In the present work, we have tested 20 of these compounds against the MurD enzymes from Staphylococcus aureus, Streptococcus pneumoniae, Borrelia burgdorferi and Mycobacterium tuberculosis. Most of the E. coli MurD inhibitors appeared less efficient against the four other orthologues. This divergent result can be explained by the differences in amino acid sequences and topologies of the active sites of the MurD ligases studied., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The cell wall of mycobacterium offers well validated targets which can be exploited for discovery of new lead compounds. MurC-MurF ligases catalyze a series of irreversible steps in the biosynthesis of peptidoglycan precursor, i.e. MurD catalyzes the ligation of D-glutamate to the nucleotide precursor UMA. The three dimensional structure of Mtb-MurD is not known and was predicted by us for the first time using comparative homology modeling technique. The accuracy and stability of the predicted Mtb-MurD structure was validated using Procheck and molecular dynamics simulation. Key interactions in Mtb-MurD were studied using docking analysis of available transition state inhibitors of E.coli-MurD. The docking analysis revealed that analogues of both L and D forms of glutamic acid have similar interaction profiles with Mtb-MurD. Further, residues His192, Arg382, Ser463, and Tyr470 are proposed to be important for inhibitor-(Mtb-MurD) interactions. We also identified few pharmacophoric features essential for Mtb-MurD ligase inhibitory activity and which can further been utilized for the discovery of putative antitubercular chemotherapy.

D-Alanine:D-alanine ligase (Ddl) is an essential ATP-dependent bacterial enzyme involved in peptidoglycan biosynthesis. Discovery of Ddl inhibitors not competitive with ATP has proven to be difficult because the Ddl bimolecular d-alanine binding pocket is very restricted, as is accessibility to the active site for larger molecules in the catalytically active closed conformation of Ddl. A molecular dynamics study of the opening and closing of the Ddl lid loop informs future structure-based design efforts that allow for the flexibility of Ddl. A virtual screen on generated enzyme conformations yielded some hit inhibitors whose bioactivity was determined.

Whole-cell screening of Mycobacterium tuberculosis (Mtb) remains a mainstay of drug discovery, but subsequent target elucidation often proves difficult. Conditional mutants that underexpress essential genes have been used to identify compounds with known mechanism of action by target-based whole-cell screening (TB-WCS). Here, the feasibility of TB-WCS in Mtb was assessed by generating mutants that conditionally express pantothenate synthetase (panC), diaminopimelate decarboxylase (lysA), and isocitrate lyase (icl1). The essentiality of panC and lysA, and conditional essentiality of icl1 for growth on fatty acids, was confirmed. Depletion of PanC and Icl1 rendered mutants hypersensitive to target-specific inhibitors. Stable reporter strains were generated for use in high-throughput screening, and their utility was demonstrated by identifying compounds that display greater potency against a PanC-depleted strain. These findings illustrate the power of TB-WCS as a tool for tuberculosis drug discovery., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Mur ligases are bacterial enzymes involved in the cytoplasmic steps of peptidoglycan biosynthesis and are viable targets for antibacterial drug discovery. We have performed virtual screening for potential ATP-competitive inhibitors targeting MurC and MurD ligases, using a protocol of consecutive hierarchical filters. Selected compounds were evaluated for inhibition of MurC and MurD ligases, and weak inhibitors possessing dual inhibitory activity have been identified. These compounds represent new scaffolds for further optimisation towards multiple Mur ligase inhibitors with improved inhibitory potency.

In a project to characterise new antibacterial chemotypes from plants, hyperenone A and hypercalin B were isolated from the hexane and chloroform extracts of the aerial parts of Hypericum acmosepalum. The structures of both compounds were characterised by extensive one- and two-dimensional nuclear magnetic resonance (NMR) spectroscopy and were confirmed by mass spectrometry. Hyperenone A and hypercalin B exhibited antibacterial activity against multidrug-resistant strains of Staphylococcus aureus, with minimum inhibition concentration ranges of 2-128 mg/L and 0.5-128 mg/L, respectively. Hyperenone A also showed growth-inhibitory activity against Mycobacterium tuberculosis H37Rv and Mycobacterium bovis BCG at 75 mg/L and 100mg/L. Neither hyperenone A nor hypercalin B inhibited the growth of Escherichia coli and both were non-toxic to cultured mammalian macrophage cells. Both compounds were tested for their ability to inhibit the ATP-dependent MurE ligase of M. tuberculosis, a crucial enzyme in the cytoplasmic steps of peptidoglycan biosynthesis. Hyperenone A inhibited MurE selectively, whereas hypercalin B did not have any effect on enzyme activity., (Copyright © 2011 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.)

Background: ATP-dependent D-alanine:D-alanine ligase (Ddl) is a part of biochemical machinery involved in peptidoglycan biosynthesis, as it catalyzes the formation of the terminal D-ala-D-ala dipeptide of the peptidoglycan precursor UDPMurNAc-pentapeptide. Inhibition of Ddl prevents bacterial growth, which makes this enzyme an attractive and viable target in the urgent search of novel effective antimicrobial drugs. To address the problem of a relentless increase in resistance to known antimicrobial agents we focused our attention to discovery of novel ATP-competitive inhibitors of Ddl., Methodology/principal Findings: Encouraged by recent successful attempts to find selective ATP-competitive inhibitors of bacterial enzymes we designed, synthesized and evaluated a library of 6-arylpyrido[2,3-d]pyrimidine-based compounds as inhibitors of Escherichia coli DdlB. Inhibitor binding to the target enzyme was subsequently confirmed by surface plasmon resonance and studied with isothermal titration calorimetry. Since kinetic analysis indicated that 6-arylpyrido[2,3-d]pyrimidines compete with the enzyme substrate ATP, inhibitor binding to the ATP-binding site was additionally studied with docking. Some of these inhibitors were found to possess antibacterial activity against membrane-compromised and efflux pump-deficient strains of E. coli., Conclusions/significance: We discovered new ATP-competitive inhibitors of DdlB, which may serve as a starting point for development of more potent inhibitors of DdlB that could include both, an ATP-competitive and D-Ala competitive moiety.

A series of optimized sulfonamide derivatives was recently reported as novel inhibitors of UDP-N-acetylmuramoyl-L-alanine:D-glutamate ligase (MurD). These are based on naphthalene-N-sulfonyl-D-glutamic acid and have the D-glutamic acid replaced with rigidified mimetics. Here we have defined the binding site of these novel ligands to MurD using (1)H/(13)C heteronuclear single quantum correlation. The MurD protein was selectively (13)C-labeled on the methyl groups of Ile (δ1 only), Leu and Val, and was isolated and purified. Crucial Ile, Leu and Val methyl groups in the vicinity of the ligand binding site were identified by comparison of chemical shift perturbation patterns among the ligands with various structural elements and known binding modes. The conformational and dynamic properties of the bound ligands and their binding interactions were examined using the transferred nuclear Overhauser effect and saturation transfer difference. In addition, the binding mode of these novel inhibitors was thoroughly examined using unrestrained molecular dynamics simulations. Our results reveal the complex dynamic behavior of ligand-MurD complexes and its influence on ligand-enzyme contacts. We further present important findings for the rational design of potent Mur ligase inhibitors.

d-boroAla was previously characterized as an inhibitor of bacterial alanine racemase and d-Ala-d-Ala ligase enzymes (Biochemistry, 28, 1989, 3541). In this study, d-boroAla was identified and characterized as an antibacterial agent. d-boroAla has activity against both Gram-positive and Gram-negative organisms, with minimal inhibitory concentrations down to 8 μg / mL. A structure-function study on the alkyl side chain (NH(2) -CHR-B(OR')(2) ) revealed that d-boroAla is the most effective agent in a series including boroGly, d-boroHomoAla, and d-boroVal. l-boroAla was much less active, and N-acetylation completely abolished activity. An LC-MS / MS assay was used to demonstrate that d-boroAla exerts its antibacterial activity by inhibition of d-Ala-d-Ala ligase. d-boroAla is bactericidal at 1× minimal inhibitory concentration against Staphylococcus aureus and Bacillus subtilis, which each encode one copy of d-Ala-d-Ala ligase, and at 4× minimal inhibitory concentration against Escherichia coli and Salmonella enterica serovar Typhimurium, which each encode two copies of d-Ala-d-Ala ligase. d-boroAla demonstrated a frequency of resistance of 8 × 10(-8) at 4× minimal inhibitory concentration in S. aureus. These results demonstrate that d-boroAla has promising antibacterial activity and could serve as the lead agent in a new class of d-Ala-d-Ala ligase targeted antibacterial agents. This study also demonstrates d-boroAla as a possible probe for d-Ala-d-Ala ligase function., (© 2011 John Wiley & Sons A/S.)

Mur ligases (MurC-MurF), a group of bacterial enzymes that catalyze four consecutive steps in the formation of cytoplasmic peptidoglycan precursor, are becoming increasingly adopted as targets in antibacterial drug design. Based on the crystal structure of MurD cocrystallized with thiazolidine-2,4-dione inhibitor I, we have designed, synthesized, and evaluated a series of improved glutamic acid containing 5-benzylidenerhodanine and 5-benzylidenethiazolidine-2,4-dione inhibitors of MurD with IC(50) values up to 28 μM. Inhibitor 37, with an IC(50) of 34 μM, displays a weak antibacterial activity against S. aureus ATCC 29213 and E. faecalis ATCC 29212 with minimal inhibitory concentrations of 128 μg/mL. High-resolution crystal structures of MurD in complex with two new inhibitors (compounds 23 and 51) reveal details of their binding modes within the active site and provide valuable information for further structure-based optimization., (Copyright © 2011 Elsevier Masson SAS. All rights reserved.)

D-Alanine:D-alanine ligase (Ddl), an intracellular bacterial enzyme essential for cell wall biosynthesis, is an attractive target for development of novel antimicrobial drugs. This study focused on an extensive evaluation of two families of Ddl inhibitors encountered in our previous research. New members of both families were obtained through similarity search and synthesis. Ellipticines and 9-acridinylamines were both found to possess inhibitory activity against Ddl from Escherichia coli and antimicrobial activity against E. coli and Staphylococcus aureus. Ellipticines with a quaternary methylpyridinium moiety were the most potent among all studied compounds, with MIC values as low as 2 mg/L in strains with intact efflux mechanisms. Antimicrobial activity of the studied compounds was connected to membrane damage, making their development as antibacterial drug candidates unlikely unless analogues devoid of this nonspecific effect can be discovered., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Mur ligases are involved in cytoplasmic steps of bacterial peptidoglycan biosynthesis and are viable targets for antibacterial drug discovery. We have designed and synthesized a focused chemical library of compounds combining the glutamic acid moiety and the 2-thioxothiazolidin-4-one, thiazolidine-2,4-dione, 2-iminothiazolidin-4-one or imidazolidine-2,4-dione ring connected by a benzylidene group. These compounds were designed to target the d-Glu- and the diphosphate-binding pockets of the MurD active site and were evaluated for inhibition of MurD ligase from Escherichia coli. The most potent compounds (R)-9 and (S)-9 inhibited MurD with IC(50) values of 45 μM and 10 μM, respectively. The specific binding mode of (R)-9 in MurD active site was established by high-resolution NMR spectroscopy., (Copyright © 2011 Elsevier Masson SAS. All rights reserved.)

Objectives: The aim of this study was to comprehensively evaluate the antibacterial activity and MurE inhibition of a set of N-methyl-2-alkenyl-4-quinolones found to inhibit the growth of fast-growing mycobacteria., Methods: Using the spot culture growth inhibition assay, MICs were determined for Mycobacterium tuberculosis H(37)Rv, Mycobacterium bovis BCG and Mycobacterium smegmatis mc(2)155. MICs were determined for Mycobacterium fortuitum, Mycobacterium phlei, methicillin-resistant Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa using microplate dilution assays. Inhibition of M. tuberculosis MurE ligase activity was determined both by colorimetric and HPLC methods. Computational modelling and binding prediction of the quinolones in the MurE structure was performed using Glide. Kinetic experiments were conducted for understanding possible competitive relations of the quinolones with the endogenous substrates of MurE ligase., Results: The novel synthetic N-methyl-2-alkenyl-4-quinolones were found to be growth inhibitors of M. tuberculosis and rapid-growing mycobacteria as well as methicillin-resistant S. aureus, while showing no inhibition for E. coli and P. aeruginosa. The quinolones were found to be inhibitory to MurE ligase of M. tuberculosis in the micromolar range (IC(50) ∼40-200 μM) when assayed either spectroscopically or by HPLC. Computational docking of the quinolones on the published M. tuberculosis MurE crystal structure suggested that the uracil recognition site is a probable binding site for the quinolones., Conclusions: N-methyl-2-alkenyl-4-quinolones are inhibitors of mycobacterial and staphylococcal growth, and show MurE ligase inhibition. Therefore, they are considered as a starting point for the development of increased affinity MurE activity disruptors.

MurD ligase is one of the key enzymes participating in the intracellular steps of peptidoglycan biosynthesis and constitutes a viable target in the search for novel antibacterial drugs to combat bacterial drug-resistance. We have designed, synthesized, and evaluated a new series of D-glutamic acid-based Escherichia coli MurD inhibitors incorporating the 5-benzylidenethiazolidin-4-one scaffold. The crystal structure of 16 in the MurD active site has provided a good starting point for the design of structurally optimized inhibitors 73-75 endowed with improved MurD inhibitory potency (IC(50) between 3 and 7 μM). Inhibitors 74 and 75 showed weak activity against Gram-positive Staphylococcus aureus and Enterococcus faecalis. Compounds 73-75, with IC(50) values in the low micromolar range, represent the most potent D-Glu-based MurD inhibitors reported to date.

Mycobacterium tuberculosis pantothenate synthetase is a potential anti-tuberculosis target, and a high-throughput screening system was previously developed to identify its inhibitors. Using a similar system, we screened a small library of compounds and identified actinomycin D (ActD) as a weak inhibitor of pantothenate synthetase. A new method was established to discover more effective inhibitors by determining the molecular mechanism of ActD inhibition followed by structure-based virtual screening. The molecular interaction of inhibition was determined by circular dichroism and tryptophan fluorescence quenching. The structure-based search and virtual screening were performed using the Molecular Operating Environment (MOE) program and SYBYL 7.5, respectively. Two inhibitors were identified with an IC(50) for pantothenate synthetase that was at least ten times better than that of ActD., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

D-Glutamic acid-adding enzyme (MurD) catalyses the essential addition of d-glutamic acid to the cytoplasmic peptidoglycan precursor UDP-N-acetylmuramoyl-l-alanine, and as such it represents an important antibacterial drug-discovery target enzyme. Based on a series of naphthalene-N-sulfonyl-d-Glu derivatives synthesised recently, we synthesised two series of new, optimised sulfonamide inhibitors of MurD that incorporate rigidified mimetics of d-Glu. The compounds that contained either constrained d-Glu or related rigid d-Glu mimetics showed significantly better inhibitory activities than the parent compounds, thereby confirming the advantage of molecular rigidisation in the design of MurD inhibitors. The binding modes of the best inhibitors were examined with high-resolution NMR spectroscopy and X-ray crystallography. We have solved a new crystal structure of the complex of MurD with an inhibitor bearing a 4-aminocyclohexane-1,3-dicarboxyl moiety. These data provide an additional step towards the development of sulfonamide inhibitors with potential antibacterial activities., (Copyright © 2011 Elsevier Masson SAS. All rights reserved.)

Many lead compounds in the search for new drugs derive from peptides and polyketides whose similar biosynthetic enzymes have been difficult to engineer for production of new derivatives. Problems with generating multiple analogs in a single experiment along with lack of high-throughput methods for structure-based screening have slowed progress in this area. Here, we use directed evolution and a multiplexed assay to screen a library of >14,000 members to generate three derivatives of the antibacterial compound, andrimid. Another limiting factor in reengineering these mega-enzymes of secondary metabolism has been that commonly used hosts such as Escherichia coli often give lower product titers, so our reengineering was performed in the native producer, Pantoea agglomerans. This integrated in vivo approach can be extended to larger enzymes to create analogs of natural products for bioactivity testing., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Background: Penicillin-binding proteins (PBPs) are well known and validated targets for antibacterial therapy. The most important clinically used inhibitors of PBPs β-lactams inhibit transpeptidase activity of PBPs by forming a covalent penicilloyl-enzyme complex that blocks the normal transpeptidation reaction; this finally results in bacterial death. In some resistant bacteria the resistance is acquired by active-site distortion of PBPs, which lowers their acylation efficiency for β-lactams. To address this problem we focused our attention to discovery of novel noncovalent inhibitors of PBPs., Methodology/principal Findings: Our in-house bank of compounds was screened for inhibition of three PBPs from resistant bacteria: PBP2a from Methicillin-resistant Staphylococcus aureus (MRSA), PBP2x from Streptococcus pneumoniae strain 5204, and PBP5fm from Enterococcus faecium strain D63r. Initial hit inhibitor obtained by screening was then used as a starting point for computational similarity searching for structurally related compounds and several new noncovalent inhibitors were discovered. Two compounds had promising inhibitory activities of both PBP2a and PBP2x 5204, and good in-vitro antibacterial activities against a panel of Gram-positive bacterial strains., Conclusions: We found new noncovalent inhibitors of PBPs which represent important starting points for development of more potent inhibitors of PBPs that can target penicillin-resistant bacteria.

D-alanine:D-alanine ligase (EC 6.3.2.4; Ddl) catalyzes the ATP-driven ligation of two D-alanine (D-Ala) molecules to form the D-alanyl:D-alanine dipeptide. This molecule is a key building block in peptidoglycan biosynthesis, making Ddl an attractive target for drug development. D-Cycloserine (DCS), an analog of D-Ala and a prototype Ddl inhibitor, has shown promise for the treatment of tuberculosis. Here, we report the crystal structure of Mycobacterium tuberculosis Ddl at a resolution of 2.1 Å. This structure indicates that Ddl is a dimer and consists of three discrete domains; the ligand binding cavity is at the intersection of all three domains and conjoined by several loop regions. The M. tuberculosis apo Ddl structure shows a novel conformation that has not yet been observed in Ddl enzymes from other species. The nucleotide and D-alanine binding pockets are flexible, requiring significant structural rearrangement of the bordering regions for entry and binding of both ATP and D-Ala molecules. Solution affinity and kinetic studies showed that DCS interacts with Ddl in a manner similar to that observed for D-Ala. Each ligand binds to two binding sites that have significant differences in affinity, with the first binding site exhibiting high affinity. DCS inhibits the enzyme, with a 50% inhibitory concentration (IC(50)) of 0.37 mM under standard assay conditions, implicating a preferential and weak inhibition at the second, lower-affinity binding site. Moreover, DCS binding is tighter at higher ATP concentrations. The crystal structure illustrates potential drugable sites that may result in the development of more-effective Ddl inhibitors.

Rational design is applied in the discovery of novel lead drugs. Its rapid development is mainly attributed to the tremendous advancements in the computer science, statistics, molecular biology, biophysics, biochemistry, medicinal chemistry, pharmacokinetics and pharmacodynamics experienced in the last few decades. The promising feature that characterizes the application of rational drug design is that it uses for developing potential leads in drug discovery all known theoretical and experimental knowledge of the system under study. The utilization of the knowledge of the molecular basis of the system ultimately aims to reduce human power cost, time saving and laboratory expenses in the drug discovery. In this review paper various strategies applied for systems which include: (i) absence of knowledge of the receptor active site; (ii) the knowledge of a homology model of a receptor, (iii) the knowledge of the experimentally determined (i.e. X-ray crystallography, NMR spectroscopy) coordinates of the active site of the protein in absence and (iv) the presence of the ligand will be analyzed.

The gene cluster from Pantoea agglomerans responsible for biosynthesis of the dapdiamide antibiotics encodes an adenylation-thiolation didomain protein, DdaD, and an Fe(II)/α-ketoglutarate-dependent dioxygenase homologue, DdaC. Here we show that DdaD, a nonribosomal peptide synthetase module, activates and sequesters N(β)-fumaramoyl-l-2,3-diaminopropionate as a covalently tethered thioester for subsequent oxidative modification of the fumaramoyl group. DdaC catalyzes Fe(II)- and α-ketoglutarate-dependent epoxidation of the covalently bound N(β)-fumaramoyl-l-2,3-diaminopropionyl-S-DdaD species to generate N(β)-epoxysuccinamoyl-DAP (DAP = 2,3-diaminopropionate) in thioester linkage to DdaD. After hydrolytic release, N(β)-epoxysuccinamoyl-DAP can be ligated to l-valine by the ATP-dependent ligase DdaF to form the natural antibiotic N(β)-epoxysuccinamoyl-DAP-Val.

The human pathogen Acinetobacter baumannii produces a siderophore called acinetobactin that is derived from one molecule each of threonine, histidine, and 2,3-dihydroxybenzoic acid (DHB). The activity of several nonribosomal peptide synthetase (NRPS) enzymes is used to combine the building blocks into the final molecule. The acinetobactin synthesis pathway initiates with a self-standing adenylation enzyme, BasE, that activates the DHB molecule and covalently transfers it to the pantetheine cofactor of an aryl-carrier protein of BasF, a strategy that is shared with many siderophore-producing NRPS clusters. In this reaction, DHB reacts with ATP to form the aryl adenylate and pyrophosphate. In a second partial reaction, the DHB is transferred to the carrier protein. Inhibitors of BasE and related enzymes have been identified that prevent growth of bacteria on iron-limiting media. Recently, a new inhibitor of BasE has been identified via high-throughput screening using a fluorescence polarization displacement assay. We present here biochemical and structural studies to examine the binding mode of this inhibitor. The kinetics of the wild-type BasE enzyme is shown, and inhibition studies demonstrate that the new compound exhibits competitive inhibition against both ATP and 2,3-dihydroxybenzoate. Structural examination of BasE bound to this inhibitor illustrates a novel binding mode in which the phenyl moiety partially fills the enzyme pantetheine binding tunnel. Structures of rationally designed bisubstrate inhibitors are also presented.

Objectives: New anti-mycobacterial entities with novel mechanisms of action are clinically needed for treating resistant forms of tuberculosis. The purpose of this study was to evaluate anti-tubercular activity and selectivity of seven recently isolated natural products from Colombian plants., Methods: MICs were determined using a liquid medium growth inhibition assay for Mycobacterium tuberculosis H(37)Rv and both solid and liquid media growth inhibition assays for Mycobacterium bovis BCG. Escherichia coli growth inhibition and mammalian macrophage cell toxicity were evaluated to establish the degree of selectivity of the natural product against whole cell organisms. Enzymatic inhibition of ATP-dependent MurE ligase from M. tuberculosis was assayed using a colorimetric phosphate detection method. The most active compound, 3-methoxynordomesticine hydrochloride, was further investigated on M. bovis BCG for its inhibition of sigmoidal growth, acid-fast staining and viability counting analysis., Results: Aporphine alkaloids were found to be potent inhibitors of slow-growing mycobacterial pathogens showing favourable selectivity and cytotoxicity. In terms of their endogenous action, the aporphine alkaloids were found inhibitory to M. tuberculosis ATP-dependent MurE ligase at micromolar concentrations. A significantly low MIC was detected for 3-methoxynordomesticine hydrochloride against both M. bovis BCG and M. tuberculosis H(37)Rv., Conclusions: Considering all the data, 3-methoxynordomesticine hydrochloride was found to be a potent anti-tubercular compound with a favourable specificity profile. The alkaloid showed MurE inhibition and is considered an initial hit for exploring related chemical space.

We have designed, synthesized, and evaluated 5-benzylidenerhodanine- and 5-benzylidenethiazolidine-2,4-dione-based compounds as inhibitors of bacterial enzyme MurD with E. coli IC(50) in the range 45-206 μM. The high-resolution crystal structure of MurD in complex with (R,Z)-2-(3-[{4-([2,4-dioxothiazolidin-5-ylidene]methyl)phenylamino}methyl)benzamido)pentanedioic acid [(R)-32] revealed details of the binding mode of the inhibitor within the active site and provides a good foundation for structure-based design of a novel generation of MurD inhibitors.

Fragment-based methods are a new and emerging approach for the discovery of protein binders that are potential new therapeutic agents. Several ways of utilizing structural information to guide the inhibitor assembly have been explored to date. One of the approaches, application of interligand Overhauser effect (ILOE) observations, is of particular interest, as it does not require the availability of a three-dimensional protein structure and is an NMR-based method that can be applied to targets that cannot be observed directly because of their size. Fragments, as small and often hydrophobic molecules, suffer from problems including compound aggregation in an aqueous environment and nonspecific binding contributions, especially when screened at higher concentrations suitable for ILOE observations. Here we report how this problem can be overcome by applying a step-by-step iterative procedure that includes the application of optimized probe molecules with known binding modes to elucidate the unknown binding modes of fragments. An enzyme substrate with well-characterized binding was used as a starting point, and the relative binding modes of modified fragments derived from ILOE observations were used to guide the fragment linking, leading to a potent inhibitor of our model system, Mycobacterium tuberculosis pantothenate synthetase, a potential drug target. We have supported our NMR data with crystal structures, thus establishing the guidelines for optimizing the ILOE observations. This model study should expand the application of the technique in drug discovery.

The structural basis for the homotropic inhibition of pantothenate synthetase by the substrate pantoate was investigated by X-ray crystallography and high-resolution NMR spectroscopic methods. The tertiary structure of the dimeric N-terminal domain of Escherichia coli pantothenate synthetase, determined by X-ray crystallography to a resolution of 1.7 A, showed a second molecule of pantoate bound in the ATP-binding pocket. Pantoate binding to the ATP-binding site induced large changes in structure, mainly for backbone and side chain atoms of residues in the ATP binding HXGH(34-37) motif. Sequence-specific NMR resonance assignments and solution secondary structure of the dimeric N-terminal domain, obtained using samples enriched in (2)H, (13)C, and (15)N, indicated that the secondary structural elements were conserved in solution. Nitrogen-15 edited two-dimensional solution NMR chemical shift mapping experiments revealed that pantoate, at 10 mm, bound at these two independent sites. The solution NMR studies unambiguously demonstrated that ATP stoichiometrically displaced pantoate from the ATP-binding site. All NMR and X-ray studies were conducted at substrate concentrations used for enzymatic characterization of pantothenate synthetase from different sources [Jonczyk R & Genschel U (2006) J Biol Chem 281, 37435-37446]. As pantoate binding to its canonical site is structurally conserved, these results demonstrate that the observed homotropic effects of pantoate on pantothenate biosynthesis are caused by competitive binding of this substrate to the ATP-binding site. The results presented here have implications for the design and development of potential antibacterial and herbicidal agents.