MurD Inhibitors Research Articles

Design, synthesis and molecular modelling of phenoxyacetohydrazide derivatives as Staphylococcus aureus MurD inhibitors

Design, synthesis and molecular modelling of phenoxyacetohydrazide derivatives as Staphylococcus aureus MurD inhibitors

MurD inhibitors as antibacterial agents: a review

Peptidoglycan is an essential component of the bacterial cell wall and is required for the survival of bacteria. ATP-dependent MurC–F ligases are responsible for the early stages of peptidoglycan biosynthesis. Second in the series, MurD catalyzes the addition of d-glutamic acid to UDP-MurNAc-l-Ala which involves acyl-phosphate and tetrahedral intermediates. Due to its high specificity for d-glutamic acid substrate and its absence in mammalian cells, MurD is considered as an attractive target for the design of novel antibacterial agents. Several MurD inhibitors have been designed and tested for their activity. These include phosphinates, N-acetylmuramic acids, pulvinones, phosphinodipeptide, cyclic nonapeptides, macrocyclic compounds, benzene-1,3-dicarboxylic acid, naphthalene sulphonamides, 2-thioxothiazolidin-4-ones, benzylidene-2,4-thiazolidindiones, etc. In the present review, an updated status of MurD enzyme inhibitors is presented which will serve as a useful source of structural information and may be utilized for the design of potent inhibitors against this enzyme.

Identification of the potential biological target of N-benzenesulfonyl-1,2,3,4-tetrahydroquinoline compounds active against gram-positive and gram-negative bacteria

The development of new antibiotics with activity towards a broad spectrum of bacteria, including multiresistant strains, is a very important topic for global public health. As part of previous works, N-benzenesulfonyl-1,2,3,4-tetrahydroquinoline (BSTHQ) derivatives were described as antimicrobial agents active against gram-positive and gram-negative pathogens. In this work, experimental and molecular modelling studies were performed in order to identify their potential biological target in the light of structure-based design efforts towards further BSTHQ derivatives. First, a carboxyfluorescein leakage assay was performed using liposomes to mimic bacterial membranes, which found no significative membrane disruption effects with respect to control samples. These results support a non-surfactant antimicrobial activity of the tested compounds. In a second stage, the inhibition of potential antimicrobial targets was screened using molecular modelling methods, taking into account previously reported druggable targets deposited in the ChEMBL database for Escherichia coli and Staphylococcus aureus. Two enzymes, namely D-glutamic acid-adding enzyme (MurD) and N-acetylglucosamine-1-phophate-uridyltransferase (GlmU), both involved in bacterial membrane synthesis, were identified as potential targets. Pharmacodynamic interaction models were developed using known MurD and GlmU inhibitors by applying state-of-the-art chemoinformatic methods (molecular docking, molecular dynamics and free energy of interaction analyses). These models were further extended to the analysis of the studied BSTHQ derivatives. Overall, our results demonstrated that the studied BSTHQ derivatives elicit their antibacterial activity by interacting with a specific molecular target, GlmU being the highly feasible one. Based on the presented results, further structure-aided design efforts towards the obtaining of novel BSTHQ derivatives are envisioned.Communicated by Ramaswamy H. Sarma

Synthesis, molecular docking, binding free energy calculation and molecular dynamics simulation studies of benzothiazol-2-ylcarbamodithioates as Staphylococcus aureus MurD inhibitors

A new series of benzothiazol-2-ylcarbamodithioate functional compounds 5a-f has been designed, synthesized and characterized by spectral data. These compounds were screened for their in vitro antibacterial activity against strains of Staphylococcus aureus (NCIM 5021, NCIM 5022 and methicillin-resistant isolate 43300), Bacillus subtilis (NCIM 2545), Escherichia coli (NCIM 2567), Klebsiella pneumoniae (NCIM 2706) and Psudomonas aeruginosa (NCIM 2036). Compounds 5a and 5d exhibited significant activity against all the tested bacterial strains. Specifically, compounds 5a and 5d showed potent activity against K. pneumoniae (NCIM 2706), while compound 5a also displayed potent activity against S. aureus (NCIM 5021). Compound 5d showed minimum IC50 value of 13.37 μM against S. aureus MurD enzyme. Further, the binding interactions of compounds 5a-f in the catalytic pocket have been investigated using the extra-precision molecular docking and binding free energy calculation by MM-GBSA approach. A 30 ns molecular dynamics simulation of 5d/modeled S. aureus MurD enzyme was performed to determine the stability of the predicted binding conformation.

Combining molecular docking and molecular dynamics studies for modelling Staphylococcus aureus MurD inhibitory activity

ABSTRACTThe ATP-dependent bacterial MurD enzyme catalyses the formation of the peptide bond between cytoplasmic intermediate UDP-N-acetylmuramoyl-L-alanine and D-glutamic acid. This is essential for bacterial cell wall peptidoglycan synthesis in both Gram-positive and Gram-negative bacteria. MurD is recognized as an important target for the development of new antibacterial agents. In the present study we prepared the 3D-stucture of the catalytic pocket of the Staphylococcus aureus MurD enzyme by homology modelling. Extra-precision docking, binding free energy calculation by the MM–GBSA approach and a 40 ns molecular dynamics (MD) simulation of 2-thioxothiazolidin-4-one based inhibitor $1 was carried out to elucidate its inhibition potential for the S. aureus MurD enzyme. Molecular docking results showed that Lys19, Gly147, Tyr148, Lys328, Thr330 and Phe431 residues are responsible for the inhibitor–protein complex stabilization. Binding free energy calculation revealed electrostatic solvation and van der Waals energy components as major contributors for the inhibitor binding. The inhibitor-modelled S. aureus protein complex had a stable conformation in response to the atomic flexibility and interaction, when subjected to MD simulation at 40 ns in aqueous solution. We designed some molecules as potent inhibitors of S. aureus MurD, and to validate the stability of the designed molecule D1-modelled protein complex we performed a 20 ns MD simulation. Results obtained from this study can be utilized for the design of potent S. aureus MurD inhibitors.

Extra precision docking, free energy calculation and molecular dynamics studies on glutamic acid derivatives as MurD inhibitors

The binding modes of well known MurD inhibitors have been studied using molecular docking and molecular dynamics (MD) simulations. The docking results of inhibitors 1-30 revealed similar mode of interaction with Escherichia coli-MurD. Further, residues Thr36, Arg37, His183, Lys319, Lys348, Thr321, Ser415 and Phe422 are found to be important for inhibitors and E. coli-MurD interactions. Our docking procedure precisely predicted crystallographic bound inhibitor 7 as evident from root mean square deviation (0.96Å). In addition inhibitors 2 and 3 have been successfully cross-docked within the MurD active site, which was pre-organized for the inhibitor 7. Induced fit best docked poses of 2, 3, 7 and 15/2Y1O complexes were subjected to 10ns MD simulations to determine the stability of the predicted binding conformations. Induce fit derived docked complexes were found to be in a state of near equilibrium as evident by the low root mean square deviations between the starting complex structure and the energy minimized final average MD complex structures. The results of molecular docking and MD simulations described in this study will be useful for the development of new MurD inhibitors with high potency.

MurD enzymes: some recent developments.

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.

Molecular dynamics simulation and linear interaction energy study of d-Glu-based inhibitors of the MurD ligase

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.

The Binding Mode of Second-Generation Sulfonamide Inhibitors of MurD: Clues for Rational Design of Potent MurD Inhibitors

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 1H/13C heteronuclear single quantum correlation. The MurD protein was selectively 13C-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.

MurD enzymes from different bacteria: Evaluation of inhibitors

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 IC50 value of 8μM. 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.

Structure-Based Design of a New Series of d-Glutamic Acid Based Inhibitors of Bacterial UDP-N-acetylmuramoyl-l-alanine:d-glutamate Ligase (MurD)

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.

Second-generation sulfonamide inhibitors of d-glutamic acid-adding enzyme: Activity optimisation with conformationally rigid analogues of d-glutamic acid

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.

Discovery of Novel 5-Benzylidenerhodanine and 5-Benzylidenethiazolidine-2,4-dione Inhibitors of MurD Ligase

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.

Virtual screening for potential inhibitors of homology modeled Leptospira interrogans MurD ligase

The life-threatening infections caused by Leptospira serovars remain a global challenge since long time. Prevention of infection by controlling environmental factors being difficult to practice in developing countries, there is a need for designing potent anti-leptospirosis drugs. ATP-dependent MurD involved in biosynthesis of peptidoglycan was identified as common drug target among pathogenic Leptospira serovars through subtractive genomic approach. Peptidoglycan biosynthesis pathway being unique to bacteria and absent in host represents promising target for antimicrobial drug discovery. Thus, MurD 3D models were generated using crystal structures of 1EEH and 2JFF as templates in Modeller9v7. Structural refinement and energy minimization of the model was carried out in Maestro 9.0 applying OPLS-AA 2001 force field and was evaluated through Procheck, ProSA, PROQ, and Profile 3D. The active site residues were confirmed from the models in complex with substrate and inhibitor. Four published MurD inhibitors (two phosphinics, one sulfonamide, and one benzene 1,3-dicarbixylic acid derivative) were queried against more than one million entries of Ligand.Info Meta-Database to generate in-house library of 1,496 MurD inhibitor analogs. Our approach of virtual screening of the best-ranked compounds with pharmacokinetics property prediction has provided 17 novel MurD inhibitors for developing anti-leptospirosis drug targeting peptidoglycan biosynthesis pathway.

New High-Throughput Fluorimetric Assay for Discovering Inhibitors of UDP-N-Acetylmuramyl-l-Alanine: d-Glutamate (MurD) Ligase

A novel assay for monitoring the activity of the bacterial enzyme UDP-N-acetylmuramyl-L-alanine:D-glutamate ligase (MurD ligase) is presented. MurD, which belongs to an enzyme family of Mur ligases, is essential for the synthesis of bacterial peptidoglycan and therefore represents an attractive target for the discovery of novel antibacterial agents. The inhibition assay described in this article is amenable to high-throughput screening. It is based on the detection of the accumulation of adenosine 5'-diphosphate (ADP), a product of the reaction catalyzed by MurD ligase, by conversion to a fluorescent signal via a coupled enzyme system, using the ADP Quest assay kit from DiscoveRx. The novel assay has been validated by obtaining KM,app values for substrates D-Glu, UDP- N-acetylmuramyl-L-alanine (UMA) and ATP that are in agreement with the data reported in the literature. A counterscreen assay was introduced to eliminate false positives, and some of the known MurD inhibitors have been retested to compare the data measured with different methods. Moreover, a focused library of around 1000 compounds was screened for the inhibition of MurD to assess the performance and robustness of the assay. Finally, a novel MurD inhibitor belonging to a new structural class, with an IC50 value of 105 microM, was discovered.

Discovery of new inhibitors of the bacterial peptidoglycan biosynthesis enzymes MurD and MurF by structure-based virtual screening

The ATP-dependent Mur ligases (MurC, MurD, MurE and MurF) successively add l-Ala, d-Glu, meso-A 2pm or l-Lys, and d-Ala- d-Ala to the nucleotide precursor UDP-Mur NAc, and they represent promising targets for antibacterial drug discovery. We have used the molecular docking programme eHiTS for the virtual screening of 1990 compounds from the National Cancer Institute ‘Diversity Set’ on MurD and MurF. The 50 top-scoring compounds from screening on each enzyme were selected for experimental biochemical evaluation. Our approach of virtual screening and subsequent in vitro biochemical evaluation of the best ranked compounds has provided four novel MurD inhibitors (best IC 50 = 10 μM) and one novel MurF inhibitor (IC 50 = 63 μM).

Novel Naphthalene-N-sulfonyl-d-glutamic Acid Derivatives as Inhibitors of MurD, a Key Peptidoglycan Biosynthesis Enzyme,

Mur ligases have essential roles in the biosynthesis of peptidoglycan, and they represent attractive targets for the design of novel antibacterials. MurD (UDP-N-acetylmuramoyl-L-alanine:D-glutamate ligase) is the second enzyme in the series of Mur ligases, and it catalyzes the addition of D-glutamic acid (D-Glu) to the cytoplasmic intermediate UDP-N-acetylmuramoyl-L-alanine (UMA). Because of the high binding affinity of D-Glu toward MurD, we synthesized and biochemically evaluated a series of N-substituted D-Glu derivatives as potential inhibitors of MurD from E. coli, which allowed us to explore the structure-activity relationships.The substituted naphthalene-N-sulfonyl-D-Glu inhibitors, which were synthesized as potential transition state analogues, displayed IC50 values ranging from 80 to 600 microM. In addition, the high-resolution crystal structures of MurD in complex with four novel inhibitors revealed details of the binding mode of the inhibitors within the active site of MurD. Structure-activity relationships and cocrystal structures constitute an excellent starting point for further development of novel MurD inhibitors of this structural class.