Identification of Potential Inhibitors of MurD Enzyme of Staphylococcus aureus from a Marine Natural Product Library.

Staphylococcus aureus is an opportunistic pathogen that can cause fatal bacterial infections. MurD catalyzes the formation of peptide bond between UDP-N-acetylehyl-l-alanine and d-glutamic acid, which plays an important role in the synthesis of peptidoglycan and the formation of cell wall by S. aureus. Because S. aureus is resistant to most existing antibiotics, it is necessary to develop new inhibitors. In this study, Schrodinger 11.5 Prime homology modeling was selected to prepare the protein model of MurD enzyme, and its structure was optimized. We used a virtual screening program and similarity screening to screen 47163 compounds from three marine natural product libraries to explore new inhibitors of S. aureus. ADME provides analysis of the physicochemical properties of the best performing compounds during the screening process. To determine the stability of the docking effect, a 100 ns molecular dynamics was performed to verify how tightly the compound was bound to the protein. By docking analysis and molecular dynamics analysis, both 46604 and 46608 have strong interaction with the docking pocket, have good pharmacological properties, and maintain stable conformation with the target protein, so they have a chance to become drugs for S. aureus. Through virtual screening, similarity screening, ADME study and molecular dynamics simulation, 46604 and 46608 were selected as potential drug candidates for S. aureus.

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.

Ugi efficient synthesis of novel N-alkylated lipopeptides, antimicrobial properties and computational studies in Staphylococcus aureus via MurD antibacterial target.

Lipopeptides are medicinally essential building blocks with strong hemolytic, antifungal and antibiotic potential. In the present research article, we are presenting our findings regarding the synthesis of N-alkylated lipopeptides via Ugi four-component approach, their antimicrobial potential against pathogenic (Gram-positive and Gram-negative) bacteria, as well as computational studies to investigate the compounds binding affinity and dynamic behavior with MurD antibacterial target. Molecular docking demonstrated the compounds have good binding ability with MurD enzyme. The FT94, FT95 and FT97 compounds revealed binding affinity scores of -8.585 kcal mol- 1, -7.660 kcal mol- 1 and -7.351 kcal mol- 1, respectively. Furthermore, dynamics analysis pointed the systems high structure dynamics. The docking and simulation results were validated by binding free energies, demonstrating solid intermolecular interactions and in the assay in vitro, the Minimal Inhibitory Concentration (MIC) of FT97 to Staphylococcus aureus (S. aureus) was 62.5 µg/mL. In conclusion, a moderate inhibitory response of peptoid FT97 was observed against the Gram-positive bacteria, S. aureus and B. cereus.

Design, Synthesis, and Application of Carbon Dots With Synergistic Antibacterial Activity.

The diversity of bacteria and their ability to acquire drug resistance lead to many challenges in traditional antibacterial methods. Photothermal therapies that convert light energy into localized physical heat to kill target microorganisms do not induce resistance and provide an alternative for antibacterial treatment. However, many photothermal materials cannot specifically target bacteria, which can lead to thermal damage to normal tissues, thus seriously affecting their biological applications. Here, we designed and synthesized bacteria-affinitive photothermal carbon dots (BAPTCDs) targeting MurD ligase that catalyzes the synthesis of peptidoglycan (PG) in bacteria. BAPTCDs presented specific recognition ability and excellent photothermal properties. BAPTCDs can bind to bacteria very tightly due to their chiral structure and inhibit enzyme activity by competing with D-glutamic acid to bind to MurD ligases, thus inhibiting the synthesis of bacterial walls. It also improves the accuracy of bacteria treatment by laser irradiation. Through the synergy of biochemical and physical effects, the material offers outstanding antibacterial effects and potentially contributes to tackling the spread of antibiotic resistance and facilitation of antibiotic stewardship.

Identification of novel natural MurD ligase inhibitors as potential antimicrobial agents targeting Acinetobacter baumannii: In silico screening and biological evaluation.

The increased multidrug resistance in Acinetobacter baumannii (A. baumannii) to the present-day known antibiotics has stimulated academic and industrial efforts globally for the development of novel antibacterial agents. Natural compounds as potential drug leads are gaining significant attention due to their less toxic and more tolerant nature. In the current study, the natural product-based compounds were explored as probable inhibitors of UDP-N-acetylmuramoyl-L-alanine:D-glutamate (MurD) ligase from A.baumannii (AbMurD) to provide a new class of drug leads. The prepared natural library of 3,16,714 compounds from ZINC database was screened into the active site of AbMurD using in silico high-throughput virtual screening which resulted in 100 compounds having high binding affinities. Further screening through flexible molecular docking yielded four potential compounds selected on the basis of estimated binding affinity (ΔG) and favorable protein-ligand interactions. MD simulation of these four compounds under physiological conditions and free binding energy calculations using MM/PBSA (molecular mechanics with Poisson- Boltzmann and surface area solvation) approach revealed three compounds ZINC08879777, ZINC30726863, and ZINC95486217 as potential binders of AbMurD. The calculated physicochemical and ADME properties of these compounds revealed that they can be exploited and modified to improve their binding affinity with the enzyme. Two compounds were purchased and tested against bacterial cell cultures of A. baumannii, Salmonella Typhi, and Staphylococcus aureus to determine their broad-spectrum antibacterial activity. The results suggest that the identified compounds can be exploited as potential herbal leads to target both Gram-positive and Gram-negative pathogens. Communicated by Ramaswamy H. Sarma.

Identification of novel multitarget antitubercular inhibitors against mycobacterial peptidoglycan biosynthetic Mur enzymes by structure-based virtual screening.

Current therapeutic strategies for several diseases, including Mycobacterium tuberculosis infection, have evolved from an initial single-target treatment to a multitarget one. A multitarget antitubercular drugs targeting different mycobacterial proteins are more effective at suppressing bacterial growth. In this study, a high throughput virtual screening was performed to identify hits to the potential antitubercular multitarget: murA, murB, murC, murD, murE, murF, murG and murI from M. tuberculosis that is involved in peptidoglycan biosynthesis. In the virtual screening, we were docked 56,400 compounds of the ChEMBL antimycobacterial library and re-scored and identified the top 10 ranked compounds as antitubercular drug candidates. Further, the best common docked complex CHEMBL446262 was subjected to molecular dynamics simulation to understand the molecule's stability in the presence of an active site environment. After that, we have calculated binding free energy the top-ranked docked complexes using the MM/PBSA method. These ligands exhibited the highest binding affinity; find out novel drug-likeness might show the M. tuberculosis effect's inhibitor by interacting with multitarget Mur enzymes. New antitubercular therapies that include multitarget drugs may have higher efficacy than single-target medicines and provide a more straightforward antitubercular therapy regimen.Communicated by Ramaswamy H. Sarma.

Recent developments on UDP-N-acetylmuramoyl-L-alanine-D-gutamate ligase (Mur D) enzyme for antimicrobial drug development: An emphasis on in-silico approaches.

Introduction: The rapid emergence of antibiotic resistance among various bacterial pathogens has been one of the major concerns of health organizations across the world. In this context, for the development of novel inhibitors against antibiotic-resistant bacterial pathogens, UDP-N-Acetylmuramoyl-L-Alanine-D-Glutamate Ligase (MurD) enzyme represents one of the most apposite targets. Body: The present review focuses on updated advancements on MurD-targeted inhibitors in recent years along with genetic regulation, structural and functional characteristics of the MurD enzyme from various bacterial pathogens. A concise account of various crystal structures of MurD enzyme, submitted into Protein Data Bank is also discussed. Discussion: MurD, an ATP dependent cytoplasmic enzyme is an important target for drug discovery. The genetic organization of MurD enzyme is well elucidated and many crystal structures of MurD enzyme are submitted into Protein Data bank. Various inhibitors against MurD enzyme have been developed so far with an increase in the use of in-silico methods in the recent past. But cell permeability barriers and conformational changes of MurD enzyme during catalytic reaction need to be addressed for effective drug development. So, a combination of in-silico methods along with experimental work is proposed to counter the catalytic machinery of MurD enzyme.

Identification of promising molecules against MurD ligase from Acinetobacter baumannii: insights from comparative protein modelling, virtual screening, molecular dynamics simulations and MM/PBSA analysis.

Acinetobacter baumannii, an opportunistic bacterium of the multidrug-resistant (MDR) ESKAPE family of pathogens, is responsible for 2-10% infections associated with all gram-negative bacteria. The hospital-acquired nosocomial infections caused by A.baumannii include deadly diseases like ventilator-associated pneumonia, bacteremia, septicemia and urinary tract infections (UTI). Over the last 3 years, it has evolved into multiple strains demonstrating high antibiotic resistance against a wide array of antibiotics. Hence, it becomes imperative to identify novel drug-like molecules to treat such infections effectively. UDP-N-acetylmuramoyl-L-alanine-D-glutamate ligase (MurD) is an essential enzyme of the Mur family which is responsible for peptidoglycan biosynthesis, making it a unique and ideal drug target. Initially, a homology modelling approach was employed to predict the three-dimensional model of MurD from A. baumannii using MurD from Escherichia coli (PDB ID: 4UAG) as a suitable structural template. Subsequently, an optimised model of MurD was subjected to virtual high-throughput screening (vHTS) against a ZINC library of ~ 642,759 commercially available molecules to identify promising lead compounds demonstrating high binding affinities towards it. From the screening process, four promising molecules were identified based on the estimated binding affinities (ΔG), estimated inhibition constants (Ki), catalytic residue interactions and drug-like properties, which were then subjected to molecular dynamics (MD) simulation studies to reflect the physiological state of protein molecules in vivo equivalently. The binding free energies of the selected MurD-ligand complexes were also calculated using MM/PBSA (molecular mechanics with Poisson-Boltzmann and surface area solvation) approach. Finally, the global dynamics along with binding free energy analysis suggested that ZINC19221101 (ΔG = - 62.6 ± 5.6 kcal/mol) and ZINC12454357 (ΔG = - 46.1 ± 2.6 kcal/mol) could act as most promising candidates for inhibiting the function of MurD ligase and aid in drug discovery and development against A.baumannii. Graphical abstract.

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

The 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.

Molecular modeling, simulation and virtual screening of MurD ligase protein from Salmonella typhimurium LT2.

The Mur enzymes of the peptidoglycan biosynthesis pathway constitute ideal targets for the design of new classes of antimicrobial inhibitors in Gram-negative bacteria. We built a homology model of MurD of Salmonella typhimurium LT2 using MODELLER (9v12) software. 'The homology model was subjected to energy minimization by molecular dynamics (MD) simulation study with GROMACS software for a simulation time of 20 ns in water environment. The model was subjected for virtual screening study from the Zinc Database using Dockblaster software. Inhibition assay for the best inhibitor, 3-(amino methyl)-n-(4-methoxyphenyl) aniline, by flow cytometric analysis revealed the effective inhibition of peptidoglycan biosynthesis. Results from this study provide new insights for the molecular understanding and development of new antibacterial drugs against the pathogen.

A novel 2-oxoindolinylidene inhibitor of bacterial MurD ligase: Enzyme kinetics, protein-inhibitor binding by NMR and a molecular dynamics study.

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.

Structure-activity relationships of new cyanothiophene inhibitors of the essential peptidoglycan biosynthesis enzyme MurF.

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.

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.