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Dual Inhibitor of MurD and MurE Ligases from Escherichia coli and Staphylococcus aureus
Abstract
MurD and MurE ligases, consecutive enzymes participating in the intracellular steps of bacterial peptidoglycan biosynthesis, are important targets for antibacterial drug discovery. We have designed, synthesized, and evaluated the first d-glutamic acid-containing dual inhibitor of MurD and MurE ligases from Escherichia coli and Staphylococcus aureus (IC50 values between 6.4 and 180 μM) possessing antibacterial activity against Gram-positive S. aureus and its methicillin-resistant strain (MRSA) with minimal inhibitory concentration (MIC) values of 8 μg/mL. The inhibitor was also found to be noncytotoxic for human HepG2 cells at concentrations below 200 μM.
... Rhodanines are also known as analytical chelating agents [5]. They showed significant biological effects, e.g., antiviral [6], and antibacterial activity linked to their ability to inhibit DNA gyrase [7], β-lactamase [8], peptide deformylase [9], glucosyltransferase MurG [10] or ligases MurD [11][12][13][14] and MurE [14]. ...
... Rhodanines are also known as analytical chelating agents [5]. They showed significant biological effects, e.g., antiviral [6], and antibacterial activity linked to their ability to inhibit DNA gyrase [7], β-lactamase [8], peptide deformylase [9], glucosyltransferase MurG [10] or ligases MurD [11][12][13][14] and MurE [14]. ...
... In the works of other authors, the biological activity of rhodanines and some related heterocycles (e.g., thiazolidine-2,4-diones, thiobarbituric acids, 2-iminothiazolidin-4-one, imidazolidine-2,4-dione) was often compared. The presence of rhodanine moiety is highlighted in some studies [8,[12][13][14][42][43][44]69], but also the side chain might be critical [8][9][10]12,53,54,67], as well as the molecule shape determined by the alkylidene rhodanine substitution [7,13]. In another study, there was no significant difference in biological activity between derivatives of rhodanine and thiazolidine-2,4-dione [11]. ...
(4-Oxo-2-thioxothiazolidin-3-yl)acetic acids exhibit a wide range of pharmacological activities. Among them, the only derivative used in clinical practice is the aldose reductase inhibitor epalrestat. Structurally related compounds, [(5Z)-(5-arylalkylidene-4-oxo-2-thioxo-1,3-thiazolidin-3-yl)]acetic acid derivatives were prepared previously as potential antifungal agents. This study was aimed at the determination of aldose reductase inhibitory action of the compounds in comparison with epalrestat and evaluation of structure-activity relationships (SAR).
The aldose reductase (ALR2) enzyme was isolated from the rat eye lenses, while aldehyde reductase (ALR1) was obtained from the kidneys. The compounds studied were found to be potent inhibitors of ALR2 with submicromolar IC50 values. (Z)-2-(5-(1-(5-butylpyrazin-2-yl)ethylidene)-4-oxo-2-thioxothiazolidin-3-yl)acetic acid (3) was identified as the most efficacious inhibitor (over five times more potent than epalrestat) with mixed-type inhibition. All the compounds also exhibited low antiproliferative (cytotoxic) activity to the HepG2 cell line. Molecular docking simulations of 3 into the binding site of the aldose reductase enzyme identified His110, Trp111, Tyr48, and Leu300 as the crucial interaction counterparts responsible for the high-affinity binding. The selectivity factor for 3 in relation to the structurally related ALR1 was comparable to that for epalrestat. SAR conclusions suggest possible modifications to improve further inhibition efficacy, selectivity, and biological availability in the group of rhodanine carboxylic acids.
... In the present study, we performed homology modelling to prepare the S. aureus MurD catalytic pocket 3D structure. For the present study we selected 2-thioxothiazolidin-4-one based MurD inhibitor $1 ( Figure 1) possessing IC 50 in the micromolar range (6.40 µM) [36]. In addition, this inhibitor showed potent inhibitory activity against S. aureus ATCC 29213 and methicillin-resistant S. aureus (MRSA) ATCC 43300 with MIC values of 8 μg/mL. ...
... The 3D-structure of inhibitor $1 (Figure 1) was sketched in the builder panel of Maestro 11.2 and optimized with the LigPrep (Schrödinger 2017-2). The S enantiomer at the chiral centre in the D-Glu structural component was chosen since it has the same chirality as the inhibitor $1 in the E. coli MurD crystal structure (PDB-ID: 2Y1O) [36]. The energy minimization was carried out with OPLS3 force field [37] until RMSD of 0.01 Å was reached. ...
... To measure the strength of inhibitor $1 and modelled protein interactions, the binding free energy (ΔG bind ) was calculated by the MM-GBSA method using the Prime (v4.8) [36]. The energy of the docked pose of the ligand-protein complex was minimized using the local optimization feature in Prime (v4.8) and then simulation was performed with the VSGB 2.0 energy model [46] using input ligand partial charges without applying any constraint on flexible residues. ...
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.
... Important developments of MurD inhibitors have been accomplished by Tomašić et al. [54][55][56] and Zidar et al. [57,58] working within the research groups of Mašič and Kikelj. Tomašić et al. [54] obtained a number of hydroxy-substituted 5-benzylidenethiazolidin-4ones. ...
... Recently, Tomašić et al. [56] have formulated, synthesized, and assessed the first D-glutamic acid- containing dual inhibitor (16) (Chart 5), of MurD and MurE ligases from E. coli, as well as S. aureus. These compounds exhibited IC 50 from 6.4 to 180 µM and presented antimicrobial activity against gram-positive Staphylococcus aureus and its methicillin-resistant strain (MRSA) with MIC of 8 µg/mL [56]. ...
... Recently, Tomašić et al. [56] have formulated, synthesized, and assessed the first D-glutamic acid- containing dual inhibitor (16) (Chart 5), of MurD and MurE ligases from E. coli, as well as S. aureus. These compounds exhibited IC 50 from 6.4 to 180 µM and presented antimicrobial activity against gram-positive Staphylococcus aureus and its methicillin-resistant strain (MRSA) with MIC of 8 µg/mL [56]. The most promising compounds had a sulfur atom at the position of X in the tiazolone ring. ...
Infectious diseases are one of the most important and urgent health problems in the world. According to the World Health Organization (WHO) statistics, infectious and parasitic diseases are a cause of about 16% of all deaths worldwide and over 40% of deaths in Africa. A considerable progress that has been made during last hundred years in the fight against infectious diseases, in particular bacterial infections, can be attributed mainly to three factors: (1) the general improvement of living conditions, in particular sanitation; (2) development of vaccines and (3) development of efficient antibacterial drugs. Although considerable progress in reduction of the number of cases of bacterial infections, especially in lethal cases, has been made, continued cases and outbreaks of these diseases persist, which is caused by different contributing factors. Indeed, during last sixty years antibacterial drugs were used against various infectious diseases caused by bacterial pathogens with an undoubtable success. The most fruitful period for antibiotic development lasted from 40's to 60's of the last century and resulted in the majority of antibiotics currently on the market, which were obtained by screening actinomycetes derived from soil. Although the market for antibacterial drugs is nowadays greater than 25 billion US dollars per year, novel antibacterial drugs are still demanded due to developed resistance of many pathogenic bacteria against current antibiotics. In last five years one can observe a dramatic increase in cases of resistant bacteria strains (eg. Klebsiella pneumoniae and E. coli) which are responsible for difficult to treat pneumonia and infections of urinary tract. The development of resistant bacteria strains is a side effect of antibiotic application for treatment: the infections become untreatable as a result of the existence of antibiotic-tolerant persisters. In this review, we discuss the challenges in antibacterial drug discovery, including the molecular basis of drug resistance, drug targets for novel antibacterial drugs, and new compounds (since year 2010) from different chemical classes with antibacterial activity, focusing on structure-activity relationships.
... A frequently used starting point in the design of MurD enzyme inhibitors was the hypothetical structure of the MurD tetrahedral reaction intermediate [4,5,18]. From this starting point, many low-molecularweight analogues of the tetrahedral intermediate structure were published, typically classified as derivatives of the Dglutamic acid (Fig. 1) [16,17,19,20]. Crystal structures of the MurD enzyme bound with these compounds have provided insight into binding modes of several inhibitors [17,19,20]. ...
... From this starting point, many low-molecularweight analogues of the tetrahedral intermediate structure were published, typically classified as derivatives of the Dglutamic acid (Fig. 1) [16,17,19,20]. Crystal structures of the MurD enzyme bound with these compounds have provided insight into binding modes of several inhibitors [17,19,20]. Binding free energies calculated by linear interaction energy (LIE) approach revealed non-polar van der Waals interactions as the main driving force responsible for the binding of these inhibitors [21,22] Recently, a drug design strategy using multiple protein structures of the MurD enzyme focusing of the ATP binding site, depicted several challenges that need to be addressed when trying to hit a flexible moving target such as the bacterial MurD enzyme for the identification of novel MurD ligase inhibitors [23]. ...
... Based on the available MurD crystal structures cocrystallized with N-sulfonyl glutamic acid inhibitors [19,20], a virtual screening campaign was performed, combining three-dimensional structure-based pharmacophores and molecular docking calculations. A novel class of glutamic acid surrogates-benzene 1,3-dicarboxylic acid derivatives-was identified: compound 1 with incorporated furan moiety was found to possess promising dual MurD and MurE inhibitory activity [24,25]. ...
Bacterial resistance to the available antibiotic agents underlines an urgent need for the discovery of novel antibacterial agents. Members of the bacterial Mur ligase family MurC-MurF involved in the intracellular stages of the bacterial peptidoglycan biosynthesis have recently emerged as a collection of attractive targets for novel antibacterial drug design. In this study, we have first extended the knowledge of the class of furan-based benzene-1,3-dicarboxylic acid derivatives by first showing a multiple MurC-MurF ligase inhibition for representatives of the extended series of this class. Steady-state kinetics studies on the MurD enzyme were performed for compound 1, suggesting a competitive inhibition with respect to ATP. To the best of our knowledge, compound 1 represents the first ATP-competitive MurD inhibitor reported to date with concurrent multiple inhibition of all four Mur ligases (MurC-MurF). Subsequent molecular dynamic (MD) simulations coupled with interaction energy calculations were performed for two alternative in silico models of compound 1 in the UMA/D-Glu- and ATP-binding sites of MurD, identifying binding in the ATP-binding site as energetically more favorable in comparison to the UMA/D-Glu-binding site, which was in agreement with steady-state kinetic data. In the final stage, based on the obtained MD data novel furan-based benzene monocarboxylic acid derivatives 8-11, exhibiting multiple Mur ligase (MurC-MurF) inhibition with predominantly superior ligase inhibition over the original series, were discovered and for compound 10 it was shown to possess promising antibacterial activity against S. aureus. These compounds represent novel leads that could by further optimization pave the way to novel antibacterial agents.
... Herein, a thiazolidinylidene-based compound (T26) was adopted as a positive control as a MurE inhibitor. The reference compound was reported with high dual inhibition activities towards MurE and MurD from S. aureus (IC 50 = 17.0 µM and 6.4 µM, respectively) [83]. Reported studies highlighted close similarity between the MurE secondary structure originating from MRSA and E. Coli microorganisms (RMSD 1.48 Å along > 450 Cα-atoms and Z-score 21.2) [60,84]. ...
... Reported studies highlighted close similarity between the MurE secondary structure originating from MRSA and E. Coli microorganisms (RMSD 1.48 Å along > 450 Cα-atoms and Z-score 21.2) [60,84]. Furthermore, T26 highlighted great antibacterial activity against MRSA and its wildtype strain with a minimum inhibition concentration of 9.0 µg/mL [83]. Concerning PBP2a, the co-crystallized cephalosporin antibiotic, ceftaroline [85], was a relevant positive control. ...
Recently, there has been a surge towards searching for primitive treatment strategies to discover novel therapeutic approaches against multi-drug-resistant pathogens. Endophytes are considered unexplored yet perpetual sources of several secondary metabolites with therapeutic significance. This study aims to isolate and identify the endophytic fungi from Annona squamosa L. fruit peels using morphological, microscopical, and transcribed spacer (ITS-rDNA) sequence analysis; extract the fungus’s secondary metabolites by ethyl acetate; investigate the chemical profile using UPLC/MS; and evaluate the potential antibacterial, antibiofilm, and antiviral activities. An endophytic fungus was isolated and identified as Aspergillus flavus L. from the fruit peels. The UPLC/MS revealed seven compounds with various chemical classes. The antimicrobial activity of the fungal ethyl acetate extract (FEA) was investigated against different Gram-positive and Gram-negative standard strains, in addition to resistant clinical isolates using the agar diffusion method. The CPE-inhibition assay was used to identify the potential antiviral activity of the crude fungal extract against low pathogenic human coronavirus (HCoV 229E). Selective Gram-positive antibacterial and antibiofilm activities were evident, demonstrating pronounced efficacy against both methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-sensitive Staphylococcus aureus (MSSA). However, the extract exhibited very weak activity against Gram-negative bacterial strains. The ethyl acetate extract of Aspergillus flavus L exhibited an interesting antiviral activity with a half maximal inhibitory concentration (IC50) value of 27.2 µg/mL against HCoV 229E. Furthermore, in silico virtual molecular docking-coupled dynamics simulation highlighted the promising affinity of the identified metabolite, orienting towards three MRSA biotargets and HCoV 229E main protease as compared to reported reference inhibitors/substrates. Finally, ADME analysis was conducted to evaluate the potential oral bioavailability of the identified metabolites.
... The reference compound was reported with high dual inhibition activities towards MurE and MurD from Staphylococcus aureus (IC50 = 17.0 µM and 6.4 µM, respectively) and Escherichia Coli (IC50 = 180.0 µM and 8.2 µM, respectively) based on radioactivity inhibition assays [88]. Reported studies highlighted the close similarity between MurE secondary structure originating from MRSA and Escherichia Coli microorganisms (RMSD 1.48 Å along > 450 Cα-atoms and Z-score 21.2) [78,89]. ...
... Reported studies highlighted the close similarity between MurE secondary structure originating from MRSA and Escherichia Coli microorganisms (RMSD 1.48 Å along > 450 Cα-atoms and Z-score 21.2) [78,89]. Furthermore, T26 highlighted great antibacterial activity against MRSA and its wild-type strain with MIC of 9.0 µg/ml [88]. Docking of T26 at MRSA MurE highlighted dominant electrostatic potentiality guiding its anchoring at the substrate site with interactions with Thr46 (2.6 Å; 159.1°), ...
Nanocomposite alginate hydrogel containing Propranolol hydrochloride (PNL) cerosomes (CERs) was prepared as a repurposed remedy for topical skin Methicillin-Resistant Staphylococcus aureus (MRSA) infection. CERs were formed via an ethanol injection technique using different ceramides, Kolliphores ® as a surfactant, and Didodecyldimethylammonium bromide (DDAB) as a positive charge inducer. CERs were optimized utilizing 1 ³ . 2 ² mixed-factorial design employing Design-Expert ® software, the assessed responses were entrapment efficiency (EE%), particle size (PS), and zeta potential (ZP). The optimum CER, composed of 5 mg DDAB, ceramide VI, and Kolliphor ® RH40 showed tubular vesicles with EE% of 92.91 ± 0.98%, PS of 388.75 ± 18.99 nm, PDI of 0.363 ± 0.01, and ZP of 30.36 ± 0.69 mV. Also, it remained stable for 90 days and manifested great mucoadhesive aspects. The optimum CER was incorporated into calcium alginate to prepare nanocomposite hydrogel. The ex-vivo evaluation illustrated that PNL was permeated in a more prolonged pattern from PNL-loaded CERs nanocomposite related to PNL-composite, optimum CER, and PNL solution. Confocal laser scanning microscopy revealed a perfect accumulation of fluorescein-labeled CERs in the skin. The in-silico investigation illustrated that the PNL was stable when mixed with other ingredients in the CERs and confirmed that PNL is a promising candidate for curing MRSA . Moreover, the PNL-loaded CERs nanocomposite revealed superiority over the PNL solution in inhibiting biofilm formation and eradication. The PNL-loaded CERs nanocomposite showed superiority over the PNL-composite for treating MRSA infection in the in-vivo mice model. Histopathological studies revealed the safety of the tested formulations. In conclusion, PNL-loaded CERs nanocomposite provided a promising, safe cure for MRSA bacterial skin infection.
Graphical Abstract
... The synthesis of peptidoglycan is divided into several steps, including the assembly of 5′-diphosphate (UDP)-MurNAc pentapeptide in cells, followed by translocation through the plasma membrane and binding to the growing peptidoglycan layer. Peptidoglycan is a unique cell wall component of prokaryotic cells; therefore, there is a growing interest in the use of enzymes involved in the biosynthesis of peptidoglycan precursors [28][29][30]. ...
... The synthesis of peptidoglycan is divided into several steps, including the assembly of 5 -diphosphate (UDP)-MurNAc pentapeptide in cells, followed by translocation through the plasma membrane and binding to the growing peptidoglycan layer. Peptidoglycan is a unique cell wall component of prokaryotic cells; therefore, there is a growing interest in the use of enzymes involved in the biosynthesis of peptidoglycan precursors [28][29][30]. ...
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.
... isatins, anhydrides of pyridine-3,4-dicarboxylic and phthalic acids etc.) [27,32e38] have been used as oxo compounds. Acetic acid or its anhydride and sodium acetate; ethanol and ammonium acetate or piperidine [39]; toluene and ammonia acetate [33,40,41]; isopropanol and potassium tert-butylate [42,43]; toluene and L-proline [44]; dimethylformamide and sodium acetate; ethanol and monoethanolamine as well as the solid carriers and phasetransfer catalysts etc. [45,46] have been widely used as the medium and catalysts in this reaction. The reaction performance was also described in the medium of the aldehyde or ketone (without solvent addition) [21], polyethyleneglycol-300 [47] or based on the green chemistry approach. ...
... Novel 2-hydrazolyl-4-thiazolidinone-5,6-a,b-unsaturated esters (41) were synthesized in the multicomponent reaction of aldehydes, thiosemicarbazides and dimethylacetylenedicarboxylate in the ethanol medium. Interestingly, the reaction doesn't depend on the presence of electron-withdrawing or electron donating groups. ...
The presented review is an attempt to summarize a huge volume of data on 5-ene-4-thiazolidinones being a widely studied class of small molecules used in modern organic and medicinal chemistry. The manuscript covers approaches to the synthesis of 5-ene-4-thiazolidinone derivatives: modification of the C5 position of the basic core; synthesis of the target compounds in the one-pot or multistage reactions or transformation of other related heterocycles. The most prominent pharmacological profiles of 5-ene derivatives of different 4-thiazolidinone subtypes belonging to hit-, lead-compounds, drug-candidates and drugs as well as the most studied targets have been discussed. Currently target compounds (especially 5-en-rhodanines) are assigned as frequent hitters or pan-assay interference compounds (PAINS) within high-throughput screening campaigns. Nevertheless, the crucial impact of the presence/nature of C5 substituent (namely 5-ene) on the pharmacological effects of 5-ene-4-thiazolidinones was confirmed by the numerous listed findings from the original articles. The main directions for active 5-ene-4-thiazolidinones optimization have been shown: i) complication of the fragment in the C5 position; ii) introduction of the substituents in the N3 position (especially fragments with carboxylic group or its derivatives); iii) annealing in complex heterocyclic systems; iv) combination with other pharmacologically attractive fragments within hybrid pharmacophore approach. Moreover, the utilization of 5-ene-4-thiazolidinones in the synthesis of complex compounds with potent pharmacological application is described. The chemical transformations cover mainly the reactions which involve the exocyclic double bond in C5 position of the main core and correspond to the abovementioned direction of the 5-ene-4-thiazolidinone modification.
... The binding modes of all of the benzylidene-2,4-thiazolidin-dione and 2-thioxothiazolidin-4-one inhibitors are very similar. The representative binding mode of compound 49 is shown in Fig. 9B [69]. This binding mode revealed that the 2,4-thiazolidin-dione and 2-thioxothiazolidin-4one rings are bound into the uracil binding site, for interactions with Arg35, Thr36 and Asp37. ...
... A comparison of the crystal structures of a thiazolidindione inhibitor (PDB entry: 2X5O) [64] and UDP-MurNAc-L-Ala-D-Glu (PDB entry: 4UAG) [70] with E. coli MurD reveals that this thiazolidin-dione inhibitor is bound to the binding site of the product. In addition to the crystal structure, the main feature of compound 49 is its dual inhibitory activity against MurD and MurE from both E. coli and S. aureus, as well as its antibacterial activity against MRSA [69]. ...
... Further, Figure 10, Compound 30 shows the most potent compound of the series, which inhibits MurD ligases from E. coli and S. aureus with IC 50 values of 8.2 and 6.4 µM, respectively. Additionally, it showed inhibitory activity against MurE ligases from E. coli and S. aureus with IC 50 values of 180 µM and 17 µM, respectively, thus acting as a dual inhibitor of the intracellular steps of peptidoglycan biosynthesis [76]. ...
Mur enzymes serve as critical molecular devices for the synthesis of UDP-MurNAc-pentapeptide, the main building block of bacterial peptidoglycan polymer. These enzymes have been extensively studied for bacterial pathogens such as Escherichia coli and Staphylococcus aureus. Various selective and mixed Mur inhibitors have been designed and synthesized in the past few years. However, this class of enzymes remains relatively unexplored for Mycobacterium tuberculosis (Mtb), and thus offers a promising approach for drug design to overcome the challenges of battling this global pandemic. This review aims to explore the potential of Mur enzymes of Mtb by systematically scrutinizing the structural aspects of various reported bacterial inhibitors and implications concerning their activity. Diverse chemical scaffolds such as thiazolidinones, pyrazole, thiazole, etc., as well as natural compounds and repurposed compounds, have been reviewed to understand their in silico interactions with the receptor or their enzyme inhibition potential. The structural diversity and wide array of substituents indicate the scope of the research into developing varied analogs and providing valuable information for the purpose of modifying reported inhibitors of other multidrug-resistant microorganisms. Therefore, this provides an opportunity to expand the arsenal against Mtb and overcome multidrug-resistant tuberculosis.
... Several drug targets are available for MRSA treatment, including a negatively charged surface, GAPDH (glyceraldehydes-3-phosphate dehydrogenase) receptor found on the cell membrane of MRSA which acts as a drug target for transferrin; PBP2A enzyme inhibition [75]; Panton valentine leukocidin toxin of CA-MRSA [76]; Muramyl ligase E or Mur E is an enzyme which is concerned with the biosynthesis of peptidoglycan of the microbial cell wall could be the most suitable hit for Methicillin-Resistant Staphylococcus aureus [77]. ...
Background
Multidrug-resistant (MDR) methicillin-resistant Staphylococcus aureus (MRSA) has become a prime health concern globally. These bacteria are found in hospital areas where they are regularly dealing with antibiotics. This brings many possibilities for its mutation, so drug resistance occurs.
Introduction
Nowadays, these nosocomial MRSA strains spread into the community and live stocks. Resistance in Staphylococcus aureus is due to mutations in their genetic elements.
Methods
As the bacteria become resistant to antibiotics, new approaches like antimicrobial peptides (AMPs) play a vital role and are more efficacious, economical, time, and energy saviours.
Result
Machine learning approaches of Artificial Intelligence are the in-silico technique which has their importance in better prediction, analysis, and fetching of important details regarding AMPs.
Conclusion
Anti-microbial peptides could be the next-generation solution to combat drug resistance among Superbugs. For better prediction and analysis, implementing the in-silico technique is beneficial for fast and more accurate results.
... In this milieu, utilizing Mur enzymes (Mur A-F) as drug targets has steadily increased as it is recognized that all these enzymes are essential for the early steps of PG biosynthesis and vital for bacterial survival. Though studies have been conducted on several Mur ligases including MurB to design inhibitors, however, currently, compounds with significant inhibition are not available [2,[6][7][8][9][10][11][12][13]. Therefore, there exists a pressing need to develop novel antibacterial agents against the MurB enzyme to overcome the hurdles of drug-resistant pathogens. ...
The increase of antibiotic‐resistant bacterial pathogens has created challenges in treatment and warranted the design of antibiotics against comparatively less exploited targets. The peptidoglycan (PG) biosynthesis delineates unique pathways for the design and development of a novel class of drugs. Mur ligases are an essential component of bacterial cell wall synthesis that play a pivotal role in peptidoglycan (PG) biosynthesis to maintain internal osmotic pressure and cell shape. Inhibition of these enzymes can interrupt bacterial replication and form attractive targets for drug discovery. In the present work, we focused on the PG biosynthesis pathway enzyme, UDP‐N‐acetylpyruvylglucosamine reductase, from Salmonella enterica serovar Typhi (stMurB). Biophysical characterization of purified StMurB was performed to gauge the molecular interactions and estimate thermodynamic stability for determination of attributes for possible therapeutic intervention. The thermal melting profile of MurB was monitored by circular dichroism (CD) and validated through Differential Scanning Calorimetry (DSC) experiment. Frequently used chemical denaturants, GdmCl and urea, were employed to study the chemical‐induced denaturation of stMurB. In the search for natural compound based inhibitors, against this important drug target, an in silico virtual screening based investigation was conducted with modeled stMurB structure. The three top hits (quercetin, berberine, and scopoletin) returned were validated for complex stability through Molecular Dynamics (MD) Simulation. Further, fluorescence binding studies were undertaken for the selected natural compounds with stMurB alone and with NADPH bound form. The compounds scopoletin and berberine, displayed lesser binding to stMurB whereas quercetin exhibited stronger binding affinity than NADPH. This study suggests that quercetin can be evolved as an inhibitor of stMurB enzyme. This article is protected by copyright. All rights reserved.
... The most powerful change is achieved by designing and synthetizing a NH-CH 2 group to connect the two inverted benzene ring compounds, which can greatly increase hydrophobic interactions and hydrogen bonds through water molecules. It can also inhibit MurE as well and showed antibacterial activity against MRSA (MIC, 8 μg/ml; Tomasic et al., 2012). 5-benzylidenethiazolidin-4-one can inhibit MurD, based on which several different compounds were designed and optimized. ...
Since the advent of penicillin, humans have known about and explored the phenomenon of bacterial inhibition via antibiotics. However, with changes in the global environment and the abuse of antibiotics, resistance mechanisms have been selected in bacteria, presenting huge threats and challenges to the global medical and health system. Thus, the study and development of new antimicrobials is of unprecedented urgency and difficulty. Bacteria surround themselves with a cell wall to maintain cell rigidity and protect against environmental insults. Humans have taken advantage of antibiotics to target the bacterial cell wall, yielding some of the most widely used antibiotics to date. The cell wall is essential for bacterial growth and virulence but is absent from humans, remaining a high-priority target for antibiotic screening throughout the antibiotic era. Here, we review the extensively studied targets, i.e., MurA, MurB, MurC, MurD, MurE, MurF, Alr, Ddl, MurI, MurG, lipid A, and BamA in the cell wall, starting from the very beginning to the latest developments to elucidate antimicrobial screening. Furthermore, recent advances, including MraY and MsbA in peptidoglycan and lipopolysaccharide, and tagO, LtaS, LspA, Lgt, Lnt, Tol-Pal, MntC, and OspA in teichoic acid and lipoprotein, have also been profoundly discussed. The review further highlights that the application of new methods such as macromolecular labeling, compound libraries construction, and structure-based drug design will inspire researchers to screen ideal antibiotics.
... Peptidoglycan is one of the main components of the bacterial cell wall, and it represents one of the most frequently used targets for antibacterial agents (Simcic et al. 2012). The mechanism of the peptidoglycan biosynthesis is one the best known and most validated drug targets for antibacterial therapy (Tomašic et al. 2012). The fact that Mur ligases are unique to prokaryotic cells and are vital for the survival of bacteria makes them promising emerging targets for novel antibacterial drug design (Kouidmi et al. 2014). ...
During the last decade there has been an alarming increase in the appearance of antibiotic-resistant bacteria. The drug-resistant microorganisms dubbed superbugs are projected to kill 10 million people a year by 2050. The annual frequency of deaths from Methicillin-resistant Staphylococcus aureus (MRSA) is rapidly increasing and surpassing those caused by human immunodeficiency virus/acquired immune deficiency syndrome (HIV/AIDS). Muramyl ligase E (MurE), an enzyme involved in the peptidoglycan biosynthesis of the bacterial cell wall, is a highly druggable target in MRSA. Six anti Methicillin-resistant Staphylococcus Aureus peptides were selected for deep bioinformatic analysis. The six anti-Methicillin-resistant Staphylococcus Aureus peptides were modeled and then docked to MurE ligase and their binding interactions were studied. The findings suggested that the interactions of three antimicrobial peptides (Flowlicidin-2, Ostricacin-2 and Ostricacin-3) with MurE could be essential for their inhibitory activity.
... From the virtual screening campaign benzene-1,3-dicarboxylic acid analogue has been identified as a conformational rigid mimetic of glutamic acid. Further modifications has been done by linking the 1,3 dicarboxylic with five member heterocyclic ring which was further linked with five or six member rings through methelene bridge [35]. The first series, where compound 11 has been identified as first dual MurD/MurE hits possess 2,5-dimethyl pyrrole moiety, where the methelene group was attached with 1-phenyl substituted dihydropyrimidine-4,6-dione and the other side was linked with 1,3-dicarboxylic acid. ...
Peptidoglycan, an essential component of the bacterial cell wall plays a critical role in protecting bacteria against osmotic lysis. The ATP-dependent MurC-F ligases are crucial for the early stages of peptidoglycan biosynthesis. MurE ligase is third in the series and catalyzes the addition of l-Lysine (l-Lys) in Gram-positive bacteria or meso-diaminopimelic acid (meso-A2pm) in most Gram-negative bacteria to form UDP-N-acetylmuramoyl-l-Ala-d-Glu-l-Lys/A2pm. The high substrate specific for l-Lys or meso-A2pm makes this enzyme an attractive target for the development of antibacterial agents. Several MurE inhibitors have been reported including phosphinates, peptidosulfonamides, napthylfuran-2-ones, benzene-1,3-dicarboxylic acids, phosphorylatedhydroxyethylamines, natural compounds, 5-benzylidenethiazolidin-4-ones, N-alkyl-2-alkynyl-4(1H)-quinolones, rhodanine substituted d-glutamic acids, 2,5-dimethyl pyrroles, 2,5-disubstitued furans, tetrahydroisoquinolines etc. In the present review we present an update status and structural information of MurE enzyme inhibitors which may be utilized for the design of potent inhibitors against this enzyme.
... Interestingly the L-glutamic derivative (54) was more potent than the corresponding Dglutamic derivative (E. coli MurD IC 50 = 10 μM and 45 μM) [70]. Inversion of the NH-CH group connecting the two phenyl rings provided a dual MurD and MurE inhibitor (55) that showed antibacterial activity against methicillin-resistant S. aureus (MRSA) (Fig. 19) [71]. ...
The introduction of antibiotics to treat bacterial infections either by killing or blocking their growth has been accompanied by the studies of mechanism that allows the drugs to kill the bacteria or to stop their proliferation. In such scenario the coming of antibacterial agents active on the bacterial cell wall has been of fundamental importance in the fight against bacterial agents responsible for severe diseases. As a matter of fact cell wall, that plays many roles during lifecycle, is an essential constituent of most bacteria. This overview focus on the intracellular steps of peptidoglycan biosynthesis and the research of new antibacterial agents based on the enzymes involved in these early steps of the formation of cell membrane components.
... Methods such as whole-cell-based screening, combinatorial synthetic chemistry, and target-based high-throughput screening (HTS) have been followed widely for the identification of potential inhibitors. These methods have led to the identification of several Mur enzyme inhibitors from other organisms 24,39,40 and many antitubercular scaffolds that are currently being evaluated in different stages of clinical trial. 41 Bedaquiline is one such example of HTS-based identification of an antitubercular drug, which works by inhibiting the ATP synthase enzyme in Mtb. ...
The rapid rise in the emergence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) strains of Mycobacterium tuberculosis (Mtb) mandates the discovery of novel tuberculosis (TB) drugs. Mur enzymes, which are identified as essential proteins in Mtb and catalyze the cytoplasmic steps in the peptidoglycan biosynthetic pathway, are considered potential drug targets. However, none of the clinical drugs have yet been developed against these enzymes. Hence, the aim of this study was to identify novel inhibitors of Mur enzymes in Mycobacterium tuberculosis. We screened an antitubercular compound library of 684 compounds, using MurB and MurE enzymes of the Mtb Mur pathway as drug targets. For experimental validation, the top hits obtained on in silico screening were screened in vitro, using Mtb Mur enzyme-specific assays. In all, seven compounds were found to show greater than 50% inhibition, with the highest inhibition observed at 77%, and the IC 50 for these compounds was found to be in the range of 28–50 μM. Compound 5175112 showed the lowest IC 50 (28.69 ± 1.17 μM), and on the basis of (1) the binding affinity, (2) the stability of interaction noted on molecular dynamics simulation, and (3) an in vitro assay, MurE appeared to be its target enzyme. We believe that the overall strategy followed in this study and the results obtained are a good starting point for developing Mur enzyme-specific Mtb inhibitors.
... The Mur ligase family provides an attractive collection of emerging drug targets, and it has been heavily investigated over the last decade, with novel and diverse classes of inhibitors discovered [11][12][13][14][15][16][17] . As all of the Mur ligases considered here (i.e. ...
The Mur ligases form a series of consecutive enzymes that participate in the intracellular steps of bacterial peptidoglycan biosynthesis. They therefore represent interesting targets for antibacterial drug discovery. MurC, D, E and F are all ATP-dependent ligases. Accordingly, with the aim being to find multiple inhibitors of these enzymes, we screened a collection of ATP-competitive kinase inhibitors, on Escherichia coli MurC, D and F, and identified five promising scaffolds that inhibited at least two of these ligases. Compounds 1, 2, 4 and 5 are multiple inhibitors of the whole MurC to MurF cascade that act in the micromolar range (IC50, 32–368 µM). NMR-assisted binding studies and steady-state kinetics studies performed on aza-stilbene derivative 1 showed, surprisingly, that it acts as a competitive inhibitor of MurD activity towards D-glutamic acid, and additionally, that its binding to the D-glutamic acid binding site is independent of the enzyme closure promoted by ATP.
... However, only a few of these compounds exhibited weak activity against S. aureus and Enterococcus faecalis. Further, 2-thioxothiazolidin-4-one-based inhibitors with IC 50 in the micromolar range against S. aureus MurD enzyme is reported [38]. One of these compounds exhibited significant inhibitory activity against methicillinresistant S. aureus (MRSA) ATCC 43300 and S. aureus ATCC 29213 with MIC values of 8 μg/mL. ...
The MurD enzyme of Staphylococcus aureus is an attractive drug target as it is essential and ubiquitous in bacteria but absent in mammalian cells. In the present study, we performed in silico high-throughput virtual screening with small molecule library of 1.60 million compounds to identify potential hits. We used S. aureus modeled MurD protein for this purpose and to find the best leads, dock complexes were further subjected to the extra-precision docking and binding free energy calculations by MM-GBSA approach. It is evident that van der Waals and Coulomb energy terms are major favorable contributors while electrostatic solvation energy term strongly disfavors the binding of ligands to the S. aureus MurD enzyme. The inhibitory activity of two selected virtual hits H5 and H10 was performed against S. aureus MurD enzyme using malachite green assay. In in vitro antibacterial screening, compound H5 inhibited the growth of S. aureus NCIM 5021, S. aureus NCIM 5022, and methicillin-resistant S. aureus (MRSA strain 43300) at high concentrations while the other tested compound H10 was inactive against all the tested strains. In order to validate the stability of inhibitor-protein complex, compound H5 with the highest inhibitory against S. aureus MurD and lowest binding free energy was subjected to 30-ns molecular dynamics simulation. Further, ADMET predictions showed the favorable pharmacokinetic profile of compounds H5 and H10.
... Instead of scanning all available chemical databases, here we employed heuristic approach starting from compounds with known activity. Penicillin (anti-bacterial) and curcumin (anti-fungal) (Fig. 4C) are known inhibitors of MurD and MurE ligases and pectate lyase, respectively (Cho et al., 2006;Tomašić et al., 2012). Their structures were used to generate the various compounds modified with the combinations of halogen elements (Br, Cl, F and I) using ACD Chemsketch (Version 11, Advanced Chemistry Development, Inc., Toronto, ON, Canada, http://www. ...
Control of plant diseases is largely dependent on use of agrochemicals. However, there are widening gaps between our knowledge on plant diseases gained from genetic/mechanistic studies and rapid translation of the knowledge into target-oriented development of effective agrochemicals. Here we propose that the time is ripe for computer-aided drug discovery/design (CADD) in molecular plant pathology. CADD has played a pivotal role in development of medically important molecules over the last three decades. Now, explosive increase in information on genome sequences and three dimensional structures of biological molecules, in combination with advances in computational and informational technologies, opens up exciting possibilities for application of CADD in discovery and development of agrochemicals. In this review, we outline two categories of the drug discovery strategies: structure- and ligand-based CADD, and relevant computational approaches that are being employed in modern drug discovery. In order to help readers to dive into CADD, we explain concepts of homology modelling, molecular docking, virtual screening, and de novo ligand design in structure-based CADD, and pharmacophore modelling, ligand-based virtual screening, quantitative structure activity relationship modelling and de novo ligand design for ligand-based CADD. We also provide the important resources available to carry out CADD. Finally, we present a case study showing how CADD approach can be implemented in reality for identification of potent chemical compounds against the important plant pathogens, Pseudomonas syringae and Colletotrichum gloeosporioides.
... Identifying new series with more favorable resistance profiles is also possible and often warranted considering the importance of such targets. Identifying more dual-target opportunities would also likely mitigate resistance development [35,44,103,104]. A greater understanding of drug resistance in a relevant infectious setting as well as potential genetic interaction circuits that can be pharmacologically interdicted to mitigate drug resistance also deserves greater consideration. ...
Antibiotics are the bedrock of modern medicine but their efficacy is rapidly eroding due to the alarming emergence of multi-drug resistant bacteria. To begin to address this crisis, novel antibacterial agents that inhibit bacterial-specific cellular functions essential for growth, viability, and/or pathogenesis are urgently needed. Although the genomics era has contributed greatly to identifying novel antibacterial targets, it has failed to appropriately characterize, prioritize, and ultimately exploit such targets to significantly impact antibiotic discovery. Here we describe a contemporary view of new antibacterial target discovery; one which complements existing genomics strategies with a deeply rooted and fundamental understanding of target biology in the context of genetic networks and environmental conditions to rigorously identify high potential targets, and cognate inhibitors, for consideration as antibacterial leads.
... Some of the well-known inhibitors that target MurE enzyme of other bacterial species were selected as the positive control molecules for docking studies. According to literature survey we have collected seven known inhibitors viz., Phosphinate inhibitor [13], (2S,3R,6S)-3-Fluoro-A 2 pm [14], N-Hydroxy-A 2 pm [14], CHEMBL564223 [15], 3methoxynordomesticine [16], thiazolidine-2,4-dione [17] and 5-Benzylidenethiazolidin-4-ones [18] for docking. Further we have also included vancomycin a broad spectrum antibi-otic which is currently being used as frontline therapeutic against Staph infections. ...
Background:
The prevalence of multi-drug resistance S. aureus is one most challenging task for treatment of nosocomial infections. Proteins and enzymes of peptidoglycan biosynthesis pathway are one among the well-studied targets, but many of the enzymes are unexplored as targets. MurE is one such enzyme featured to be a promising target. As MurE plays an important role in ligating the L-lys to stem peptide at third position that is crucial for peptidoglycan synthesis.
Objective:
To screen the potential MurE inhibitor.
Method:
In the current study, we have employed structure based virtual screening targeting the active site of MurE, followed by Molecular dynamics and in vitro studies.
Results:
Virtual screening resulted in successful screening of potential lead molecule ((2R)-2-[[1-[(2R)-2-(benzyloxycarbonylamino) propanoyl] piperidine-4-carbonyl]amino]-5-guanidino-pentan). The molecular dynamics of the MurE and Lead molecule complex emphasizes that lead molecule has shown stable interactions with active site residues Asp 406 and with Glu 460. In vitro studies demonstrate that the lead molecule shows antibacterial activity close to standard antibiotic Vancomycin and higher than that of Ampicillin, Streptomycin and Rifampicin. The MIC of lead molecule at 50µg/mL was observed to be 3.75 µg/mL, MBC being bactericidal with value of 6.25 µg/mL, cytotoxicity showing 34.44% and IC50 of 40.06µg/mL.
Conclusion:
These results suggest, ((2R)-2-[[1-[(2R)-2-(benzyloxycarbonylamino) propanoyl] piperidine-4-carbonyl]amino]-5-guanidino-pentan) as a promising lead molecule for developing a MurE inhibitor against treatment of S. aureus infections.
... The γ-carboxyl group interacts with Ser415, Leu416 and Phe422, while the α-carboxyl group is held in position through interactions with Thr321 and Lys348 directly. In the MurD/21 complex (PDB entry: 2Y1O), the α-carboxyl group interacts with Lys115 and Lys348 via water molecules ( Figure 3B), which is a unique property of the binding mode of inhibitor 21 (93). No polar contacts between the protein and the central linker part of the inhibitors exist. ...
Abstract 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 difficulty in evaluating the conformational distribution of proteins in solution often hinders mechanistic insights. One possible strategy for visualizing conformational distribution is distance distribution measurement by single-pair small-angle X-ray scattering (SAXS), in which the scattering interference from only a specific pair of atoms in the target molecule is extracted. Despite this promising concept, with few applications in synthetic small molecules and DNA, technical difficulties have prevented its application in protein conformational studies. This study used a synthetic tag to fix the lanthanide ion at desired sites on the protein and used single-pair SAXS with contrast matching to evaluate the conformational distribution of the multidomain protein enzyme MurD. These data highlighted the broad conformational and ligand-driven distribution shifts of MurD in solution. This study proposes an important strategy in solution structural biology that targets dynamic proteins, including multidomain and intrinsically disordered proteins.
The emergence of Multidrug Resistance (MDR) strains of bacteria has accelerated the search for new antibacterials. The specific bacterial peptidoglycan biosynthetic pathway represents opportunities for the development of novel antibacterial agents. Among the enzymes involved, Mur ligases, described herein, and especially the amide ligases MurC-F are key targets for the discovery of multi-inhibitors, as they share common active sites and structural features.
An increase in the number of antibiotic-resistant bacterial pathogens, in recent times, has posed a great challenge for treating the affected patients. This has paved the way for the development and design of antibiotics against the previously less explored newer targets. Among these, peptidoglycan (PG) biosynthesis serves as a promising target for the design and development of novel drugs. The peptidoglycan cell wall synthesis in bacteria is essential for its viability. The enzyme class, Mur ligases, plays a key role in PG biosynthesis. Therefore, compounds with the ability to inhibit these enzymes (Mur ligase) can serve as potential candidates for developing small modulators. The enzyme, UDP-N-acetyl pyruvyl-glucosamine reductase (MurB), is essential for PG biosynthesis, a crucial part of the bacterial cell wall. The development of novel drugs to treat infections may thus focus on inhibiting MurB function. Understanding the mechanism of action of Mur B is central to developing efficient inhibitors. For the treatment of S. typhi infections, it is also critical to find therapeutic drugs that specifically target MurB. The enzyme Mur B from Salmonella enterica serovar Typhi (stMurB) was expressed and purified for biophysical characterization to gauge the molecular interactions and estimate thermodynamic stability, for determining attributes for possible therapeutic intervention. The thermal melting profile of MurB was monitored by circular dichroism (CD) and validated by performing differential scanning calorimetry (DSC). An in silico virtual screening of various natural inhibitors was conducted with modelled stMurB structure. The three top hits (quercetin, berberine, and scopoletin) obtained from in silico screening were validated for complex stability through molecular dynamics (MD) simulation. Further, fluorescence binding studies were undertaken for the selected natural inhibitors with stMurB alone and with its NADPH-bound form. The natural inhibitors, scopoletin and berberine, displayed lesser binding to stMurB compared to quercetin. Also, a stronger binding affinity was exhibited between quercetin and stMurB compared to NADPH and stMurB. Based on the above two findings, quercetin can be developed as an inhibitor of stMurB enzyme.
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.
Microbial resistance towards antibiotics has become a global threat to human health. There is currently an urgent need to develop novel antibacterial and antifungal agents with new mechanisms of antimicrobial action and lower levels of toxicity. This paper reviews the structure-activity relationship as well as the antimicrobial effect of substituted rhodanine derivatives. The inhibitory effects of the substituted rhodanines on different, specific anti-bacterial targets, and the potential that rhodanine-derived compounds have to be new anti-bacterial compounds have been discussed in detail.
Antibiotic resistance is one of the biggest challenges that is escalating and affecting humanity across the globe. To overcome this increasing burden of resistance, discovering novel hits by targeting the enzymes involved in peptidoglycan (murein) biosynthesis has always been considered better in antimicrobial drug discovery. UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) enzyme has been identified as essential for Escherichia coli survival and catalyzes the early-stage step in bacterial cell wall synthesis. The present article gives a brief overview of the role of enzymes in peptidoglycan synthesis and MurA enzyme (previously known as MurZ in E. coli), in particular, including its structural and active site features. This review also provides an insight into the current knowledge of the reported MurA inhibitors, their mechanism of action and drawbacks of these hits that hinder their clinical trials, which would be helpful for synthesis and discovering potent molecules.
Supplementary information:
The online version contains supplementary material available at 10.1007/s12088-021-00988-6.
Despite their importance in function, the conformational state of proteins and its changes are often poorly understood mainly because of the lack of an efficient tool. MurD, a 47-kDa protein enzyme responsible for peptidoglycan biosynthesis, is one of those proteins whose conformational states and changes during their catalytic cycle are not well understood. Although it has been considered that MurD takes a single conformational state in solution as shown by a crystal structure, the solution NMR study suggested the existence of multiple conformational state of apo MurD in solution. However, the conformational distribution has not been evaluated. In this work we investigate the conformational states of MurD by the use of electron paramagnetic resonance (EPR), especially inter-gadolinium distance measurement using double electron–electron resonance (DEER) measurement. The gadolinium ions are fixed on specific positions on MurD via a rigid double-arm paramagnetic lanthanide tag that has been originally developed for paramagnetic nuclear magnetic resonance (NMR). The combined use of NMR and EPR enables accurate interpretation of the DEER distance information to the structural information of MurD. The DEER distance measurement for apo MurD shows a broad distance distribution, whereas the presence of the inhibitor narrows the distance distribution. The results suggest that MurD exists in a wide variety of conformational states in the absence of ligands, whereas binding of the inhibitor eliminates variation in conformational states. The multiple conformational states of MurD were previously implied by NMR experiments, but our DEER data provided structural characterization of the conformational variety of MurD.
A series of newer derivatives 10a–n containing a 2,4-thiazolidinedione and 2-aryl-4-thiazolidinone moieties of pharmacological significance have been synthesized. The compounds were screened for their in vitro antimicrobial activity against three Gram-positive bacteria Staphylococcus aureus, Bacillus cereus, and Micrococcus luteus and three Gram-negative bacteria Pseudomonas fluorescens, Escherichia coli, and Flavobacterium devorans. Among them,10c, 10d, 10i, 10j, 10k, 10l, and 10n displayed equipotent antibacterial activity against the tested strains. All synthesized compounds were also tested for their cytotoxic activity against HeLa and MCF-7 cell lines. This study shows that all compounds were non-cytotoxicin nature, and confirmed their antimicrobial specificity apart from any general cytotoxicity. All these synthesized compounds were characterized by ¹H NMR, ¹³C NMR, and HRMS spectral techniques.
In the present work we synthesized a new series of phenoxyacetohydrazide functional compounds 4a-k and characterized by spectral data. Synthesized compounds were screened in vitro for their antibacterial activity. Compounds 4a, 4j and 4k exhibited inhibitory activity against S. aureus NCIM 5022 with MIC value of 64 µg/ml These compounds also exhibited activity against methicillin resistant S. aureus ATCC 43300 with MIC of 128 µg/ml. Among all the tested compounds 4c and 4j showed highest activity, respectively against B. subtilis NCIM 2545 and K. pneumoniae NCIM 2706. Only one compound i.e. 4d showed activity against another Gram-negative bacteria P. aeruginosa NCIM 2036 with MIC value of 64 µg/ml. Among three tested compounds, 4k exhibited highest inhibitory activity against S. aureus MurD enzyme with IC50 value of 35.80 µM. Further binding interactions of 4a-k with the modelled S. aureus MurD catalytic pocket residues is investigated with the extra-precision molecular docking and binding free energy calculation by MM-GBSA approach. The van der Waals energy term was observed to be the driving force for binding. Further, 50 ns molecular dynamics simulations were performed to validate the stabilities of 4j- and 4k-modelled S. aureus MurD.Graphic abstract
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.
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.
Staphyloccocus aureus MurE enzyme catalyzes the addition of l-lysine as third residue of the peptidoglycan peptide moiety. Due to the high substrate specificity and its ubiquitous nature among bacteria, MurE enzyme is considered as one of the potential target for the development of new therapeutic agents. In the present work, induced fit docking (IFD), binding free energy calculation, and molecular dynamics (MD) simulation were carried out to elucidate the inhibition potential of 2-thioxothiazolidin-4-one based inhibitor 1 against S. aureus MurE enzyme. The inhibitor 1 formed majority of hydrogen bonds with the C-terminal domain residues Asn151, Thr152, Ser180, Arg187, and Lys219. Binding free-energy calculation by MM-GBSA approach showed that van der Waals (ΔGvdW, −57.30 kcal/mol) and electrostatic solvation (ΔGsolv, −36.86 kcal/mol) energy terms are major contributors for the inhibitor binding. Further, 30-ns MD simulation was performed to validate the stability of ligand–protein complex and also to get structural insight into mode of binding. Based on the IFD and MD simulation results, we designed four new compounds D1–D4 with promising binding affinity for the S. aureus MurE enzyme. The designed compounds were subjected to the extra-precision docking and binding free energy was calculated for complexes. Further, a 30-ns MD simulation was performed for D1/4C13 complex.
The rise of superbugs is a serious public health concern. It is estimated to kill around 10 million people a year by 2050 and will overtake cancer as the number one cause of death worldwide. One of the most prevalent drug-resistant pathogen is Methicillin-resistant Staphylococcus aureus (MRSA). Intense efforts have been devoted to the discovery and development of anti-MRSA drug. Muramyl ligase E (MurE), an enzyme involved in the peptidoglycan biosynthesis of the bacterial cell wall, is a highly druggable target in MRSA. In this study, virtual screening of approved and experimental drugs in the Drug Bank database was performed based on a pharmacophore derived from the structure of MurE. Molecular docking was subsequently done with the top hits. The top hits and their derivatives were further evaluated for their predicted pharmacokinetics properties. This drug repurposing effort has identified four experimental drugs that are predicted to bind more strongly to MurE than the drug Fosfomycin. One of the top hits, DB01758, exhibited all the characteristics of a good drug candidate, albeit it is likely to be non-biodegradable.
Acinetobacter baumannii belongs to the most critical group of bacterial pathogens that are resistant to large number of antibiotics, including the highly effective carbapenems and third generation cephalosporins. Novel antibiotics targeting unique pathways of the said pathogen could reduce the mortality rate due to its infections that are impervious to the current antibiotics. Herein, we explored a library of natural compounds that was filtered first for drug-like, followed by lead-like molecules, which were finally utilized in structure based virtual screening to identify the most promising inhibitor for the Ligand binding (LB) domain of MurC ligase enzyme. The inhibitor (1′-((2H-imidazol-2-yl)methyl)-N-(pyridin-2-yl)-1′,2′-dihydro-[4,4′-bipyridin]-2-amine) was observed with a conformation to target most conserved and catalytically critical residues of both the intended LB as well as the ATP binding domain of MurC. This multi-domain inhibitor revealed to have an excellent pharmacokinetics profile thus likely to have safe and effective therapeutic applications for the future. Molecular dynamics simulation in aqueous solution further supported the high affinity of the compound for the target site involving strong hydrogen bonding. At residue level, radial distribution function (RDF) and axial distribution function (AFD) illustrated Asp334 as the most critical amino acid that drives recognition, binding, and activity of the compound. The complex stability was validated by subjecting it to Molecular Mechanics Poisson-Boltzmann Surface Area (MMPBSA), Molecular Mechanics Generalized Born Surface Area (MMGBSA) and WaterSwap based binding free energy calculations. The system was observed with high stability: total binding energy in MMGBSA (−48.45 kcal/mol) and MMPBSA (−3.62 kcal/mol). The columbic interactions were noticed to dominate (−336.90 kcal/mol), followed by van der Waals energies (−45.52 kcal/mol) in MurC-inhibitor binding. The absolute binding free energy estimated by WaterSwap was −43.2 kcal/mol, depicting higher complex stability. The screened scaffold might be used in functional groups substitution to achieve further lead optimization.
Depuis une vingtaine d’années, l’utilisation massive des antibiotiques a provoqué l’apparition de souches bactériennes résistantes contre la plupart des familles d’antibiotiques disponibles sur le marché pharmaceutique. L’apparition de souches multirésistantes voire totorésistantes notamment dans le milieu hospitalier, pose de manière croissante des difficultés thérapeutiques et constitue un grave problème de santé publique. Lors de mon doctorat, deux approches ont été abordées.La première approche consiste à inhiber les enzymes DapF et MurE impliquées dans la voie de biosynthèse du peptidoglycane, composant principal de la paroi des cellules bactériennes. Nous avons donc préparé des acides aminés comportant des groupes fonctionnels triazolyle ou alcynyle, analogues stériquement contraints de l’acide 2,6-diaminopimélique (méso-DAP).La deuxième approche développée au laboratoire repose sur le développement d’adjuvants d’antibiotiques, permettant de cibler spécifiquement les protéines responsables des mécanismes de résistances (Histidines kinases, HKs). Ce travail a permis l’obtention de trente-trois molécules dérivées du thiophène, dont huit présentent une activité biologique contre trois HKs différentes (1,63 < CI50 (μM) < 243,9). De plus, sur les huit molécules biologiquement actives, deux ont présenté une inhibition de la croissance bactérienne contre des bactéries à gram-positif et/ou à gram-négatif (B. subtilis, S. aureus, B. anthracis, E. coli…) et une restaure la sensibilité de souches bactériennes (E. coli productrice de β-lactamases à spectre large (BLSE) et S. aureus résistant à la méticilline (SARM)) à l’antibiotique approprié (céfotaxime).
This chapter illustrates the two strategies that are employed to inhibit multiple targets with a single chemical entity: single-pharmacophore molecules designed to address multitarget inhibition and design of hybrid molecules as single chemical entities obtained by fusing or linking two antibacterial pharmacophores. In any bacterium, a β-lactam antibiotic inhibits several transpeptidases penicillin-binding proteins (PBPs) and any fluoroquinolones (FQs) potentially blocks two DNA topoisomerases. The design contributions involve a chemical or an enzymatic transformation in order for both pharmacophores to be operative. The chapter presents the examples of designed multitarget-directed ligands addressing the targets like Mur ligases and type II fatty acids synthases. Type II fatty acid biosynthesis is an essential process that ensures bacterial cell viability. A large number of clinically used antibacterial drugs act on the peptidoglycan biosynthesis. Peptidoglycan is an essential component of the cell envelope of Gram-positive and Gram-negative bacteria.
The discovery of a new zinc binding chemotype from screening a non-biased fragment library is reported. Using the orthogonal fragment screening methods of native state mass spectrometry and surface plasmon resonance a 3-unsubstituted-2,4-oxazolidinedione fragment was found to have low micromolar binding affinity to the zinc metalloenzyme carbonic anhydrase II (CA II). This affinity approached that of fragment sized primary benzene sulfonamides, the classical zinc binding group found in most CA II inhibitors. Protein X-ray crystallography established that 3-unsubstituted-2,4-oxazolidinediones bound to CA II via an interaction of the acidic ring nitrogen with the CA II active site zinc, as well as a hydrogen bond between the oxazolidinedione ring oxygen and the CA II protein backbone. Furthermore, 3-unsubstituted-2,4-oxazolidinediones appear to be a viable starting point for the development of an alternative class of CA inhibitor, wherein the medicinal chemistry pedigree of primary sulfonamides has dominated for several decades.
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 (0.96 Å). 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 10 ns 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.
The biosynthetic pathway of peptidoglycan, an essential component of bacterial cell wall, is a well-recognized target for antibiotic development. Peptidoglycan precursors are synthesized in the bacterial cytosol by various enzymes including the ATP-hydrolyzing Mur ligases, which catalyze the stepwise addition of amino acids to a UDP-MurNAc precursor to yield UDP-MurNAc-pentapeptide. MurD catalyzes the addition of D-glutamic acid to UDP-MurNAc-L-Ala in the presence of ATP; structural and biochemical studies have suggested the binding of the substrates with an ordered kinetic mechanism in which ligand binding inevitably closes the active site. In this work, we challenge this assumption by reporting the crystal structures of intermediate forms of MurD either in the absence of ligands or in the presence of small molecules. A detailed analysis provides insight into the events that lead to the closure of MurD and reveals that minor structural modifications contribute to major overall conformation alterations. These novel insights will be instrumental in the development of new potential antibiotics designed to target the peptidoglycan biosynthetic pathway.
Despite existing experimental and computational tools to assess the risk, the non-specific chemical modification of protein thiol groups remains a significant source of false-positive hits, particularly in academic drug discovery. Herein, we describe the application of a simple NMR method in a systematic study on the reactivity of 5-benzylidenebarbiturates, 5-benzylidenerhodanines, and their related oxo-heterocycles, which have been associated with numerous biological activities and have recently gained a reputation as unselective promiscuous binders. Using this method, we confirmed the reactivity of 5-benzylidenebarbiturates, which are known to easily form Michael adducts with nucleophiles. In contrast, 5-benzylidene five-membered oxo-heterocycles revealed almost insignificant reactivity. We can conclude that the distinct binding profile of the most controversial compounds, 5-benzylidenerhodanines, is not necessarily related to their unspecific Michael acceptor reactivity.
The bacterial peptidoglycan biosynthesis pathway provides multiple targets for antibacterials as proven by the clinical success of β-lactam and glycopeptide classes of antibiotics. The Mur ligases play an essential role in the biosynthesis of the peptidoglycan building block, N-acetyl-muramic acid-pentapeptide. MurC, the first of four Mur ligases, ligates L-alanine to UDP-N-acetylmuramic acid, initiating the synthesis of pentapeptide precursor. Therefore inhibiting the MurC enzyme should result in bacterial cell death. Herein, we report a novel class of pyrazolopyrimidines with sub-nanomolar potency against both Escherichia coli and Pseudomonas aeruginosa MurC enzymes which demonstrates a concomitant bactericidal activity against efflux pump deficient strains. Radio-labeled precursor incorporation showed these compounds selectively inhibited peptidoglycan biosynthesis and genetic studies confirmed the target of pyrazolopyrimidines to be MurC. In the presence of permeability enhancers like colistin, pyrazolopyrimidines exhibited low micromolar MIC against the wild-type bacteria, thereby, indicating permeability and efflux as major challenges for this chemical series. Our studies provide biochemical and genetic evidence to support the essentiality of MurC and serve to validate the attractiveness of target for antibacterial discovery.
Inspired by the core fragment of antibacterial natural products such as streptolydigin, 3-acyltetramic acids and 3-acylpiperidine-2,4-diones have been synthesised from the core heterocycle by direct acylation with the substituted carboxylic acids using a strategy which permits ready access to a structurally diverse compound library. The antibacterial activity of these systems has been established against a panel of Gram-positive and Gram-negative bacteria, with activity mostly against the former, which in some cases is very potent. Data consistent with modes of action against undecaprenylpyrophosphate synthase (UPPS) and/or RNA polymerase (RNAP) for a small subset of the library has been obtained. The most active compounds have been shown to exhibit binding at known binding sites of streptolydigin and myxopyronin at UPPS and RNAP. These systems offer potential for their antibacterial activity, and further demonstrate the use of natural products as biologically validated starting points for drug discovery.
An uncatalyzed aldol reaction of N-substituted thiazolidinediones with isatin derivatives has been developed "on water" to afford a new class of pharmacologically important thiazolidinedione-isatin conjugates in excellent yields and diastereoselectivities. The isatin-thiazolidine conjugate undergoes a catalyst free stereoselective transfer aldol reaction on-water. Single crystal X-ray studies reveal that the aldol products can self-assemble to form supramolecular DNA "zipper" like structures through intermolecular hydrogen bonds and aromatic pi-pi interactions.
Introduction:
Resistance to current antibacterial therapies is an inevitability that represents a significant global health concern. Bacteria have the capacity to render all current drug treatments ineffective, which places a demand on the drug discovery community to constantly develop new antibacterial agents. Compounds that inhibit multiple biological targets, often referred to as multitarget ligands, are an inviting prospect in antibacterial research because, although they will not solve the issue of resistance, they might help to delay the onset.
Areas covered:
This review covers some of the recent progress in identifying new ligands that deliberately interact with more than one essential biological target in bacteria. The two principal areas covered are inhibitors of DNA replication and cell wall biosynthesis.
Expert opinion:
Antibacterial programs for the design of multitarget ligands present an important opportunity for production of antibacterial agents. Their longevity, due to slow development of resistance, is comparable to that seen with other successful agents - but is much improved over single-targeted agents for which resistance can appear in vitro overnight. The preclinical development of these agents will have to overcome the standard problems of antibacterial discovery. Such problems include optimization of characteristics favoring cell entry and particularly the demonstration of selectivity of inhibition of the desired multiple targets without inhibition of other bacterial or any mammalian functions.
The discovery of novel small-molecule antibacterial drugs has been stalled for many years. The purpose of this review is to underscore and illustrate those scientific problems unique to the discovery and optimization of novel antibacterial agents that have adversely affected the output of the effort. The major challenges fall into two areas: (i) proper target selection, particularly the necessity of pursuing molecular targets that are not prone to rapid resistance development, and (ii) improvement of chemical libraries to overcome limitations of diversity, especially that which is necessary to overcome barriers to bacterial entry and proclivity to be effluxed, especially in Gram-negative organisms. Failure to address these problems has led to a great deal of misdirected effort.
For bacteria, the structural integrity of its cell wall is of utmost importance for survival, and to this end, a rigid scaffold called peptidoglycan, comprised of sugar molecules and peptides, is synthesized and located outside the cytoplasmic membrane of the cell. Disruption of this peptidoglycan layer has for many years been a prime target for effective antibiotics, namely the penicillins and cephalosporins. Because this rigid layer is synthesized by a multi-step pathway numerous additional targets also exist that have no counterpart in the animal cell. Central to this pathway are four similar ligase enzymes, which add peptide groups to the sugar molecules, and interrupting these steps would ultimately prove fatal to the bacterial cell. The mechanisms of these ligases are well understood and the structures of all four of these ligases are now known. A detailed comparison of these four enzymes shows that considerable conformational changes are possible and that these changes, along with the recruitment of two different N-terminal binding domains, allows these enzymes to bind a substrate which at one end is identical and at the other has the growing polypeptide tail. Some insights into the structure-function relationships in these enzymes is presented.
Determination of nucleotide sequence of a 2.8kb DNA fragment involving the murG and murC genes has completed the sequencing of the total 12kb mra region at 2min on the Escherichia coli chromosome map, which functions in the growth and division of the cell. Product proteins of the genes in the mra region have also been identified, of which the MurC and MurG proteins are reported here. Considerable homologies were found in the deduced amino acid sequences of four ligases, products of the murC, murD, murE and murF genes in the mra region. These synthesize UDP-N-acetylmuramyl-pentapeptide from UDP-N-acetylmuramic acid in peptidoglycan synthesis. The MurG protein, also involved in the cell growth of E. coli, showed considerable homology of the deduced amino acid sequence with that of a peptide coded for by an open reading frame in the spoVE-ftsZ region of the B. subtilis chromosome.
Peptidosulfonamides are an emerging group of peptidomimetics with a variety of applications in medicinal chemistry. We present a novel approach to the synthesis of peptidosulfonamides, and apply it to a series of new potential inhibitors of the bacterial peptidoglycan biosynthesis enzymes MurD and MurE. The synthesis was conducted via N-phthalimido β-aminoethanesulfonyl chlorides, which are new building blocks for the synthesis of peptidosulfonamides. In the most crucial step, sulfonic acids or their sodium salts were converted into the corresponding sulfonyl chlorides using an excess of either SOCl2 or SOCl2/DMF, and then coupled to the C-protected amino acid. None of the compounds significantly inhibited MurD, however, some inhibited MurE; one had an IC50 below 200 μM, which constitutes a promising starting point for further development. Molecular modelling simulations were performed on two analogues to investigate the absence of inhibitory activity of the sulfonamide compounds on MurD.Graphical abstract
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.
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.
Figure
Structure and predicted binding mode of dual 1,3,5-triazine-based inhibitor in E. coli MurC and MurD active sites
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.
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.
Mur ligases participate in the intracellular path of bacterial peptidoglycan biosynthesis and constitute attractive, although so far underexploited, targets for antibacterial drug discovery. A series of hydroxy-substituted 5-benzylidenethiazolidin-4-ones were synthesized and tested as inhibitors of Mur ligases. The most potent compound 5 a was active against MurD-F with IC(50) values between 2 and 6 microm, making it a promising multitarget inhibitor of Mur ligases. Antibacterial activity against different strains, inhibitory activity against protein kinases, mutagenicity and genotoxicity of 5 a were also investigated, and kinetic and NMR studies were conducted.
Enzymes involved in the biosynthesis of bacterial peptidoglycan represent important targets for development of new antibacterial drugs. Among them, Mur ligases (MurC to MurF) catalyze the formation of the final cytoplasmic precursor UDP-N-acetylmuramyl-pentapeptide from UDP-N-acetylmuramic acid. We present the design, synthesis and biological evaluation of a series of phosphorylated hydroxyethylamines as new type of small-molecule inhibitors of Mur ligases. We show that the phosphate group attached to the hydroxyl moiety of the hydroxyethylamine core is essential for good inhibitory activity. The IC(50) values of these inhibitors were in the micromolar range, which makes them a promising starting point for the development of multiple inhibitors of Mur ligases as potential antibacterial agents. In addition, 1-(4-methoxyphenylsulfonamido)-3-morpholinopropan-2-yl dihydrogen phosphate 7a was discovered as one of the best inhibitors of MurE described so far.
The peptidoglycan biosynthetic pathway provides an array of potential targets for antibacterial drug design, attractive especially with respect to selective toxicity. Within this pathway, the members of the Mur ligase family are considered as promising emerging targets for novel antibacterial drug design. Based on the available MurD crystal structures co-crystallised with N-sulfonyl glutamic acid inhibitors, a virtual screening campaign was performed, combining three-dimensional structure-based pharmacophores and molecular docking calculations. A novel class of glutamic acid surrogates-benzene 1,3-dicarboxylic acid derivatives-were identified and compounds 14 and 16 found to possess dual MurD and MurE inhibitory activity.
The presented series of naphthalene-N-sulfonyl-D-glutamic acid derivatives are novel MurD ligase inhibitors with moderate affinity that occupy the D-Glu binding site. We performed an NMR study including transfer NOE to determine the ligand bound conformation, as well as saturation transfer difference experiments to obtain ligand epitope maps. The difference in overall appearance of the epitope maps highlights the importance of hydrophobic interactions and shows the segments of molecular structure that are responsible for them. Transfer NOE experiments indicate the conformational flexibility of bound ligands, which were then further examined by unrestrained molecular dynamics calculations. The results revealed the differing degrees of ligand flexibility and their effect on particular ligand-enzyme contacts. Conformational flexibility not evident in the crystal structures may have an effect on ligand-binding site adaptability, and this is probably one of the important reasons for the only moderate activity of novel derivatives.
Mur ligases catalyze the biosynthesis of the UDP-MurNAc-pentapeptide precursor of peptidoglycan, an essential polymer of bacterial cell-wall. They constitute attractive targets for the development of novel antibacterial agents. Here we report on the synthesis of a series of 2,4-diaminoquinazolines, quinazoline-2,4(1H,3H)-diones, 5-benzylidenerhodanines and 5-benzylidenethiazolidine-2,4-diones and their inhibitory activities against MurD from Escherichia coli. Compounds (R)-27 and (S)-27 showed inhibitory activity against MurD with IC(50) values of 174 and 206 microM, respectively, which makes them promising starting points for optimization.
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.
The D-alanyl-D-alanine-adding enzyme encoded by the murF gene catalyzes the ATP-dependent formation of UDP-N-acetylmuramyl-L-gamma-D-Glu-meso-diaminopimelyl-D-Ala-D-Ala (UDP-MurNAc-tripeptide). MurF has been cloned from Escherichia coli and expressed as a glutathione S-transferase (GST) fusion using the tac promoter-based pGEX-KT vector. From induced, broken cell preparations, highly active fusion was recovered and purified in one step by affinity chromatography. The purified fusion protein was strongly inhibited by substrate UDPMurNAc-tripeptide, a response unaltered by changes in assay pH or by cleavage from the fusion partner. However, this effect was suppressed by the addition of 0.5 M NaCl. Initial velocity and dead-end inhibitor studies with the fusion enzyme were most consistent with a sequential ordered kinetic mechanism for the forward reaction in which ATP binds to free enzyme, followed by tripeptide and D-Ala-D-Ala in sequence prior to product release. Reported homologies between the MurF protein and the three preceding steps of cytoplasmic murein biosynthesis, MurC, -D, and -E, [Ikeda et al. (1990) J. Gen. Appl. Microbiol. 36, 179-187], raise the prospect that all of these enzymes will be found to proceed via this mechanism.
Bacterial peptidoglycan biosynthesis includes four enzymatic reactions in which successive amino acid residues are ligated to uridine diphospho-N-acetylmuramic acid (UDP-MurNAc). By comparing the amino acid sequences of MurC, -D, -E, and -F proteins from various bacterial genera, four regions of homology were identified. A profile search of Swissprot for related sequences revealed that these regional similarities were present in the folyl-gamma-polyglutamate ligases. These sequence homologies appear to track with catalytic function: both enzyme families proceed through an ordered kinetic mechanism and form product via an acyl phosphate intermediate. Two highly conserved residues in region II were examined through site-directed mutagenesis of the murein D-alanyl-D-alanine-adding enzyme from Escherichia coli (murF; E158 and H188). All mutations were highly detrimental to activity with enzyme specific activity reductions of 200-4500-fold, validating the critical nature of these residues. DNA sequence analysis from three E. coli mutants harboring the murC3 (G344D), murE1 (G344K, A495S), and murF2 (A288T) mutations revealed the presence of point mutation(s) closely associated with the fourth of these aligned regions. The murF2 allele, expressed and purified as a glutathione S-transferase::MurF2 fusion, was 181-fold less catalytically active at 30 degrees C and was further reduced at the nonpermissive temperature (42 degrees C). Thus the murF2 temperature-sensitive phenotype arises from a point mutation within a highly conserved region within this protein family. These data argue that these proteins comprise a superfamily of three substrate amide ligases that share significant structural and catalytic homologies.
Initial velocity methods were used to probe the kinetic mechanism of Escherichia coli uridine diphosphate-N-acetylmuramate:L-alanine ligase (UNAM:L-Ala ligase). When the activity (in the forward direction) versus substrate concentration data were plotted in double-reciprocal form, all line patterns were intersecting. The best fit of these data was to the equation for an ordered mechanism with the following parameters: k(cat), 1000 +/- 100 min(-1); Kma, 210 +/- 40 microM; Kmb, 84 +/- 20 microM; Kmc, 70 +/- 15 microM; Kia, 180 +/- 50 microM; Kib, 68 +/- 24 microM. Initial velocity line patterns were also determined when the concentration of one substrate was varied at different fixed concentrations of a second substrate while the third substrate was held at a concentration more than 100 times its Km value. Reciprocal plots of data collected with either ATP or L-alanine present at more than 100 times their Km values resulted in intersecting line patterns. Data collected with UNAM present at 100 times its Km value gave a set of parallel lines. These data are consistent with UNAM binding as the second substrate in an ordered mechanism. ADP, uridine diphosphate-N-acetylmuramoyl-L-alanine (UNAMA), and phosphate were tested as product inhibitors versus substrates. None of the products were competitive inhibitors versus L-alanine or UNAM, while the only observed competitive inhibition was ADP versus ATP. These results are consistent with an ordered kinetic mechanism wherein ATP binds first, UNAM binds second, and ADP is the last product released. Rapid quench experiments were performed in the presence of all three substrates or in the presence of ATP and UNAM. The production of acid-labile phosphate as a function of time is characterized by a burst phase followed by a slower linear phase with the rate close to k(cat) in the presence of all three substrates. Only a burst phase was observed for the time course of the reaction in the presence of ATP and UNAM. In both cases, the burst rate was identical. These observations are consistent with L-alanine being the third substrate to bind in a sequential mechanism involving a putative acyl-phosphate intermediate.
The comparison of the amino acid sequences of 20 cytoplasmic peptidoglycan synthetases (MurC, MurD, MurE, MurF, and Mpl) from various bacterial organisms has allowed us to detect common invariants: seven amino acids and the ATP-binding consensus sequence GXXGKT/S all at the same position in the alignment. The Mur synthetases thus appeared as a well-defined class of closely functionally related proteins. The conservation of a constant backbone length between certain invariants suggested common structural motifs. Among the other enzymes catalyzing a peptide bond formation driven by ATP hydrolysis to ADP and Pi, only folylpoly-gamma-l-glutamate synthetases presented the same common conserved amino acid residues, except for the most N-terminal invariant D50. Site-directed mutageneses were carried out to replace the K130, E174, H199, N293, N296, R327, and D351 residues by alanine in the MurC protein from Escherichia coli taken as model. For this purpose, plasmid pAM1005 was used as template, MurC being highly overproduced in this genetic setting. Analysis of the Vmax values of the mutated proteins suggested that residues K130, E174, and D351 are essential for the catalytic process whereas residues H199, N293, N296, and R327 were not. Mutations K130A, H199A, N293A, N296A, and R327A led to important variations of the Km values for one or more substrates, thereby indicating that these residues are involved in the structure of the active site and suggesting that the binding order of the substrates could be ATP, UDP-MurNAc, and alanine. The various mutated murC plasmids were tested for their effects on the growth, cell morphology, and peptidoglycan cell content of a murC thermosensitive strain at 42 degrees C. The observed effects (complementation, altered morphology, and reduced peptidoglycan content) paralleled more or less the decreased values of the MurC activity of each mutant.
UDP -N- acetylmuramoyl- L -alanine: D -glutamate (MurD) ligase catalyses the addition of d -glutamate to the nucleotide precursor UDP -N- acetylmuramoyl- L -alanine (UMA). The crystal structures of three complexes of Escherichia coli MurD with a variety of substrates and products have been determined to high resolution. These include (1) the quaternary complex of MurD, the substrate UMA, the product ADP, and Mg2+, (2) the quaternary complex of MurD, the substrate UMA, the product ADP, and Mn2+, and (3) the binary complex of MurD with the product UDP - N- acetylmuramoyl- L -alanine- D -glutamate (UMAG). The reaction mechanism supported by these structures proceeds by the phosphorylation of the C-terminal carboxylate group of UMA by the gamma-phosphate group of ATP to form an acyl-phosphate intermediate, followed by the nucleophilic attack by the amino group of D-glutamate to produce UMAG. A key feature in the reaction intermediate is the presence of two magnesium ions bridging negatively charged groups.
To evaluate their role in the active site of the UDP-N-acetylmuramoyl-L-alanine:D-glutamate ligase (MurD) from Escherichia coli, 12 residues conserved either in the Mur superfamily [Eveland, S. S., Pompliano, D. L., and Anderson, M. S. (1997) Biochemistry 36, 6223-6229; Bouhss, A., Mengin-Lecreulx, D., Blanot, D., van Heijenoort, J., and Parquet, C. (1997) Biochemistry 36, 11556-11563] or in the sequences of 26 MurD orthologs were submitted to site-directed mutagenesis. All these residues lay within the cleft of the active site of MurD as defined by its 3D structure [Bertrand, J. A., Auger, D., Fanchon, E., Martin, L., Blanot, D., van Heijenoort, J., and Dideberg, O. (1997) EMBO J. 16, 3416-3425]. Fourteen mutant proteins (D35A, K115A, E157A/K, H183A, Y194F, K198A/F, N268A, N271A, H301A, R302A, D317A, and R425A) containing a C-terminal (His)(6) extension were prepared and their steady-state kinetic parameters determined. All had a reduced enzymatic activity, which in many cases was very low, but no mutation led to a total loss of activity. Examination of the specificity constants k(cat)/K(m) for the three MurD substrates indicated that most mutations affected both the binding of one substrate and the catalytic process. These kinetic results correlated with the assigned function of the residues based on the X-ray structures.
This review focuses on target-based approaches for developing new chemical classes of antibacterial agents aimed at the bacterial cell wall. The clinical success of antibiotics such as beta-lactams and glycopeptides validates this chemotherapeutic strategy and emerging resistance to these agents warrants the development of new antibacterial drugs. Understanding the mechanism of action and resistance to beta-lactams and glycopeptides at a molecular level has supported the development of new agents that prevent transpeptidation and transglycosylation reactions of peptidoglycan polymerisation. The enzymes involved in the synthesis of the peptidoglycan structural unit have also been targeted for antibacterial discovery. The influence of bacterial genetics and genomics, structural biology, assay development and the properties of known inhibitors on these approaches will be discussed in the context of drug discovery.
Acyl phosphates can be identified by their chemical trapping into stable derivatives. In the present work, the alcohol derivatives originating from the chemical reduction of the acyl phosphates for MurC and MurD are detected, thereby firmly establishing the formation of such compounds. The possibility that these acyl phosphates are off-pathway, dead-end adducts is ruled out by the observation that isotope exchange reactions occur when the amino acid substrate (L-alanine for MurC, D-glutamic acid for MurD) is present. The comparison of 20 Mur synthetases from various bacterial species has revealed seven invariant amino acids and the same ATP-binding sequence at the same position in the alignment. The Mur synthetases thus appear to be a well-defined class of closely functionally related proteins originating presumably from a common ancestor. Moreover, the conservation of a constant backbone length between certain invariants suggests common 3-D structural motifs. Therefore, it is reasonable to assume that they all share the same reaction mechanism, which involves an acyl phosphate and a tetrahedral compound as reaction intermediates. The crystallographic and site-directed mutagenesis studies of MurD have allowed the assignment of a role in acyl phosphate formation to some invariant residues.
One of the biggest challenges for recent medical research is the continuous development of new antibiotics interacting with bacterial essential mechanisms. The machinery for peptidoglycan biosynthesis is a rich source of crucial targets for antibacterial chemotherapy. The cytoplasmic steps of the biosynthesis of peptidoglycan precursor, catalysed by a series of Mur enzymes, are excellent candidates for drug development. There has been growing interest in these bacterial enzymes over the last decade. Many studies attempted to understand the detailed mechanisms and structural features of the key enzymes MurA to MurF. Only MurA is inhibited by a known antibiotic, fosfomycin. Several attempts made to develop novel inhibitors of this pathway are discussed in this review. Three novel inhibitors of MurA were identified recently. 4-Thiazolidinone compounds were designed as MurB inhibitors. Many phosphinic acid derivatives and substrate analogues were identified as inhibitors of the MurC to MurF amino acid ligases.
Promiscuous small molecules plague screening libraries and hit lists. Previous work has found that several nonspecific compounds form submicrometer aggregates, and it has been suggested that this aggregate species is responsible for the inhibition of many different enzymes. It is not understood how aggregates inhibit their targets. To address this question, biophysical, kinetic, and microscopy methods were used to study the interaction of promiscuous, aggregate-forming inhibitors with model proteins. By use of centrifugation and gel electrophoresis, aggregates and protein were found to directly interact. This is consistent with a subsequent observation from confocal fluorescence microscopy that aggregates concentrate green fluorescent protein. beta-Lactamase mutants with increased or decreased thermodynamic stability relative to wild-type enzyme were equally inhibited by an aggregate-forming compound, suggesting that denaturation by unfolding was not the primary mechanism of interaction. Instead, visualization by electron microscopy revealed that enzyme associates with the surface of inhibitor aggregates. This association could be reversed or prevented by the addition of Triton X-100. These observations suggest that the aggregates formed by promiscuous compounds reversibly sequester enzyme, resulting in apparent inhibition. They also suggest a simple method to identify or reverse the action of aggregate-based inhibitors, which appear to be widespread.
The increasing emergence of pathogenic bacterial strains with high resistance to antibiotic therapy has created an urgent need for the development of new antibacterial agents that are directed towards novel targets. We have focused our attention on the Mur ligases (MurC-F), which catalyze the early steps of bacterial peptidoglycan biosynthesis, and which to date represent under-exploited targets for antibacterial drug design. We show that some of our phosphinate inhibitors of UDP-N-acetylmuramoyl-L-alanyl:D-glutamate ligase (MurD) also inhibits UDP-N-acetylmuramoyl-L-alanyl-D-glutamate:L-lysine ligase (MurE). To obtain new information on their structure-activity relationships, three new, structurally related phosphinates were synthesized and evaluated for inhibition of MurD and MurE.
Mur ligases play an essential role in the intracellular biosynthesis of bacterial peptidoglycan, the main component of the bacterial cell wall, and represent attractive targets for the design of novel antibacterials. UDP-N-acetylmuramoyl-L-alanine:D-glutamate ligase (MurD) catalyses the addition of D-glutamic acid to the cytoplasmic intermediate UDP-N-acetylmuramoyl-L-alanine (UMA) and is the second in the series of Mur ligases. MurD ligase is highly stereospecific for its substrate, D-glutamic acid (D-Glu). Here, we report the high resolution crystal structures of MurD in complexes with two novel inhibitors designed to mimic the transition state of the reaction, which contain either the D-Glu or the L-Glu moiety. The binding modes of N-sulfonyl-D-Glu and N-sulfonyl-L-Glu derivatives were also characterised kinetically. The results of this study represent an excellent starting point for further development of novel inhibitors of this enzyme.
The peptidoglycan (murein) sacculus is a unique and essential structural element in the cell wall of most bacteria. Made of glycan strands cross-linked by short peptides, the sacculus forms a closed, bag-shaped structure surrounding the cytoplasmic membrane. There is a high diversity in the composition and sequence of the peptides in the peptidoglycan from different species. Furthermore, in several species examined, the fine structure of the peptidoglycan significantly varies with the growth conditions. Limited number of biophysical data on the thickness, elasticity and porosity of peptidoglycan are available. The different models for the architecture of peptidoglycan are discussed with respect to structural and physical parameters.
The biosynthesis of bacterial cell wall peptidoglycan is a complex process that involves enzyme reactions that take place in the cytoplasm (synthesis of the nucleotide precursors) and on the inner side (synthesis of lipid-linked intermediates) and outer side (polymerization reactions) of the cytoplasmic membrane. This review deals with the cytoplasmic steps of peptidoglycan biosynthesis, which can be divided into four sets of reactions that lead to the syntheses of (1) UDP-N-acetylglucosamine from fructose 6-phosphate, (2) UDP-N-acetylmuramic acid from UDP-N-acetylglucosamine, (3) UDP-N-acetylmuramyl-pentapeptide from UDP-N-acetylmuramic acid and (4) D-glutamic acid and dipeptide D-alanyl-D-alanine. Recent data concerning the different enzymes involved are presented. Moreover, special attention is given to (1) the chemical and enzymatic synthesis of the nucleotide precursor substrates that are not commercially available and (2) the search for specific inhibitors that could act as antibacterial compounds.
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