RESUMO
The cytoplasmatic biosynthesis of the stem peptide from the peptidoglycan in bacteria involves six steps, which have the role of three ATP-dependent Mur ligases that incorporate three consecutive amino acids to a substrate precursor. MurE is the last Mur ligase to incorporate a free amino acid. Although the structure of MurE from Mycobacterium tuberculosis (MtbMurE) was determined at 3.0 Å, the binding mode of meso-Diaminopimelate (m-DAP) and the effect of substrate absence is unknown. Herein, we show the structure of MurE from M. thermoresistibile (MthMurE) in complex with ADP and m-DAP at 1.4 Å resolution. The analysis of the structure indicates key conformational changes that the substrate UDP-MurNAc-L-Ala-D-Glu (UAG) and the free amino acid m-DAP cause on the MthMurE conformation. We observed several movements of domains or loop regions that displace their position in order to perform enzymatic catalysis. Since MthMurE has a high similarity to MtbMurE, this enzyme could also guide strategies for structure-based antimicrobial discovery to fight against tuberculosis or other mycobacterial infections.
Assuntos
Mycobacterium tuberculosis , Peptídeo Sintases , Peptídeo Sintases/química , Proteínas de Bactérias/química , Mycobacterium tuberculosis/metabolismo , AminoácidosRESUMO
Archaea have diverse cell wall types, yet none are identical to bacterial peptidoglycan (murein). Methanogens Methanobacteria and Methanopyrus possess cell walls of pseudomurein, a structural analogue of murein. Pseudomurein differs from murein in containing the unique archaeal sugar N-acetyltalosaminuronic acid instead of N-acetylmuramic acid, ß-1,3 glycosidic bonds in place of ß-1,4 bonds and only l-amino acids in the peptide cross-links. We have determined crystal structures of methanogen pseudomurein peptide ligases (termed pMurE) from Methanothermus fervidus (Mfer762) and Methanothermobacter thermautotrophicus (Mth734) that are structurally most closely related to bacterial MurE peptide ligases. The homology of the archaeal pMurE and bacterial MurE enzymes is clear both in the overall structure and at the level of each of the three domains. In addition, we identified two UDP-binding sites in Mfer762 pMurE, one at the exterior surface of the interface of the N-terminal and middle domains, and a second site at an inner surface continuous with the highly conserved interface of the three domains. Residues involved in ATP binding in MurE are conserved in pMurE, suggesting that a similar ATP-binding pocket is present at the interface of the middle and the C-terminal domains of pMurE. The presence of pMurE ligases in members of the Methanobacteriales and Methanopyrales, that are structurally related to bacterial MurE ligases, supports the idea that the biosynthetic origins of archaeal pseudomurein and bacterial peptidoglycan cell walls are evolutionarily related.
Assuntos
Euryarchaeota , Peptidoglicano , Trifosfato de Adenosina/metabolismo , Aminoácidos/metabolismo , Archaea/metabolismo , Bactérias/metabolismo , Parede Celular/metabolismo , Euryarchaeota/metabolismo , Ligases/metabolismo , Peptídeo Sintases/metabolismo , Peptidoglicano/metabolismo , Açúcares/metabolismo , Difosfato de Uridina/análise , Difosfato de Uridina/metabolismoRESUMO
Cordyceps spp. are widely healthy foods around the world with several traditional uses and bio-functionalities. The chemical characterization of ethyl acetate-soluble extract of the entomopathogenic fungus Cordyceps tenuipes NBRC 111,630 afforded two new metabolites with 1,6-dioxaspiro[4.4]nonane motif, tenuipesone A (1) and tenuipesone B (2), along with four well-known metabolites (3-6). The elucidation of the chemical structures was carried out via extensive spectroscopic experiments including FTIR, HRMS, 1D-NMR, and 2D-NMR. The probable biosynthetic pathway of 1 and 2 was hypothesized. From the six isolates, beauvericin (6) exhibited antimicrobial activity against Bacillus subtilis and Staphylococcus aureus with respective MIC of 6.25 and 12.5 µM. Docking results exhibited that beauvericin (6) has significant binding affinities against MurE and HK proteins with ΔG = - 8.021 and - 8.585 kcal/mol, respectively. KEY POINTS: ⢠Six compounds, including two new, were isolated from the entomopathogenic fungus Cordyceps tenuipes. ⢠Plausible biosynthetic pathway of compounds 1, 2, 4, and 5 was hypothesized. ⢠Beauvericin (6) exhibited significant antimicrobial activity against Bacillus subtilis and Staphylococcus aureus alongside binding affinities against MurE and HK proteins in MOE study.
Assuntos
Anti-Infecciosos , Cordyceps , Antibacterianos/química , Antibacterianos/farmacologia , Anti-Infecciosos/farmacologia , Bacillus subtilis , Biologia Computacional , Cordyceps/química , Histidina Quinase , Ligases , Testes de Sensibilidade Microbiana , Extratos Vegetais , Staphylococcus aureusRESUMO
Staphylococcus 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 central 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.
Assuntos
Proteínas de Bactérias/antagonistas & inibidores , Inibidores Enzimáticos/metabolismo , Simulação de Dinâmica Molecular , Peptídeo Sintases/antagonistas & inibidores , Peptídeo Sintases/metabolismo , Staphylococcus aureus/efeitos dos fármacos , Domínio Catalítico , Desenho de Fármacos , Inibidores Enzimáticos/química , Ligação Proteica , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
The rigidity of bacterial cell walls synthesized by a complicated pathway limit the cell shapes as coccus, bar or ellipse or even fibers. A less rigid bacterium could be beneficial for intracellular accumulation of poly-3-hydroxybutyrate (PHB) as granular inclusion bodies. To understand how cell rigidity affects PHB accumulation, E. coli cell wall synthesis pathway was reinforced and weakened, respectively. Cell rigidity was achieved by thickening the cell walls via insertion of a constitutive gltA (encoding citrate synthase) promoter in front of a series of cell wall synthesis genes on the chromosome of several E. coli derivatives, resulting in 1.32-1.60 folds increase of Young's modulus in mechanical strength for longer E. coli cells over-expressing fission ring FtsZ protein inhibiting gene sulA. Cell rigidity was weakened by down regulating expressions of ten genes in the cell wall synthesis pathway using CRISPRi, leading to elastic cells with more spaces for PHB accumulation. The regulation on cell wall synthesis changes the cell rigidity: E. coli with thickened cell walls accumulated only 25% PHB while cell wall weakened E. coli produced 93% PHB. Manipulation on cell wall synthesis mechanism adds another possibility to morphology engineering of microorganisms.
Assuntos
Parede Celular , Escherichia coli , Hidroxibutiratos/metabolismo , Poliésteres/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Parede Celular/genética , Parede Celular/metabolismo , Citrato (si)-Sintase/genética , Citrato (si)-Sintase/metabolismo , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Engenharia MetabólicaRESUMO
The endosymbiotic theory states that plastids are derived from a single cyanobacterial ancestor that possessed a cell wall. Peptidoglycan (PG), the main component of the bacteria cell wall, gradually degraded during plastid evolution. PG-synthesizing Mur genes have been found to be retained in the genomes of basal streptophyte plants, although many of them have been lost from the genomes of angiosperms. The enzyme encoded by bacterial MurE genes catalyzes the formation of the UDP-N-acetylmuramic acid (UDP-MurNAc) tripeptide in bacterial PG biosynthesis. Knockout of the MurE gene in the moss Physcomitrella patens resulted in defects of chloroplast division, whereas T-DNA-tagged mutants of Arabidopsis thaliana for MurE revealed inhibition of chloroplast development but not of plastid division, suggesting that AtMurE is functionally divergent from the bacterial and moss MurE proteins. Here, we could identify 10 homologs of bacterial Mur genes, including MurE, in the recently sequenced genomes of Picea abies and Pinus taeda, suggesting the retention of the plastid PG system in gymnosperms. To investigate the function of gymnosperm MurE, we isolated an ortholog of MurE from the larch, Larix gmelinii (LgMurE) and confirmed its presence as a single copy per genome, as well as its abundant expression in the leaves of larch seedlings. Analysis with a fusion protein combining green fluorescent protein and LgMurE suggested that it localizes in chloroplasts. Cross-species complementation assay with MurE mutants of A. thaliana and P. patens showed that the expression of LgMurE cDNA completely rescued the albefaction defects in A. thaliana but did not rescue the macrochloroplast phenotype in P. patens. The evolution of plastid PG and the mechanism behind the functional divergence of MurE genes are discussed in the context of information about plant genomes at different evolutionary stages.
Assuntos
Arabidopsis/genética , Cycadopsida/genética , Larix/genética , Proteínas de Ligação às Penicilinas/genética , Peptidoglicano/genética , Fenótipo , Proteínas de Plantas/genética , Arabidopsis/enzimologia , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Bactérias/genética , Bryopsida/genética , Bryopsida/metabolismo , Parede Celular/genética , Parede Celular/metabolismo , Cloroplastos/genética , Cloroplastos/metabolismo , Cianobactérias/genética , Cianobactérias/metabolismo , Cycadopsida/metabolismo , DNA Bacteriano , Regulação da Expressão Gênica de Plantas , Técnicas de Inativação de Genes , Genes Bacterianos , Genes de Plantas , Genoma de Planta , Proteínas de Fluorescência Verde , Larix/metabolismo , Magnoliopsida/genética , Mutação , Proteínas de Ligação às Penicilinas/metabolismo , Peptidoglicano/metabolismo , Picea/genética , Pinus taeda/genética , Folhas de Planta/genética , Proteínas de Plantas/metabolismo , Plastídeos/genética , Plastídeos/metabolismo , Sementes/genética , Sementes/metabolismoRESUMO
Formation of the peptidoglycan stem pentapeptide requires the insertion of both L and D amino acids by the ATP-dependent ligase enzymes MurC, -D, -E, and -F. The stereochemical control of the third position amino acid in the pentapeptide is crucial to maintain the fidelity of later biosynthetic steps contributing to cell morphology, antibiotic resistance, and pathogenesis. Here we determined the x-ray crystal structure of Staphylococcus aureus MurE UDP-N-acetylmuramoyl-L-alanyl-D-glutamate:meso-2,6-diaminopimelate ligase (MurE) (E.C. 6.3.2.7) at 1.8 Šresolution in the presence of ADP and the reaction product, UDP-MurNAc-L-Ala-γ-D-Glu-L-Lys. This structure provides for the first time a molecular understanding of how this Gram-positive enzyme discriminates between L-lysine and D,L-diaminopimelic acid, the predominant amino acid that replaces L-lysine in Gram-negative peptidoglycan. Despite the presence of a consensus sequence previously implicated in the selection of the third position residue in the stem pentapeptide in S. aureus MurE, the structure shows that only part of this sequence is involved in the selection of L-lysine. Instead, other parts of the protein contribute substrate-selecting residues, resulting in a lysine-binding pocket based on charge characteristics. Despite the absolute specificity for L-lysine, S. aureus MurE binds this substrate relatively poorly. In vivo analysis and metabolomic data reveal that this is compensated for by high cytoplasmic L-lysine concentrations. Therefore, both metabolic and structural constraints maintain the structural integrity of the staphylococcal peptidoglycan. This study provides a novel focus for S. aureus-directed antimicrobials based on dual targeting of essential amino acid biogenesis and its linkage to cell wall assembly.
Assuntos
Proteínas de Bactérias/química , Parede Celular/enzimologia , Lisina/química , Peptídeo Sintases/química , Peptidoglicano/química , Staphylococcus aureus/enzimologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Parede Celular/genética , Cristalografia por Raios X , Lisina/genética , Lisina/metabolismo , Metabolômica , Peptídeo Sintases/genética , Peptídeo Sintases/metabolismo , Peptidoglicano/biossíntese , Peptidoglicano/genética , Estrutura Terciária de Proteína , Staphylococcus aureus/genéticaRESUMO
The opportunistic bacterium Acinetobacter baumannii, which belongs to ESKAPE group of pathogenic bacteria, is leading cause of infections associated with gram-negative bacteria. Acinetobacter baumannii causes severe diseases, such as VAP (ventilator-associated pneumonia), meningitis, and UTI (urinary tract infections) among the nosocomial infections contracted in hospitals. The high infection rate and growing resistance to the vast array of antibiotics makes it paramount to look for new therapeutic strategies against this pathogen. The most promising therapeutic targets are the proteins involved in the synthesis of peptidoglycan which is chief component of bacterial cell wall, MurE is one of those enzymes and is responsible for the addition of one unit of meso-diaminopimelic acid (meso-A2pm) to the nucleotide precursor, UDPMurNAc-L-Ala-D-Glu, and aids in the formation of crosslinker pentapeptide chain. The three-dimensional structure of MurE was modelled using homology modelling technique and then vHTS was performed using this model against Approved Drug Library on DrugRep server using AutoDock Vina. Out of 500 drug molecules, two were selected based on estimated binding affinity, interaction pattern, interacting residues, etc. The selected drug molecules are DB12887 (Tazemetostat) and DB13879 (Glecaprevir). Then, MD simulations were performed on native MurE and its complexes with ligands to examine their dynamical behaviour, stability, integrity, compactness, and folding properties. The protein-ligand complexes were then subjected to binding free energy calculations using the MM/PBSA-based binding free energy analysis and the values are -109.788 ± 8.03 and -152.753 ± 11.98 kcal for MurE-DB12887 and MurE-DB13879 complex, respectively. All the analysis performed on MD trajectories for the complexes of these ligands with protein provided plenty of dependable evidences to consider these molecules for inhibition of MurE enzyme from A. baumannii. Communicated by Ramaswamy H. Sarma.
RESUMO
Acinetobacter baumannii is an opportunistic pathogen with ability to cause serious infection such as bacteremia, ventilator associated pneumonia, and wound infections. As strains of A. baumannii are resistant to almost all clinically used antibiotics and with the emergence of carbapenems resistant phenotypes warrants the search for novel antibiotics. Considering this, herein, a series of computer aided drug designing approach was utilized to search novel chemical scaffolds that bind stronger to MurE ligase enzyme of A. baumannii, which is involved peptidoglycan synthesis. The work identified LAS_22461675, LAS_34000090 and LAS_51177972 compounds as promising binding molecules with MurE enzyme having binding energy score of -10.5 kcal/mol, -9.3 kcal/mol and -8.6 kcal/mol, respectively. The compounds were found to achieve docked inside the MurE substrate binding pocket and established close distance chemical interactions. The interaction energies were dominated by van der Waals and less contributions were seen from hydrogen bonding energy. The dynamic simulation assay predicted the complexes stable with no major global and local changes noticed. The docked stability was also validated by MM/PBSA and MM/GBSA binding free energy methods. The net MM/GBSA binding free energy of LAS_22461675 complex, LAS_34000090 complex and LAS_51177972 complex is -26.25 kcal/mol, -27.23 kcal/mol and -29.64 kcal/mol, respectively. Similarly in case of MM-PBSA, the net energy value was in following order; LAS_22461675 complex (-27.67 kcal/mol), LAS_34000090 complex (-29.94 kcal/mol) and LAS_51177972 complex (-27.32 kcal/mol). The AMBER entropy and WaterSwap methods also confirmed stable complexes formation. Further, molecular features of the compounds were determined that predicted compounds to have good druglike properties and pharmacokinetic favorable. The study concluded the compounds to good candidates to be tested by in vivo and in vitro experimental assays.Communicated by Ramaswamy H. Sarma.
Assuntos
Acinetobacter baumannii , Simulação de Dinâmica Molecular , Antibacterianos/farmacologia , Antibacterianos/química , Carbapenêmicos , Ligases , Simulação de Acoplamento MolecularRESUMO
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.
RESUMO
Current therapeutic strategies for several diseases, including Mycobacterium tuberculosis infection, have evolved from an initial single-target treatment to a multitarget one. A multitarget antitubercular drugs targeting different mycobacterial proteins are more effective at suppressing bacterial growth. In this study, a high throughput virtual screening was performed to identify hits to the potential antitubercular multitarget: murA, murB, murC, murD, murE, murF, murG and murI from M. tuberculosis that is involved in peptidoglycan biosynthesis. In the virtual screening, we were docked 56,400 compounds of the ChEMBL antimycobacterial library and re-scored and identified the top 10 ranked compounds as antitubercular drug candidates. Further, the best common docked complex CHEMBL446262 was subjected to molecular dynamics simulation to understand the molecule's stability in the presence of an active site environment. After that, we have calculated binding free energy the top-ranked docked complexes using the MM/PBSA method. These ligands exhibited the highest binding affinity; find out novel drug-likeness might show the M. tuberculosis effect's inhibitor by interacting with multitarget Mur enzymes. New antitubercular therapies that include multitarget drugs may have higher efficacy than single-target medicines and provide a more straightforward antitubercular therapy regimen.Communicated by Ramaswamy H. Sarma.
Assuntos
Mycobacterium tuberculosis , Tuberculose , Antituberculosos/química , Humanos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , PeptidoglicanoRESUMO
MurE ligase catalyzes the attachment of meso-diaminopimelic acid to the UDP-MurNAc-l -Ala-d -Glu using ATP and producing UDP-MurNAc-l -Ala-d -Glu-meso-A2 pm during bacterial cell wall biosynthesis. Owing to the critical role of this enzyme, MurE is considered an attractive target for antibacterial drugs. Despite extensive studies on MurE ligase, the structural dynamics of its conformational changes are still elusive. In this study, we present the substrate-free structure of MurE from Acinetobacter baumannii, which is an antibiotic-resistant superbacterium that has threatened global public health. The structure revealed that MurE has a wide-open conformation and undergoes wide-open, intermediately closed, and fully closed dynamic conformational transition. Unveiling structural dynamics of MurE will help to understand the working mechanism of this ligase and to design next-generation antibiotics targeting MurE.
Assuntos
Acinetobacter baumannii/enzimologia , Peptídeo Sintases/química , Peptídeo Sintases/metabolismo , Acinetobacter baumannii/efeitos dos fármacos , Antibacterianos/química , Antibacterianos/farmacologia , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Cristalografia por Raios X , Desenho de Fármacos , Modelos Moleculares , Conformação Proteica , Relação Estrutura-AtividadeRESUMO
Infections due to resistant bacteria are the life-threatening and leading cause of mortality worldwide. The current therapy for bacterial infections includes treatment with various drugs and antibiotics. The misuse and over usage of these antibiotics leads to bacterial resistance. There are several mechanisms by which bacteria exhibit resistance to some antibiotics. These include drug inactivation or modification, elimination of antibiotics through efflux pumps, drug target alteration, and modification of metabolic pathway. However, it is difficult to treat infections caused by resistant bacteria by conventional existing therapy. In the present study binding affinities of some glitazones against ParE and MurE bacterial enzymes are investigated by in silico methods. As evident by extra-precision docking and binding free energy calculation (MM-GBSA) results, rivoglitazone exhibited higher binding affinity against both ParE and MurE enzymes compared to all other selected compounds. Further molecular dynamic (MD) simulations were performed to validate the stability of rivoglitazone/4MOT and rivoglitazone/4C13 complexes and to get insight into the binding mode of inhibitor. Thus, we hypothesize that structural modifications of the rivoglitazone scaffold can be useful for the development of an effective antibacterial agent.
Assuntos
Antibacterianos/farmacologia , Infecções Bacterianas/tratamento farmacológico , DNA Topoisomerase IV/antagonistas & inibidores , Peptídeo Sintases/antagonistas & inibidores , Tiazolidinedionas/farmacologia , Tiazolidinas/farmacologia , Antibacterianos/química , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/química , DNA Topoisomerase IV/química , Resistência Microbiana a Medicamentos , Humanos , Ligação de Hidrogênio , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Peptídeo Sintases/química , Relação Estrutura-Atividade , Tiazolidinedionas/química , Tiazolidinas/químicaRESUMO
MurE ligase catalyzes the assembly of peptide moiety, an essential component of bacterial cell wall. We have explored the conformational stability and unfolding equilibrium behaviour of the protein MurE ligase by determining the conformational free energy, entropy and enthalpy parameters under stress conditions. MurE from Salmonella enterica Serovar Typhi was cloned, expressed and purified. Conformational changes associated with increasing concentration of GdmCl- and urea-induced denaturation of MurE were monitored using Circular Dichroism (CD) and fluorescence spectroscopies. The secondary structural content of protein estimated by CD experiment is in close agreement with the predicted MurE ligase structure by homology modeling. Denaturant-induced transition curve was analyzed for thermodynamic parameters. Average values for MurE ligase of ΔGD0â¯=â¯3.13â¯kcalâ¯mol-1, mâ¯=â¯1.52â¯kcalâ¯mol-1â¯M-1 and Cm (=ΔGD0/m)â¯=â¯2.05â¯M were calculated in the presence of GdmCl whereas in the case of urea these were ΔGD0â¯=â¯3.04â¯kcalâ¯mol-1, mâ¯=â¯1.20â¯kcalâ¯mol-1â¯M-1 and Cm (=ΔGD0/m)â¯=â¯2.53â¯M. The observed superposition of normalized transition curve of two independent optical properties suggested that GdmCl- and urea-induced denaturation follow a two-state process.
Assuntos
Estrutura Molecular , Peptídeo Sintases/química , Peptídeo Sintases/metabolismo , Salmonella typhi/enzimologia , Dicroísmo Circular , Ativação Enzimática , Estabilidade Enzimática , Modelos Moleculares , Peptídeo Sintases/genética , Peptídeo Sintases/isolamento & purificação , Conformação Proteica , Desnaturação Proteica , Salmonella typhi/genética , Análise Espectral , Relação Estrutura-AtividadeRESUMO
Repurposing has gained momentum globally and become an alternative avenue for drug discovery because of its better success rate, and reduced cost, time and issues related to safety than the conventional drug discovery process. Several drugs have already been successfully repurposed for other clinical conditions including drug resistant tuberculosis (DR-TB). Though TB can be cured completely with the use of currently available anti-tubercular drugs, emergence of drug resistant strains of Mycobacterium tuberculosis and the huge death toll globally, together necessitate urgently newer and effective drugs for TB. Therefore, we performed virtual screening of 1554 FDA approved drugs against murE, which is essential for peptidoglycan biosynthesis of M. tuberculosis. We used Glide and AutoDock Vina for virtual screening and applied rigid docking algorithm followed by induced fit docking algorithm in order to enhance the quality of the docking prediction and to prioritize drugs for repurposing. We found 17 drugs binding strongly with murE and three of them, namely, lymecycline, acarbose and desmopressin were consistently present within top 10 ranks by both Glide and AutoDock Vina in the induced fit docking algorithm, which strongly indicates that these three drugs are potential candidates for further studies towards repurposing for TB.
RESUMO
Erysipelothrix rhusiopathiae is a Gram-positive bacterium pathogenic to many species of birds and mammals, including humans. The main feature of its peptidoglycan is the presence of l-alanine at position 3 of the peptide stem. In the present work, we cloned the murE gene from E. rhusiopathiae and purified the corresponding protein as His6-tagged form. Enzymatic assays showed that E. rhusiopathiae MurE was indeed an l-alanine-adding enzyme. Surprisingly, it was also able, although to a lesser extent, to add meso-diaminopimelic acid, the amino acid found at position 3 in many Gram-negative bacteria, Bacilli and Mycobacteria. Sequence alignment of MurE enzymes from E. rhusiopathiae and Escherichia coli revealed that the DNPR motif that is characteristic of meso-diaminopimelate-adding enzymes was replaced by HDNR. The role of the latter motif in the interaction with l-alanine and meso-diaminopimelic acid was demonstrated by site-directed mutagenesis experiments and the construction of a homology model. The overexpression of the E. rhusiopathiae murE gene in E. coli resulted in the incorporation of l-alanine at position 3 of the peptide part of peptidoglycan.
Assuntos
Erysipelothrix/enzimologia , Peptídeo Sintases/genética , Peptídeo Sintases/metabolismo , Escherichia coli/enzimologia , Escherichia coli/genética , Peptidoglicano/metabolismo , Especificidade por SubstratoRESUMO
An in-depth evaluation was undertaken of a new antibacterial natural product (1) recently isolated and characterised from the plant Hypericum olympicum L. cf. uniflorum. Minimum inhibitory concentrations (MICs) were determined for a panel of bacteria, including: meticillin-resistant and -susceptible strains of Staphylococcus aureus, Staphylococcus epidermidis and Staphylococcus haemolyticus; vancomycin-resistant and -susceptible Enterococcus faecalis and Enterococcus faecium; penicillin-resistant and -susceptible Streptococcus pneumoniae; group A streptococci (Streptococcus pyogenes); and Clostridium difficile. MICs were 2-8 mg/L for most staphylococci and all enterococci, but were ≥16 mg/L for S. haemolyticus and were >32 mg/L for all species in the presence of blood. Compound 1 was also tested against Gram-negative bacteria, including Escherichia coli, Pseudomonas aeruginosa and Salmonella enterica serovar Typhimurium but was inactive. The MIC for Mycobacterium bovis BCG was 60 mg/L, and compound 1 inhibited the ATP-dependent Mycobacterium tuberculosis MurE ligase [50% inhibitory concentration (IC(50)) = 75 µM]. In a radiometric accumulation assay with a strain of S. aureus overexpressing the NorA multidrug efflux pump, the presence of compound 1 increased accumulation of (14)C-enoxacin in a concentration-dependent manner, implying inhibition of efflux. Only moderate cytotoxicity was observed, with IC50 values of 12.5, 10.5 and 8.9 µM against human breast, lung and fibroblast cell lines, respectively, highlighting the potential value of this chemotype as a new antibacterial agent and efflux pump inhibitor.
Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Hypericum/química , Proteínas Associadas à Resistência a Múltiplos Medicamentos/antagonistas & inibidores , Extratos Vegetais/farmacologia , Antibacterianos/isolamento & purificação , Antibacterianos/toxicidade , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Inibidores Enzimáticos/isolamento & purificação , Inibidores Enzimáticos/toxicidade , Humanos , Concentração Inibidora 50 , Testes de Sensibilidade Microbiana , Extratos Vegetais/isolamento & purificaçãoRESUMO
Peptidoglycan is an essential component of the bacterial cell wall, and enzymes involved in its biosynthesis represent validated targets for antibacterial drug discovery. MurF catalyzes the final intracellular peptidoglycan biosynthesis step: the addition of D-Ala-D-Ala to the nucleotide precursor UDP-MurNAc-L-Ala-γ-D-Glu-meso-DAP (or L-Lys). As MurF has no human counterpart, it represents an attractive target for the development of new antibacterial drugs. Using recently published cyanothiophene inhibitors of MurF from Streptococcus pneumoniae as a starting point, we designed and synthesized a series of structurally related derivatives and investigated their inhibition of MurF enzymes from different bacterial species. Systematic structural modifications of the parent compounds resulted in a series of nanomolar inhibitors of MurF from S. pneumoniae and micromolar inhibitors of MurF from Escherichia coli and Staphylococcus aureus. Some of the inhibitors also show antibacterial activity against S. pneumoniae R6. These findings, together with two new co-crystal structures, represent an excellent starting point for further optimization toward effective novel antibacterials.