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1.
Chem Biol Drug Des ; 99(4): 513-526, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-34918458

RESUMO

Proline racemases (PRAC), catalyzing the l-proline and d-proline interconversion, are essential factors in eukaryotic pathogens such as Trypanosoma cruzi, Trypanosoma vivax, and Clostridioides difficile. If the discovery of irreversible inhibitors of T. cruzi PRAC (TcPRAC) led to innovative therapy of the Chagas disease, no inhibitors of CdPRAC have been discovered to date. However, C. difficile, due to an increased incidence in recent years, is considered as a major cause of health threat. In this work, we have taken into account the similarity between TcPRAC and CdPRAC enzymes to design new inhibitors of CdPRAC. Starting from (E) 4-oxopent-2-enoic acid TcPRAC irreversible inhibitors, we synthesized 4-aryl substituted analogs and evaluated their CdPRAC enzymatic inhibition against eleven strains of C. difficile. This study resulted in promising candidates and allowed for identification of (E)-4-(3-bromothiophen-2-yl)-4-oxobut-2-enoic acid 20 that was chosen for complementary in vivo studies and did not reveal in vivo toxicity.


Assuntos
Isomerases de Aminoácido , Antibacterianos , Clostridioides difficile , Isomerases de Aminoácido/antagonistas & inibidores , Antibacterianos/farmacologia , Clostridioides difficile/efeitos dos fármacos , Prolina
2.
J Mol Recognit ; 34(9): e2894, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-33719110

RESUMO

Enterococcus faecalis (E. faecalis) is a Gram-positive coccoid, non-sporulating, facultative anaerobic, multidrug resistance bacterium responsible for almost 65% to 80% of all enterococcal nosocomial infections. It usually causes infective endocarditis, urinary tract and surgical wound infections. The increase in E. faecalis resistance to conventionally available antibiotic has rekindled intense interest in developing useful antibacterial drugs. In E. faecalis, diaminopimelate epimerase (DapF) is involved in the lysine biosynthetic pathway. The product of this pathway is precursors of peptidoglycan synthesis, which is a component of bacterial cell wall. Also, because mammals lack this enzyme, consequently E. faecalis diaminopimelate epimerase (EfDapF) represents a potential target for developing novel class of antibiotics. In this regard, we have successfully cloned, overexpressed the gene encoding DapF in BL-21(DE3) and purified with Ni-NTA Agarose resin. In addition to this, binding studies were performed using fluorescence spectroscopy in order to confirm the bindings of the identified lead compounds (acetaminophen and dexamethasone) with EfDapF. Docking studies revealed that acetaminophen found to make hydrogen bonds with Asn72 and Asn13 while dexamethasone interacted by forming hydrogen bonds with Asn205 and Glu223. Thus, biochemical studies indicated acetaminophen and dexamethasone, as potential inhibitors of EfDapF and eventually can reduce the catalytic activity of EfDapF.


Assuntos
Acetaminofen/farmacologia , Isomerases de Aminoácido/antagonistas & inibidores , Dexametasona/farmacologia , Enterococcus faecalis/enzimologia , Simulação de Acoplamento Molecular , Isomerases de Aminoácido/química , Isomerases de Aminoácido/metabolismo , Proteínas de Bactérias/antagonistas & inibidores , Sítios de Ligação , Reposicionamento de Medicamentos , Enterococcus faecalis/efeitos dos fármacos , Conformação Proteica
3.
Biochemistry (Mosc) ; 85(2): 248-256, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-32093601

RESUMO

Streptococcus iniae is a pathogenic and zoonotic bacterium responsible for human diseases and mortality of many fish species. Recently, this bacterium has demonstrated an increasing trend for antibiotics resistance, which has warranted a search for new approaches to tackle its infection. Glutamate racemase (MurI) is a ubiquitous enzyme of the peptidoglycan synthesis pathway that plays an important role in the cell wall integrity maintenance; however, the significance of this enzyme differs in different species. In this study, we knocked out the MurI gene in S. iniae in order to elucidate the role of glutamate racemase in maintaining cell wall integrity in this bacterial species. We also cloned, expressed, and purified MurI and determined its biochemical characteristics. Biochemical analysis revealed that the MurI gene in S. iniae encodes a functional enzyme with a molecular weight of 30 kDa, temperature optimum at 35°C, and pH optimum at 8.5. Metal ions, such as Cu2+, Mn2+, Co2+ and Zn2+, inhibited the enzyme activity. MurI was found to be essential for the viability and cell wall integrity of S. iniae. The optimal growth of the MurI-deficient S. iniae mutant can be achieved only by adding a high concentration of D-glutamate to the medium. Membrane permeability assay of the mutant showed an increasing extent of the cell wall damage with time upon D-glutamate starvation. Moreover, the mutant lost its virulence when incubated in fish blood. Our results demonstrated that the MurI knockout leads to the generation of S. iniae auxotroph with damaged cell walls.


Assuntos
Isomerases de Aminoácido/metabolismo , Parede Celular , Viabilidade Microbiana , Streptococcus iniae/enzimologia , Isomerases de Aminoácido/antagonistas & inibidores , Isomerases de Aminoácido/genética , Parede Celular/efeitos dos fármacos , Parede Celular/metabolismo , Concentração de Íons de Hidrogênio , Metais Pesados/farmacologia , Viabilidade Microbiana/efeitos dos fármacos , Mutação , Streptococcus iniae/efeitos dos fármacos , Streptococcus iniae/metabolismo
4.
ChemMedChem ; 15(4): 376-384, 2020 02 17.
Artigo em Inglês | MEDLINE | ID: mdl-31876113

RESUMO

Glutamate racemases (GR) are members of the family of bacterial enzymes known as cofactor-independent racemases and epimerases and catalyze the stereoinversion of glutamate. D-amino acids are universally important for the proper construction of viable bacterial cell walls, and thus have been repeatedly validated as attractive targets for novel antimicrobial drug design. Significant aspects of the mechanism of this challenging stereoinversion remain unknown. The current study employs a combination of MD and QM/MM computational approaches to show that the GR from H. pylori must proceed via a pre-activation step, which is dependent on the enzyme's flexibility. This mechanism is starkly different from previously proposed mechanisms. These findings have immediate pharmaceutical relevance, as the H. pylori GR enzyme is a very attractive allosteric drug target. The results presented in this study offer a distinctly novel understanding of how AstraZeneca's lead series of inhibitors cripple the H. pylori GR's native motions, via prevention of this critical chemical pre-activation step. Our experimental studies, using SPR, fluorescence and NMR WaterLOGSY, show that H. pylori GR is not inhibited by the uncompetitive mechanism originally put forward by Lundqvist et al.. The current study supports a deep connection between native enzyme motions and chemical reactivity, which has strong relevance to the field of allosteric drug discovery.


Assuntos
Isomerases de Aminoácido/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Helicobacter pylori/efeitos dos fármacos , Simulação de Dinâmica Molecular , Regulação Alostérica/efeitos dos fármacos , Isomerases de Aminoácido/metabolismo , Antibacterianos/química , Antibacterianos/farmacologia , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/química , Helicobacter pylori/enzimologia , Testes de Sensibilidade Microbiana , Estrutura Molecular , Relação Estrutura-Atividade
5.
Nat Prod Rep ; 36(12): 1687-1705, 2019 12 11.
Artigo em Inglês | MEDLINE | ID: mdl-30994146

RESUMO

Covering: up to March 2019 Amino acid racemases and epimerases are key enzymes that invert the configuration of common amino acids and supply many corresponding d-isomers in living organisms. Some d-amino acids are inherently bioactive, whereas others are building blocks for important biomolecules, for example lipid II, the bacterial cell wall precursor. Peptides containing them have enhanced proteolytic stability and can act as important recognition elements in mammalian systems. Selective inhibition of certain amino acid racemases (e.g. glutamate racemase) is believed to offer a promising target for new antibacterial drugs effective against pathogens resistant to current antibiotics. Many amino acid racemases employ imine formation with pyridoxal phosphate (PLP) as a cofactor to accelerate the abstraction of the alpha proton. However, the group reviewed herein achieves racemization of free amino acids without the use of cofactors or metals, and uses a thiol/thiolate pair for deprotonation and reprotonation. All bacteria and higher plants contain such enzymes, for example diaminopimelate epimerase, which is required for lysine biosynthesis in these organisms. This process cannot be accomplished without an enzyme catalyst as the acidities of a thiol and the substrate α-hydrogen are inherently mismatched by at least 10 orders of magnitude. This review describes the structural and mechanistic studies on PLP-independent racemases and the evolving view of key enzymatic machinery that accomplishes these remarkable transformations.


Assuntos
Inibidores Enzimáticos/farmacologia , Fosfato de Piridoxal/metabolismo , Racemases e Epimerases/química , Racemases e Epimerases/metabolismo , Isomerases de Aminoácido/antagonistas & inibidores , Isomerases de Aminoácido/química , Isomerases de Aminoácido/metabolismo , Inibidores Enzimáticos/química , Conformação Proteica , Racemases e Epimerases/antagonistas & inibidores , Compostos de Sulfidrila/metabolismo
6.
Appl Microbiol Biotechnol ; 103(2): 843-851, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30456576

RESUMO

Increasing drug resistance in pathogens including Mycobacterium tuberculosis (MTB) has been ascribed to mutations in the known target genes. However, many of these drugs have multiple targets; some of which have not been identified so far. Understanding the mechanism of action of these drugs holds a great promise in better management of disease especially by drug-resistant strains. In this study, we report glutamate racemase (MurI), a crucial enzyme of phase I peptidoglycan (PG) biosynthesis pathway of MTB, as an additional target of ethambutol (EMB). The effect on EMB on the MurI protein at structural and functional level was studied using different spectroscopic, biochemical, and insilico approaches. Spectroscopic analysis revealed that EMB-modified protein undergoes conformational alterations. Furthermore, in vitro racemization studies of the MurI protein suggest that EMB decreases its functional activity. Docking studies revealed that EMB interacts with most of the active residues at the binding site and blocks the binding pocket. Overall, data suggests that EMB, a primary drug used for the treatment of tuberculosis (TB), acts as a competitive inhibitor of substrate for binding to mycobacterial MurI protein. The study also points out to our lacunae in understanding the site and mechanism of action of existing drugs. Furthermore, glutamate racemase is a conserved protein of the bacterial kingdom; therefore, ethambutol could be a promising candidate as a broad-spectrum antibiotic for many other bacterial diseases.


Assuntos
Isomerases de Aminoácido/antagonistas & inibidores , Antituberculosos/farmacologia , Inibidores Enzimáticos/farmacologia , Etambutol/farmacologia , Mycobacterium tuberculosis/efeitos dos fármacos , Peptidoglicano/biossíntese , Isomerases de Aminoácido/química , Isomerases de Aminoácido/metabolismo , Sítios de Ligação , Parede Celular/metabolismo , Simulação de Acoplamento Molecular , Mycobacterium tuberculosis/enzimologia , Ligação Proteica , Conformação Proteica , Análise Espectral
7.
PLoS Negl Trop Dis ; 12(10): e0006853, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30372428

RESUMO

Chagas disease, caused by Trypanosoma cruzi, affects millions of people in South America and no satisfactory therapy exists, especially for its life threatening chronic phase. We targeted the Proline Racemase of T. cruzi, which is present in all stages of the parasite life cycle, to discover new inhibitors against this disease. The first published crystal structures of the enzyme revealed that the catalytic site is too small to allow any relevant drug design. In previous work, to break through the chemical space afforded to virtual screening and drug design, we generated intermediate models between the open (ligand free) and closed (ligand bound) forms of the enzyme. In the present work, we co-crystallized the enzyme with the selected inhibitors and found that they were covalently bound to the catalytic cysteine residues in the active site, thus explaining why these compounds act as irreversible inhibitors. These results led us to the design of a novel, more potent specific inhibitor, NG-P27. Co-crystallization of this new inhibitor with the enzyme allowed us to confirm the predicted protein functional motions and further characterize the chemical mechanism. Hence, the catalytic Cys300 sulfur atom of the enzyme attacks the C2 carbon of the inhibitor in a coupled, regiospecific-stereospecific Michael reaction with trans-addition of a proton on the C3 carbon. Strikingly, the six different conformations of the catalytic site in the crystal structures reported in this work had key similarities to our intermediate models previously generated by inference of the protein functional motions. These crystal structures span a conformational interval covering roughly the first quarter of the opening mechanism, demonstrating the relevance of modeling approaches to break through chemical space in drug design.


Assuntos
Isomerases de Aminoácido/antagonistas & inibidores , Isomerases de Aminoácido/química , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Trypanosoma cruzi/enzimologia , Domínio Catalítico , Cristalografia por Raios X , Desenho de Fármacos , Humanos , Modelos Moleculares , Ligação Proteica , Conformação Proteica
8.
ChemMedChem ; 13(23): 2514-2521, 2018 12 06.
Artigo em Inglês | MEDLINE | ID: mdl-30264520

RESUMO

The application of covalent inhibitors has experienced a renaissance within drug discovery programs in the last decade. To leverage the superior potency and drug target residence time of covalent inhibitors, there have been extensive efforts to develop highly specific covalent modifications to decrease off-target liabilities. Herein, we present a series of covalent inhibitors of an antimicrobial drug target, glutamate racemase, discovered through structure-based virtual screening. A combination of enzyme kinetics, mass spectrometry, and surface-plasmon resonance experiments details a highly specific 1,4-conjugate addition of a small-molecule inhibitor with a catalytic cysteine of glutamate racemase. Molecular dynamics simulations and quantum mechanics-molecular mechanics geometry optimizations reveal the chemistry of the conjugate addition. Two compounds from this series of inhibitors display antimicrobial potency similar to ß-lactam antibiotics, with significant activity against methicillin-resistant S. aureus strains. This study elucidates a detailed chemical rationale for covalent inhibition and provides a platform for the development of antimicrobials with a novel mechanism of action against a target in the cell wall biosynthesis pathway.


Assuntos
Isomerases de Aminoácido/antagonistas & inibidores , Antibacterianos/química , Antibacterianos/farmacologia , Bactérias/enzimologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Isomerases de Aminoácido/metabolismo , Bacillus subtilis/efeitos dos fármacos , Bacillus subtilis/enzimologia , Bactérias/efeitos dos fármacos , Infecções Bacterianas/tratamento farmacológico , Infecções Bacterianas/microbiologia , Descoberta de Drogas , Humanos , Simulação de Acoplamento Molecular , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia
9.
Eur J Med Chem ; 145: 23-34, 2018 Feb 10.
Artigo em Inglês | MEDLINE | ID: mdl-29310027

RESUMO

In the present study, we attempted to develop novel class of Mycobacterium tuberculosis (Mtb) inhibitors by exploring the pharmaceutically underexploited enzyme targets which are majorly involved in cell wall biosynthesis of mycobacteria. For this purpose glutamate racemase was selected which racemizes d-glutamate from l-glutamate, a key step in peptidoglycan synthesis. Furthermore, enzyme is neither expressed nor its product, d-glutamate is produced in mammals, and hence inhibiting this enzyme will have no vulnerable effect in host organism. A library of our in-house compounds were screened against glutamate racemase using a biophysical technique; thermal shift assay and further by enzyme inhibition assay to identify Lead 1 molecule. Lead 1 optimization and expansion resulted in twenty four compounds. Among the synthesized compounds twelve compounds shown good enzyme inhibition than Lead 1 (IC50 20.07 ±â€¯0.29 µM). Among all the compounds; compound 22 (IC50 1.1 ±â€¯0.52 µM) showed potent non-competitive mode of inhibition in enzyme assay. Further showed good susceptibility (in replicating bacteria) of MIC 8.72 µM and bactericidal time dependant kill on dormant culture. It also exhibited significant activity in Mtb nutrient starvation model (2.5) and Mtb biofilm model (2.4) and in vivo M. marinum infected Zebra fish model studies (3.6) reduction at logarithmic scale.


Assuntos
Isomerases de Aminoácido/antagonistas & inibidores , Antibacterianos/farmacologia , Benzoxazóis/farmacologia , Inibidores Enzimáticos/farmacologia , Mycobacterium/efeitos dos fármacos , Isomerases de Aminoácido/metabolismo , Animais , Antibacterianos/síntese química , Antibacterianos/química , Benzoxazóis/síntese química , Benzoxazóis/química , Biofilmes/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Cinética , Camundongos , Testes de Sensibilidade Microbiana , Simulação de Acoplamento Molecular , Estrutura Molecular , Mycobacterium/enzimologia , Células RAW 264.7 , Relação Estrutura-Atividade , Peixe-Zebra
10.
Bioorg Med Chem ; 26(1): 177-190, 2018 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-29239770

RESUMO

Mycobacterium tuberculosis glutamate racemase is an essential enzyme involved in peptidoglycan synthesis and conserved in most bacteria. Small molecule inhibitors were reported on other bacterial species whereas in M. tuberculosis it wasn't explored much. In this study we have screened in house compound library using fluorescence thermal shift assay and enzyme inhibition assay, form this (1-(3-(benzo[d]thiazol-2-yl)phenyl)-3-(p-tolyl)thiourea) was identified as lead compound with IC50 19.47 ±â€¯0.81 µM. Further lead optimization by synthesis resulted in twenty-three compounds, of which Compound 25 has shown more efficacy compared to lead 1 showing non-competitive mode of inhibition with IC50 1.32 ±â€¯0.43 µM. It also showed significant activity (represented in log reduction) in nutrient starved dormant M. tuberculosis model (2.1), M. tuberculosis biofilm assay (2.0) and in vivo M. marinum infected zebrafish model (3.5).


Assuntos
Isomerases de Aminoácido/antagonistas & inibidores , Antituberculosos/farmacologia , Inibidores Enzimáticos/farmacologia , Mycobacterium tuberculosis/efeitos dos fármacos , Isomerases de Aminoácido/metabolismo , Animais , Antituberculosos/síntese química , Antituberculosos/química , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Camundongos , Testes de Sensibilidade Microbiana , Estrutura Molecular , Mycobacterium tuberculosis/enzimologia , Mycobacterium tuberculosis/crescimento & desenvolvimento , Células RAW 264.7 , Relação Estrutura-Atividade
11.
ACS Infect Dis ; 3(5): 349-359, 2017 05 12.
Artigo em Inglês | MEDLINE | ID: mdl-28215073

RESUMO

In 2013, the Centers for Disease Control highlighted Clostridium difficile as an urgent threat for antibiotic-resistant infections, in part due to the emergence of highly virulent fluoroquinolone-resistant strains. Limited therapeutic options currently exist, many of which result in disease relapse. We sought to identify molecules specifically targeting C. difficile in high-throughput screens of our diversity-oriented synthesis compound collection. We identified two scaffolds with apparently novel mechanisms of action that selectively target C. difficile while having little to no activity against other intestinal anaerobes; preliminary evidence suggests that compounds from one of these scaffolds target the glutamate racemase. In vivo efficacy data suggest that both compound series may provide lead optimization candidates.


Assuntos
Isomerases de Aminoácido/antagonistas & inibidores , Antibacterianos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Clostridioides difficile/efeitos dos fármacos , Enterocolite Pseudomembranosa/tratamento farmacológico , Compostos Heterocíclicos com 2 Anéis/farmacologia , Compostos de Fenilureia/farmacologia , Pirróis/farmacologia , Quinolinas/farmacologia , Isomerases de Aminoácido/genética , Isomerases de Aminoácido/metabolismo , Animais , Antibacterianos/síntese química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Clostridioides difficile/enzimologia , Clostridioides difficile/genética , Clostridioides difficile/crescimento & desenvolvimento , Desenho de Fármacos , Enterocolite Pseudomembranosa/microbiologia , Enterocolite Pseudomembranosa/mortalidade , Enterocolite Pseudomembranosa/patologia , Feminino , Expressão Gênica , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Negativas/crescimento & desenvolvimento , Bactérias Gram-Positivas/efeitos dos fármacos , Bactérias Gram-Positivas/crescimento & desenvolvimento , Compostos Heterocíclicos com 2 Anéis/síntese química , Camundongos , Camundongos Endogâmicos C57BL , Testes de Sensibilidade Microbiana , Compostos de Fenilureia/síntese química , Pirróis/síntese química , Quinolinas/síntese química , Especificidade da Espécie , Relação Estrutura-Atividade , Análise de Sobrevida
12.
PLoS One ; 11(11): e0167350, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27898711

RESUMO

The greatest obstacle for the treatment of cystic fibrosis patients infected with the Burkholderia species is their intrinsic antibiotic resistance. For this reason, there is a need to develop new effective compounds. Glutamate racemase, an essential enzyme for the biosynthesis of the bacterial cell wall, is an excellent candidate target for the design of new antibacterial drugs. To this aim, we recombinantly produced and characterized glutamate racemase from Burkholderia cenocepacia J2315. From the screening of an in-house library of compounds, two Zn (II) and Mn (III) 1,3,5-triazapentadienate complexes were found to efficiently inhibit the glutamate racemase activity with IC50 values of 35.3 and 10.0 µM, respectively. Using multiple biochemical approaches, the metal complexes have been shown to affect the enzyme activity by binding to the enzyme-substrate complex and promoting the formation of an inhibited dimeric form of the enzyme. Our results corroborate the value of glutamate racemase as a good target for the development of novel inhibitors against Burkholderia.


Assuntos
Isomerases de Aminoácido/antagonistas & inibidores , Isomerases de Aminoácido/metabolismo , Burkholderia cenocepacia/enzimologia , Complexos de Coordenação/farmacologia , Inibidores Enzimáticos/farmacologia , Antibacterianos/química , Antibacterianos/isolamento & purificação , Antibacterianos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/metabolismo , Infecções por Burkholderia/microbiologia , Burkholderia cenocepacia/efeitos dos fármacos , Burkholderia cenocepacia/isolamento & purificação , Complexos de Coordenação/química , Complexos de Coordenação/metabolismo , Sistemas de Liberação de Medicamentos , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/isolamento & purificação , Humanos , Concentração Inibidora 50 , Cinética , Manganês/química , Testes de Sensibilidade Microbiana , Ligação Proteica , Estabilidade Proteica , Zinco/química
13.
Antimicrob Agents Chemother ; 60(10): 6091-9, 2016 10.
Artigo em Inglês | MEDLINE | ID: mdl-27480853

RESUMO

The increasing global prevalence of drug resistance among many leading human pathogens necessitates both the development of antibiotics with novel mechanisms of action and a better understanding of the physiological activities of preexisting clinically effective drugs. Inhibition of peptidoglycan (PG) biosynthesis and cross-linking has traditionally enjoyed immense success as an antibiotic target in multiple bacterial pathogens, except in Mycobacterium tuberculosis, where it has so far been underexploited. d-Cycloserine, a clinically approved antituberculosis therapeutic, inhibits enzymes within the d-alanine subbranch of the PG-biosynthetic pathway and has been a focus in our laboratory for understanding peptidoglycan biosynthesis inhibition and for drug development in studies of M. tuberculosis During our studies on alternative inhibitors of the d-alanine pathway, we discovered that the canonical alanine racemase (Alr) inhibitor ß-chloro-d-alanine (BCDA) is a very poor inhibitor of recombinant M. tuberculosis Alr, despite having potent antituberculosis activity. Through a combination of enzymology, microbiology, metabolomics, and proteomics, we show here that BCDA does not inhibit the d-alanine pathway in intact cells, consistent with its poor in vitro activity, and that it is instead a mechanism-based inactivator of glutamate racemase (MurI), an upstream enzyme in the same early stage of PG biosynthesis. This is the first report to our knowledge of inhibition of MurI in M. tuberculosis and thus provides a valuable tool for studying this essential and enigmatic enzyme and a starting point for future MurI-targeted antibacterial development.


Assuntos
Isomerases de Aminoácido/química , Antituberculosos/farmacologia , Proteínas de Bactérias/química , Inibidores Enzimáticos/farmacologia , Mycobacterium tuberculosis/efeitos dos fármacos , beta-Alanina/análogos & derivados , Isomerases de Aminoácido/antagonistas & inibidores , Isomerases de Aminoácido/genética , Isomerases de Aminoácido/metabolismo , Sequência de Aminoácidos , Antituberculosos/química , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Clonagem Molecular , Inibidores Enzimáticos/química , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Cinética , Testes de Sensibilidade Microbiana , Mycobacterium tuberculosis/enzimologia , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/crescimento & desenvolvimento , Peptidoglicano/biossíntese , Ligação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Especificidade por Substrato , beta-Alanina/química , beta-Alanina/farmacologia
14.
Chem Commun (Camb) ; 51(87): 15764-7, 2015 Nov 11.
Artigo em Inglês | MEDLINE | ID: mdl-26365322

RESUMO

We describe competitive activity-based protein profiling (ABPP) to accelerate the functional prediction and assessment of adenylation (A) domains in nonribosomal peptide synthetases (NRPSs) in proteomic environments. Using a library of sulfamoyloxy-linked aminoacyl-AMP analogs, the competitive ABPP technique offers a simple and rapid assay system for adenylating enzymes and provides insight into enzyme substrate candidates and enzyme active-site architecture.


Assuntos
Peptídeo Sintases/análise , Monofosfato de Adenosina/análogos & derivados , Monofosfato de Adenosina/síntese química , Isomerases de Aminoácido/antagonistas & inibidores , Bacillales , Domínio Catalítico , Inibidores Enzimáticos/síntese química , Cinética , Sondas Moleculares/síntese química , Peptídeo Sintases/química , Análise Serial de Proteínas , Estrutura Terciária de Proteína , Proteoma , Especificidade por Substrato , Sulfonamidas/síntese química
15.
Antimicrob Agents Chemother ; 59(4): 2337-42, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25645840

RESUMO

A pyridodiazepine amine inhibitor of Helicobacter pylori glutamate racemase (MurI) was characterized. The compound was selectively active against H. pylori, and growth suppression was shown to be mediated through the inhibition of MurI by several methods. In killing kinetics experiments, the compound showed concentration-independent activity, with about a 2-log loss of viability in 24 h. A demonstration of efficacy in a mouse infection model was attempted but not achieved, and this was attributed to the failure to attain extended exposure levels above the MIC for >95% of the time. This index and magnitude were derived from pharmacokinetic-pharmacodynamic (PK-PD) studies with amoxicillin, another inhibitor of peptidoglycan biosynthesis that showed slow killing kinetics similar to those of the pyridodiazepine amines. These studies indicate that MurI and other enzymes involved in peptidoglycan biosynthesis may be less desirable targets for monotherapy directed against H. pylori if once-a-day dosing is required.


Assuntos
Isomerases de Aminoácido/antagonistas & inibidores , Antibacterianos/uso terapêutico , Azepinas/uso terapêutico , Infecções por Helicobacter/tratamento farmacológico , Helicobacter pylori/efeitos dos fármacos , Helicobacter pylori/enzimologia , Piridinas/uso terapêutico , Amoxicilina/farmacocinética , Amoxicilina/farmacologia , Animais , Antibacterianos/farmacocinética , Azepinas/farmacocinética , Feminino , Mucosa Gástrica/metabolismo , Infecções por Helicobacter/sangue , Infecções por Helicobacter/microbiologia , Cinética , Camundongos , Testes de Sensibilidade Microbiana , Peptidoglicano/metabolismo , Piridinas/farmacocinética , Ratos , Ratos Sprague-Dawley
16.
Amino Acids ; 47(5): 975-85, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25646960

RESUMO

D-Aspartate (D-Asp) has important physiological functions, and recent studies have shown that substantial amounts of free D-Asp are present in a wide variety of mammalian tissues and cells. Biosynthesis of D-Asp has been observed in several cultured rat cell lines, and a murine gene (glutamate-oxaloacetate transaminase 1-like 1, Got1l1) that encodes Asp racemase, a synthetic enzyme that produces D-Asp from L-Asp, was proposed recently. The product of this gene is homologous to mammalian glutamate-oxaloacetate transaminase (GOT). Here, we tested the hypothesis that rat and human homologs of mouse GOT1L1 are involved in Asp synthesis. The following two approaches were applied, since the numbers of attempts were unsuccessful to prepare soluble GOT1L1 recombinant proteins. First, the relationship between the D-Asp content and the expression levels of the mRNAs encoding GOT1L1 and D-Asp oxidase, a primary degradative enzyme of D-Asp, was examined in several rat and human cell lines. Second, the effect of knockdown of the Got1l1 gene on D-Asp biosynthesis during culture of the cells was determined. The results presented here suggest that the rat and human homologs of mouse GOT1L1 are not involved in D-Asp biosynthesis. Therefore, D-Asp biosynthetic pathway in mammals is still an urgent issue to be resolved.


Assuntos
Isomerases de Aminoácido/metabolismo , D-Aspartato Oxidase/metabolismo , Ácido D-Aspártico/biossíntese , RNA Mensageiro/metabolismo , Isomerases de Aminoácido/antagonistas & inibidores , Isomerases de Aminoácido/genética , Animais , Linhagem Celular Tumoral , D-Aspartato Oxidase/genética , Expressão Gênica , Técnicas de Silenciamento de Genes , Células HeLa , Células Hep G2 , Humanos , Rim/enzimologia , Rim/patologia , Camundongos , Células PC12 , Hipófise/enzimologia , Hipófise/patologia , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Ratos , Homologia de Sequência de Aminoácidos , Especificidade da Espécie
17.
Bioorg Med Chem Lett ; 24(5): 1432-6, 2014 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-24507924

RESUMO

D-Glutamate is an essential biosynthetic building block of the peptidoglycans that encapsulate the bacterial cell wall. Glutamate racemase catalyzes the reversible formation of D-glutamate from L-glutamate and, hence, the enzyme is a potential therapeutic target. We show that the novel cyclic substrate-product analogue (R,S)-1-hydroxy-1-oxo-4-amino-4-carboxyphosphorinane is a modest, partial noncompetitive inhibitor of glutamate racemase from Fusobacterium nucleatum (FnGR), a pathogen responsible, in part, for periodontal disease and colorectal cancer (Ki=3.1±0.6 mM, cf. Km=1.41±0.06 mM). The cyclic substrate-product analogue (R,S)-4-amino-4-carboxy-1,1-dioxotetrahydro-thiopyran was a weak inhibitor, giving only ∼30% inhibition at a concentration of 40 mM. The related cyclic substrate-product analogue 1,1-dioxo-tetrahydrothiopyran-4-one was a cooperative mixed-type inhibitor of FnGR (Ki=18.4±1.2 mM), while linear analogues were only weak inhibitors of the enzyme. For glutamate racemase, mimicking the structure of both enantiomeric substrates (substrate-product analogues) serves as a useful design strategy for developing inhibitors. The new cyclic compounds developed in the present study may serve as potential lead compounds for further development.


Assuntos
Isomerases de Aminoácido/metabolismo , Proteínas de Transporte/química , Inibidores Enzimáticos/química , Ácido Glutâmico/química , Proteolipídeos/química , Isomerases de Aminoácido/antagonistas & inibidores , Bacillus subtilis/enzimologia , Proteínas de Transporte/metabolismo , Domínio Catalítico , Inibidores Enzimáticos/metabolismo , Fusobacterium/enzimologia , Ácido Glutâmico/metabolismo , Ligação Proteica , Proteolipídeos/metabolismo , Especificidade por Substrato
18.
Bioorg Med Chem Lett ; 24(1): 390-3, 2014 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-24314397

RESUMO

d-Amino acids can play important roles as specific biosynthetic building blocks required by organisms or act as regulatory molecules. Consequently, amino acid racemases that catalyze the formation of d-amino acids are potential therapeutic targets. Serine racemase catalyzes the reversible formation of d-serine (a modulator of neurotransmission) from l-serine, while proline racemase (an essential enzymatic and mitogenic protein in trypanosomes) catalyzes the reversible conversion of l-proline to d-proline. We show the substrate-product analogue α-(hydroxymethyl)serine is a modest, linear mixed-type inhibitor of serine racemase from Schizosaccharomyces pombe (Ki=167±21mM, Ki'=661±81mM, cf. Km=19±2mM). The bicyclic substrate-product analogue of proline, 7-azabicyclo[2.2.1]heptan-7-ium-1-carboxylate is a weak inhibitor of proline racemase from Clostridium sticklandii, giving only 29% inhibition at 142.5mM. However, the more flexible bicyclic substrate-product analogue tetrahydro-1H-pyrrolizine-7a(5H)-carboxylate is a noncompetitive inhibitor of proline racemase from C. sticklandii (Ki=111±15mM, cf. Km=5.7±0.5mM). These results suggest that substrate-product analogue inhibitors of racemases may only be effective when the active site is capacious and/or plastic, or when the inhibitor is sufficiently flexible.


Assuntos
Isomerases de Aminoácido/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Prolina/farmacologia , Racemases e Epimerases/antagonistas & inibidores , Serina/análogos & derivados , Isomerases de Aminoácido/metabolismo , Clostridium sticklandii/enzimologia , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Modelos Moleculares , Estrutura Molecular , Prolina/análogos & derivados , Prolina/química , Racemases e Epimerases/metabolismo , Schizosaccharomyces/enzimologia , Serina/síntese química , Serina/química , Serina/farmacologia , Relação Estrutura-Atividade
19.
Curr Top Med Chem ; 13(24): 3118-30, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24200359

RESUMO

Imidazole, a five-membered heterocycle having three carbon atoms, and two double bonds, having efficient antibacterial Escherichia coli, Bacillus subtili, Bacillus proteus, Staphylococcus aureus, Pseudomonas aeruginosa, and Helicobacter pyloriurease etc, shows a broad-spectrum of antibacterial activities. To Search new antibacterial drugs to overcome resistance of microorganisms to antibiotics, to date hundreds of this sort of derivatives have been synthesized and possess potent antibacterial activity. As the structure of imidazole derivatives is various, the target of antibacterial is also diverse including ß-Lactamases, ß-ketoacyl-acyl carrier protein synthase III (FabH), DNA gyrase and topoisomerase, glutamate racemase and urease. In this review, we will discuss the emergence of resistance to antibiotics and attempt to summarize the main developments of imidazole derivatives in the past ten years. We hope that increasing knowledge of the structure-activity relationship (SAR) will be beneficial to the rational design of new generation of small molecule antibacterial drugs.


Assuntos
Antibacterianos/química , Antibacterianos/farmacologia , Imidazóis/química , 3-Oxoacil-(Proteína de Transporte de Acila) Sintase , Acetiltransferases/antagonistas & inibidores , Isomerases de Aminoácido/antagonistas & inibidores , DNA Girase/metabolismo , DNA Topoisomerase IV/antagonistas & inibidores , Desenho de Fármacos , Farmacorresistência Bacteriana Múltipla , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Proteínas de Escherichia coli/antagonistas & inibidores , Ácido Graxo Sintase Tipo II/antagonistas & inibidores , Helicobacter pylori/efeitos dos fármacos , Helicobacter pylori/enzimologia , Testes de Sensibilidade Microbiana , Relação Quantitativa Estrutura-Atividade , Inibidores da Topoisomerase II/química , Inibidores da Topoisomerase II/farmacologia , Urease/antagonistas & inibidores , Inibidores de beta-Lactamases
20.
J Chem Inf Model ; 53(9): 2349-59, 2013 Sep 23.
Artigo em Inglês | MEDLINE | ID: mdl-24111836

RESUMO

Glutamate racemase (GR) is a cofactor independent amino acid racemase that has recently garnered increasing attention as an antimicrobial drug target. There are numerous high resolution crystal structures of GR, yet these are invariably bound to either D-glutamate or very weakly bound oxygen-based salts. Recent in silico screens have identified a number of new competitive inhibitor scaffolds, which are not based on D-Glu, but exploit many of the same hydrogen bond donor positions. In silico studies on 1-H-benzimidazole-2-sulfonic acid (BISA) show that the sulfonic acid points to the back of the GR active site, in the most buried region, analogous to the C2-carboxylate binding position in the GR-d-glutamate complex. Furthermore, BISA has been shown to be the strongest nonamino acid competitive inhibitor. Previously published computational studies have suggested that a portion of this binding strength is derived from complexation with a more closed active site, relative to weaker ligands, and in which the internal water network is more isolated from the bulk solvent. In order to validate key contacts between the buried sulfonate moiety of BISA and moieties in the back of the enzyme active site, as well as to probe the energetic importance of the potentially large number of interstitial waters contacted by the BISA scaffold, we have designed several mutants of Asn75. GR-N75A removes a key hydrogen bond donor to the sulfonate of BISA, but also serves to introduce an additional interstitial water, due to the newly created space of the mutation. GR- N75L should also show the loss of a hydrogen bond donor to the sulfonate of BISA, but does not (a priori) seem to permit an additional interstitial water contact. In order to investigate the dynamics, structure, and energies of this water-mediated complexation, we have employed the extended linear response (ELR) approach for the calculation of binding free energies to GR, using the YASARA2 knowledge based force field on a set of ten GR complexes, and yielding an R-squared value of 0.85 and a RMSE of 2.0 kJ/mol. Surprisingly, the inhibitor set produces a uniformly large interstitial water contribution to the electrostatic interaction energy (), ranging from 30 to >50%, except for the natural substrate (D-glutamate), which has only a 7% contribution of from water. The broader implications for predicting and exploiting significant interstitial water contacts in ligand-enzyme complexation are discussed.


Assuntos
Isomerases de Aminoácido/química , Isomerases de Aminoácido/metabolismo , Simulação de Dinâmica Molecular , Água/metabolismo , Isomerases de Aminoácido/antagonistas & inibidores , Isomerases de Aminoácido/genética , Bacillus subtilis/enzimologia , Domínio Catalítico , Inibidores Enzimáticos/farmacologia , Cinética , Ligantes , Mutação , Ligação Proteica , Termodinâmica
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