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1.
Biochemistry ; 59(14): 1428-1441, 2020 Apr 14.
Artigo em Inglês | MEDLINE | ID: mdl-32207968

RESUMO

Osimertinib is a covalent, third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) approved for treating non-small cell lung cancer patients with activating EGFR mutations (Exon19del or L858R) or with the T790M resistance mutation following disease progression on first- or second-generation EGFR TKIs. The aim of this work is to rationalize and understand how osimertinib achieves mutant EGFR selectivity over the wild-type (WT) by evaluating its kinetic mechanism of action. In doing so, we developed methodologies combining steady-state and pre-steady-state kinetics to determine the covalent inactivation rates (kinact) and reversible binding affinities (Ki) for osimertinib against WT, L858R, and L858R/T790M EGFR and compared these data to the inhibition kinetics of earlier generations of EGFR TKIs. The kinact/KI values indicate osimertinib inactivates L858R and L858R/T790M with 20- and 50-fold higher overall efficiencies, respectively, compared to that for WT. The Ki and kinact values reveal that osimertinib binds 3-fold tighter to and reacts 3-fold faster with L858R than WT EGFR and binds 17-fold tighter to and reacts 3-fold faster with L858R/T790M than with the WT EGFR. We conclude that osimertinib overcomes the T790M mutation through improved affinities from stronger hydrophobic interactions with Met790 versus Thr790 and an improved rate of covalent bond formation via better positioning of the acrylamide warhead, while osimertinib targets the L858R mutation through better affinities and reactivities with the mutant in the context of differential binding affinities of the competing substrate ATP. This work highlights the importance of optimizing both reversible drug-target interactions and inactivation rates for covalent inhibitors to achieve selectivity in targeting mutant EGFRs.

2.
J Med Chem ; 63(5): 2557-2576, 2020 Mar 12.
Artigo em Inglês | MEDLINE | ID: mdl-31922409

RESUMO

Decaprenylphosphoryl-ß-d-ribose 2'-epimerase (DprE1) is an essential enzyme in Mycobacterium tuberculosis and has recently been studied as a potential drug target, with inhibitors progressing to clinical studies. Here we describe the identification of a novel series of morpholino-pyrimidine DprE1 inhibitors. These were derived from a phenotypic high-throughput screening (HTS) hit with suboptimal physicochemical properties. Optimization strategies included scaffold-hopping, synthesis, and evaluation of fragments of the lead compounds and property-focused optimization. The resulting optimized compounds had much improved physicochemical properties and maintained enzyme and cellular potency. These molecules demonstrated potent efficacy in an in vivo tuberculosis murine infection model.

3.
Cell Chem Biol ; 27(1): 41-46.e17, 2020 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-31786184

RESUMO

Deregulation of the PRC2 complex, comprised of the core subunits EZH2, SUZ12, and EED, drives aberrant hypermethylation of H3K27 and tumorigenicity of many cancers. Although inhibitors of EZH2 have shown promising clinical activity, preclinical data suggest that resistance can be acquired through secondary mutations in EZH2 that abrogate drug target engagement. To address these limitations, we have designed several hetero-bifunctional PROTACs (proteolysis-targeting chimera) to efficiently target EED for elimination. Our PROTACs bind to EED (pKD ∼ 9.0) and promote ternary complex formation with the E3 ubiquitin ligase. The PROTACs potently inhibit PRC2 enzyme activity (pIC50 ∼ 8.1) and induce rapid degradation of not only EED but also EZH2 and SUZ12 within the PRC2 complex. Furthermore, the PROTACs selectively inhibit proliferation of PRC2-dependent cancer cells (half maximal growth inhibition [GI50] = 49-58 nM). In summary, our data demonstrate a therapeutic modality to target PRC2-dependent cancer through a PROTAC-mediated degradation mechanism.

4.
J Med Chem ; 61(24): 11221-11249, 2018 12 27.
Artigo em Inglês | MEDLINE | ID: mdl-30500189

RESUMO

Tuberculosis is the leading cause of death worldwide from infectious diseases. With the development of drug-resistant strains of Mycobacterium tuberculosis, there is an acute need for new medicines with novel modes of action. Herein, we report the discovery and profiling of a novel hydantoin-based family of antimycobacterial inhibitors of the decaprenylphospho-ß-d-ribofuranose 2-oxidase (DprE1). In this study, we have prepared a library of more than a 100 compounds and evaluated them for their biological and physicochemical properties. The series is characterized by high enzymatic and whole-cell activity, low cytotoxicity, and a good overall physicochemical profile. In addition, we show that the series acts via reversible inhibition of the DprE1 enzyme. Overall, the novel compound family forms an attractive base for progression to further stages of optimization and may provide a promising drug candidate in the future.


Assuntos
Oxirredutases do Álcool/antagonistas & inibidores , Antituberculosos/química , Antituberculosos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Hidantoínas/química , Actinobacteria/efeitos dos fármacos , Oxirredutases do Álcool/metabolismo , Proteínas de Bactérias/metabolismo , Estabilidade de Medicamentos , Inibidores Enzimáticos/química , Células Hep G2 , Ensaios de Triagem em Larga Escala/métodos , Humanos , Macrófagos/microbiologia , Testes de Sensibilidade Microbiana , Mycobacterium tuberculosis/efeitos dos fármacos , Reprodutibilidade dos Testes , Relação Estrutura-Atividade , Tuberculose/tratamento farmacológico , Tuberculose/microbiologia
5.
Sci Rep ; 8(1): 13473, 2018 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-30194385

RESUMO

Nitro-substituted 1,3-benzothiazinones (nitro-BTZs) are mechanism-based covalent inhibitors of Mycobacterium tuberculosis decaprenylphosphoryl-ß-D-ribose-2'-oxidase (DprE1) with strong antimycobacterial properties. We prepared a number of oxidized and reduced forms of nitro-BTZs to probe the mechanism of inactivation of the enzyme and to identify opportunities for further chemistry. The kinetics of inactivation of DprE1 was examined using an enzymatic assay that monitored reaction progress up to 100 min, permitting compound ranking according to kinact/Ki values. The side-chain at the 2-position and heteroatom identity at the 1-position of the BTZs were found to be important for inhibitory activity. We obtained crystal structures with several compounds covalently bound. The data suggest that steps upstream from the covalent end-points are likely the key determinants of potency and reactivity. The results of protein mass spectrometry using a 7-chloro-nitro-BTZ suggest that nucleophilic reactions at the 7-position do not operate and support a previously proposed mechanism in which BTZ activation by a reduced flavin intermediate is required. Unexpectedly, a hydroxylamino-BTZ showed time-dependent inhibition and mass spectrometry corroborated that this hydroxylamino-BTZ is a mechanism-based suicide inhibitor of DprE1. With this BTZ derivative, we propose a new covalent mechanism of inhibition of DprE1 that takes advantage of the oxidation cycle of the enzyme.


Assuntos
Oxirredutases do Álcool , Antituberculosos/química , Proteínas de Bactérias , Inibidores Enzimáticos/química , Mycobacterium tuberculosis/enzimologia , Oxirredutases do Álcool/antagonistas & inibidores , Oxirredutases do Álcool/química , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/química , Cristalografia por Raios X , Espectrometria de Massas
6.
Biochemistry ; 57(24): 3387-3401, 2018 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-29684272

RESUMO

The biosynthetic pathway of peptidoglycan is essential for Mycobacterium tuberculosis. We report here the acetyltransferase substrate specificity and catalytic mechanism of the bifunctional N-acetyltransferase/uridylyltransferase from M. tuberculosis (GlmU). This enzyme is responsible for the final two steps of the synthesis of UDP- N-acetylglucosamine, which is an essential precursor of peptidoglycan, from glucosamine 1-phosphate, acetyl-coenzyme A, and uridine 5'-triphosphate. GlmU utilizes ternary complex formation to transfer an acetyl from acetyl-coenzyme A to glucosamine 1-phosphate to form N-acetylglucosamine 1-phosphate. Steady-state kinetic studies and equilibrium binding experiments indicate that GlmU follows a steady-state ordered kinetic mechanism, with acetyl-coenzyme A binding first, which triggers a conformational change in GlmU, followed by glucosamine 1-phosphate binding. Coenzyme A is the last product to dissociate. Chemistry is partially rate-limiting as indicated by pH-rate studies and solvent kinetic isotope effects. A novel crystal structure of a mimic of the Michaelis complex, with glucose 1-phosphate and acetyl-coenzyme A, helps us to propose the residues involved in deprotonation of glucosamine 1-phosphate and the loop movement that likely generates the active site required for glucosamine 1-phosphate to bind. Together, these results pave the way for the rational discovery of improved inhibitors against M. tuberculosis GlmU, some of which might become candidates for antibiotic discovery programs.


Assuntos
Proteínas de Bactérias/metabolismo , Biocatálise , Complexos Multienzimáticos/metabolismo , Uridina Difosfato N-Acetilglicosamina/biossíntese , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/química , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Concentração de Íons de Hidrogênio , Cinética , Cloreto de Magnésio/química , Cloreto de Magnésio/farmacologia , Estrutura Molecular , Complexos Multienzimáticos/antagonistas & inibidores , Complexos Multienzimáticos/química , Mycobacterium tuberculosis/enzimologia , Especificidade por Substrato , Uridina Difosfato N-Acetilglicosamina/química
7.
Neurosci Lett ; 673: 44-50, 2018 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-29499308

RESUMO

Expression of mutant Huntingtin (HTT) protein is central to the pathophysiology of Huntington's Disease (HD). The E3 ubiquitin ligase MID1 appears to have a key role in facilitating translation of the mutant HTT mRNA suggesting that interference with the function of this complex could be an attractive therapeutic approach. Here we describe a peptide that is able to disrupt the interaction between MID1 and the α4 protein, a regulatory subunit of protein phosphatase 2A (PP2A). By fusing this peptide to a sequence from the HIV-TAT protein we demonstrate that the peptide can disrupt the interaction within cells and show that this results in a decrease in levels of ribosomal S6 phosphorylation and HTT expression in cultures of cerebellar granule neurones derived from HdhQ111/Q7 mice. This data serves to validate this pathway and paves the way for the discovery of small molecule inhibitors of this interaction as potential therapies for HD.


Assuntos
Proteína Huntingtina/metabolismo , Neurônios/metabolismo , Proteínas/metabolismo , Animais , Células HEK293 , Humanos , Proteína Huntingtina/genética , Camundongos , Mutação , Cultura Primária de Células , Ligação Proteica , Proteína Fosfatase 2/metabolismo , Ubiquitina-Proteína Ligases
8.
Nat Commun ; 7: 12581, 2016 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-27581223

RESUMO

Phenotypic screens for bactericidal compounds are starting to yield promising hits against tuberculosis. In this regard, whole-genome sequencing of spontaneous resistant mutants generated against an indazole sulfonamide (GSK3011724A) identifies several specific single-nucleotide polymorphisms in the essential Mycobacterium tuberculosis ß-ketoacyl synthase (kas) A gene. Here, this genomic-based target assignment is confirmed by biochemical assays, chemical proteomics and structural resolution of a KasA-GSK3011724A complex by X-ray crystallography. Finally, M. tuberculosis GSK3011724A-resistant mutants increase the in vitro minimum inhibitory concentration and the in vivo 99% effective dose in mice, establishing in vitro and in vivo target engagement. Surprisingly, the lack of target engagement of the related ß-ketoacyl synthases (FabH and KasB) suggests a different mode of inhibition when compared with other Kas inhibitors of fatty acid biosynthesis in bacteria. These results clearly identify KasA as the biological target of GSK3011724A and validate this enzyme for further drug discovery efforts against tuberculosis.


Assuntos
3-Oxoacil-(Proteína de Transporte de Acila) Sintase/antagonistas & inibidores , 3-Oxoacil-(Proteína de Transporte de Acila) Sintase/genética , Antituberculosos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/genética , Indazóis/farmacologia , Mycobacterium tuberculosis/efeitos dos fármacos , Sulfonamidas/farmacologia , Tuberculose Pulmonar/tratamento farmacológico , Animais , Farmacorresistência Bacteriana/genética , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Testes de Sensibilidade Microbiana , Mycobacterium tuberculosis/genética , Polimorfismo de Nucleotídeo Único/genética , Tuberculose Pulmonar/microbiologia , Tuberculose Pulmonar/prevenção & controle
9.
Proc Natl Acad Sci U S A ; 113(31): E4523-30, 2016 08 02.
Artigo em Inglês | MEDLINE | ID: mdl-27432954

RESUMO

The rising incidence of antimicrobial resistance (AMR) makes it imperative to understand the underlying mechanisms. Mycobacterium tuberculosis (Mtb) is the single leading cause of death from a bacterial pathogen and estimated to be the leading cause of death from AMR. A pyrido-benzimidazole, 14, was reported to have potent bactericidal activity against Mtb. Here, we isolated multiple Mtb clones resistant to 14. Each had mutations in the putative DNA-binding and dimerization domains of rv2887, a gene encoding a transcriptional repressor of the MarR family. The mutations in Rv2887 led to markedly increased expression of rv0560c. We characterized Rv0560c as an S-adenosyl-L-methionine-dependent methyltransferase that N-methylates 14, abolishing its mycobactericidal activity. An Mtb strain lacking rv0560c became resistant to 14 by mutating decaprenylphosphoryl-ß-d-ribose 2-oxidase (DprE1), an essential enzyme in arabinogalactan synthesis; 14 proved to be a nanomolar inhibitor of DprE1, and methylation of 14 by Rv0560c abrogated this activity. Thus, 14 joins a growing list of DprE1 inhibitors that are potently mycobactericidal. Bacterial methylation of an antibacterial agent, 14, catalyzed by Rv0560c of Mtb, is a previously unreported mechanism of AMR.


Assuntos
Antituberculosos/metabolismo , Proteínas de Bactérias/metabolismo , Farmacorresistência Bacteriana , Mycobacterium tuberculosis/metabolismo , Antituberculosos/química , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Benzimidazóis/química , Benzimidazóis/metabolismo , Regulação Bacteriana da Expressão Gênica , Metilação , Metiltransferases/química , Metiltransferases/genética , Metiltransferases/metabolismo , Modelos Moleculares , Estrutura Molecular , Mutação , Mycobacterium tuberculosis/genética , Domínios Proteicos , Proteínas Repressoras/química , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , S-Adenosilmetionina/metabolismo
10.
ACS Infect Dis ; 1(12): 615-26, 2015 Dec 11.
Artigo em Inglês | MEDLINE | ID: mdl-27623058

RESUMO

We have targeted the Mycobacterium tuberculosis decaprenylphosphoryl-ß-d-ribose oxidase (Mt-DprE1) for potential chemotherapeutic intervention of tuberculosis. A multicopy suppression strategy that overexpressed Mt-DprE1 in M. bovis BCG was used to profile the publically available GlaxoSmithKline antimycobacterial compound set, and one compound (GSK710) was identified that showed an 8-fold higher minimum inhibitory concentration relative to the control strain. Analogues of GSK710 show a clear relationship between whole cell potency and in vitro activity using an enzymatic assay employing recombinant Mt-DprE1, with binding affinity measured by fluorescence quenching of the flavin cofactor of the enzyme. M. bovis BCG spontaneous resistant mutants to GSK710 and a closely related analogue were isolated and sequencing of ten such mutants revealed a single point mutation at two sites, E221Q or G248S within DprE1, providing further evidence that DprE1 is the main target of these compounds. Finally, time-lapse microscopy experiments showed that exposure of M. tuberculosis to a compound of this series arrests bacterial growth rapidly followed by a slower cytolysis phase.

11.
J Biol Chem ; 289(48): 33456-68, 2014 Nov 28.
Artigo em Inglês | MEDLINE | ID: mdl-25301950

RESUMO

HCV infection is an urgent global health problem that has triggered a drive to discover therapies that specifically target the virus. BMS-791325 is a novel direct antiviral agent specifically targeting HCV NS5B, an RNA-dependent RNA polymerase. Robust viral clearance of HCV was observed in infected patients treated with BMS-791325 in combination with other anti-HCV agents in Phase 2 clinical studies. Biochemical and biophysical studies revealed that BMS-791325 is a time-dependent, non-competitive inhibitor of the polymerase. Binding studies with NS5B genetic variants (WT, L30S, and P495L) exposed a two-step, slow binding mechanism, but details of the binding mechanism differed for each of the polymerase variants. For the clinically relevant resistance variant (P495L), the rate of initial complex formation and dissociation is similar to WT, but the kinetics of the second step is significantly faster, showing that this variant impacts the final tight complex. The resulting shortened residence time translates into the observed decrease in inhibitor potency. The L30S variant has a significantly different profile. The rate of initial complex formation and dissociation is 7-10 times faster for the L30S variant compared with WT; however, the forward and reverse rates to form the final complex are not significantly different. The impact of the L30S variant on the inhibition profile and binding kinetics of BMS-791325 provides experimental evidence for the dynamic interaction of fingers and thumb domains in an environment that supports the formation of active replication complexes and the initiation of RNA synthesis.


Assuntos
Antivirais/química , Benzazepinas/química , Hepacivirus/enzimologia , Indóis/química , RNA Replicase/antagonistas & inibidores , Proteínas não Estruturais Virais/antagonistas & inibidores , Substituição de Aminoácidos , Antivirais/farmacologia , Benzazepinas/uso terapêutico , Hepatite C/tratamento farmacológico , Hepatite C/enzimologia , Humanos , Indóis/uso terapêutico , Mutação de Sentido Incorreto , Ligação Proteica , RNA Replicase/química , RNA Replicase/metabolismo , RNA Viral/biossíntese , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/metabolismo
12.
J Biomol Screen ; 19(4): 508-15, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24381207

RESUMO

Kynurenine 3-monooxygenase (KMO) is a therapeutically important target on the eukaryotic tryptophan catabolic pathway, where it converts L-kynurenine (Kyn) to 3-hydroxykynurenine (3-HK). We have cloned and expressed the human form of this membrane protein as a full-length GST-fusion in a recombinant baculovirus expression system. An enriched membrane preparation was used for a directed screen of approximately 78,000 compounds using a RapidFire mass spectrometry (RF-MS) assay. The RapidFire platform provides an automated solid-phase extraction system that gives a throughput of approximately 7 s per well to the mass spectrometer, where direct measurement of both the substrate and product allowed substrate conversion to be determined. The RF-MS methodology is insensitive to assay interference, other than where compounds have the same nominal mass as Kyn or 3-HK and produce the same mass transition on fragmentation. These instances could be identified by comparison with the product-only data. The screen ran with excellent performance (average Z' value 0.8) and provided several tractable hit series for further investigation.


Assuntos
Descoberta de Drogas , Inibidores Enzimáticos/farmacologia , Ensaios de Triagem em Larga Escala , Quinurenina 3-Mono-Oxigenase/antagonistas & inibidores , Espectrometria de Massas , Animais , Domínio Catalítico , Linhagem Celular , Avaliação Pré-Clínica de Medicamentos , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/química , Humanos , Cinética , Quinurenina 3-Mono-Oxigenase/química , Quinurenina 3-Mono-Oxigenase/metabolismo , Espectrometria de Massas/métodos , Ligação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo
13.
J Biomol Screen ; 19(2): 278-86, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23896685

RESUMO

Using mass spectrometry to detect enzymatic activity offers several advantages over fluorescence-based methods. Automation of sample handling and analysis using platforms such as the RapidFire (Agilent Technologies, Lexington, MA) has made these assays amenable to medium-throughput screening (of the order of 100,000 wells). However, true high-throughput screens (HTS) of large compound collections (>1 million) are still considered too time-consuming to be feasible. Here we propose a simple multiplexing strategy that can be used to increase the throughput of RapidFire, making it viable for HTS. The method relies on the ability to analyze pooled samples from several reactions simultaneously and to deconvolute their origin using "mass-tagged" substrates. Using the JmjD2d H3K9me3 demethylase as a model system, we demonstrate the practicality of this method to achieve a 4-fold increase in throughput. This was achieved without any loss of assay quality. This multiplex strategy could easily be scaled to give even greater reductions in analysis time.


Assuntos
Ensaios de Triagem em Larga Escala , Histona Desmetilases com o Domínio Jumonji/metabolismo , Espectrometria de Massas/métodos , Epigenômica , Humanos , Especificidade por Substrato
14.
J Biomol Screen ; 17(1): 39-48, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21859681

RESUMO

A high-throughput RapidFire mass spectrometry assay is described for the JMJD2 family of Fe(2+), O(2), and α-ketoglutarate-dependent histone lysine demethylases. The assay employs a short amino acid peptide substrate, corresponding to the first 15 amino acid residues of histone H3, but mutated at two positions to increase assay sensitivity. The assay monitors the direct formation of the dimethylated-Lys9 product from the trimethylated-Lys9 peptide substrate. Monitoring the formation of the monomethylated and des-methylated peptide products is also possible. The assay was validated using known inhibitors of the histone lysine demethylases, including 2,4-pyridinedicarboxylic acid and an α-ketoglutarate analogue. With a sampling rate of 7 s per well, the RapidFire technology permitted the single-concentration screening of 101 226 compounds against JMJD2C in 10 days using two instruments, typically giving Z' values of 0.75 to 0.85. Several compounds were identified of the 8-hydroxyquinoline chemotype, a known series of inhibitors of the Lys9-specific histone demethylases. The peptide also functions as a substrate for JMJD2A, JMJD2D, and JMJD2E, thus enabling the development of assays for all 3 enzymes to monitor progress in compound selectivity. The assay represents the first report of a RapidFire mass spectrometry assay for an epigenetics target.


Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Inibidores Enzimáticos/farmacologia , Ensaios de Triagem em Larga Escala/métodos , Histona Desmetilases/metabolismo , Histona Desmetilases com o Domínio Jumonji/metabolismo , Espectrometria de Massas/métodos , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/metabolismo , Epigênese Genética/efeitos dos fármacos , Histona Desmetilases/antagonistas & inibidores , Histona Desmetilases com o Domínio Jumonji/antagonistas & inibidores , Cinética , Lisina/metabolismo , Oxiquinolina/metabolismo , Oxiquinolina/farmacologia , Peptídeos/metabolismo , Piridinas/metabolismo , Piridinas/farmacologia , Especificidade por Substrato
15.
Biochemistry ; 48(9): 1996-2004, 2009 Mar 10.
Artigo em Inglês | MEDLINE | ID: mdl-19166329

RESUMO

The enzyme alpha-isopropylmalate synthase from Mycobacterium tuberculosis (MtIPMS) has been identified as a possible target for the design of new antitubercular therapeutics. Recently, it was shown that MtIPMS is subject to slow-onset, feedback inhibition by l-leucine, the first instance of an allosteric regulator utilizing this mechanism. Structural studies are inconsistent with canonical allosteric mechanisms, including changes to the quaternary structure or large, rigid-body conformational changes to the enzyme upon l-leucine binding. Thus, the allosteric regulation may result from a discrete inhibitory signal transmitted to the active site upon l-leucine binding in the regulatory domain, a distance of more than 50 A. To test this mechanism, site-directed mutagenesis was employed to construct enzymes with substitutions at phylogenetically conserved active site residues near the interface of the catalytic and linker domains. The substitutions had wide-ranging effects on the kinetics of l-leucine inhibition, with some modest effects on the kinetic parameters of catalysis. The most dramatic result was the finding that the Y410F mutant form of MtIPMS is insensitive to l-leucine inhibition, suggesting that this residue has completely uncoupled the inhibitory signal to the active site. Overall, the data are consistent with a mechanism of allosteric regulation described by the interdomain communication of the inhibitory signal from the regulatory to catalytic domain and implicate the interactions between the linker and catalytic domains as critical determinants of inhibitory signal transmission.


Assuntos
2-Isopropilmalato Sintase/química , Proteínas de Bactérias/química , Mycobacterium tuberculosis/enzimologia , 2-Isopropilmalato Sintase/genética , 2-Isopropilmalato Sintase/metabolismo , Regulação Alostérica , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação/genética , Calorimetria/métodos , Catálise/efeitos dos fármacos , Cinética , Leucina/metabolismo , Leucina/farmacologia , Espectroscopia de Ressonância Magnética , Malatos/química , Malatos/metabolismo , Modelos Moleculares , Estrutura Molecular , Mutagênese Sítio-Dirigida , Mutação de Sentido Incorreto , Mycobacterium tuberculosis/genética , Conformação Proteica , Estrutura Terciária de Proteína , Titulometria
16.
Biochemistry ; 47(24): 6499-507, 2008 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-18500821

RESUMO

Riboflavin is biosynthesized by most microorganisms and plants, while mammals depend entirely on the absorption of this vitamin from the diet to meet their metabolic needs. Therefore, riboflavin biosynthesis appears to be an attractive target for drug design, since appropriate inhibitors of the pathway would selectively target the microorganism. We have cloned and solubly expressed the bifunctional ribD gene from Escherichia coli, whose three-dimensional structure was recently determined. We have demonstrated that the rate of deamination (370 min (-1)) exceeds the rate of reduction (19 min (-1)), suggesting no channeling between the two active sites. The reductive ring opening reaction occurs via a hydride transfer from the C 4- pro-R hydrogen of NADPH to C'-1 of ribose and is the rate-limiting step in the overall reaction, exhibiting a primary kinetic isotope effect ( (D) V) of 2.2. We also show that the INH-NADP adduct, one of the active forms of the anti-TB drug isoniazid, inhibits the E. coli RibD. On the basis of the observed patterns of inhibition versus the two substrates, we propose that the RibD-catalyzed reduction step follows a kinetic scheme similar to that of its structural homologue, DHFR.


Assuntos
Proteínas de Escherichia coli/química , Escherichia coli/enzimologia , Nucleotídeo Desaminases/química , Riboflavina/biossíntese , Riboflavina/química , Desidrogenase do Álcool de Açúcar/química , Catálise , Desaminação , Medição da Troca de Deutério , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Cinética , NADP/química , NADP/metabolismo , Nucleotídeo Desaminases/genética , Nucleotídeo Desaminases/metabolismo , Oxirredução , Pentosefosfatos/química , Pentosefosfatos/metabolismo , Estrutura Terciária de Proteína/genética , Bases de Schiff , Solventes , Especificidade por Substrato/genética , Desidrogenase do Álcool de Açúcar/genética , Desidrogenase do Álcool de Açúcar/metabolismo
18.
Biochemistry ; 45(47): 13947-53, 2006 Nov 28.
Artigo em Inglês | MEDLINE | ID: mdl-17115689

RESUMO

Isoniazid (INH) is an essential drug used to treat tuberculosis. The mycobactericidal agents are INH adducts [INH-NAD(P)] of the pyridine nucleotide coenzymes, which are generated in vivo after INH activation and which bind to, and inhibit, essential enzymes. The NADH-dependent enoyl-ACP reductase (InhA) and the NADPH-dependent dihydrofolate reductase (DfrA) have both been shown to be inhibited by INH-NAD(P) adducts with nanomolar affinity. In this paper, we profiled the Mycobacterium tuberculosis proteome using both the INH-NAD and INH-NADP adducts coupled to solid supports and identified, in addition to InhA and DfrA, 16 other proteins that bind these adducts with high affinity. The majority of these are predicted to be pyridine nucleotide-dependent dehydrogenases/reductases. They are involved in many cellular processes, including S-adenosylmethionine-dependent methyl transfer reactions, pyrimidine and valine catabolism, the arginine degradative pathway, proton and potassium transport, stress response, lipid metabolism, and riboflavin biosynthesis. The targeting of multiple enzymes could, thus, account for the pleiotropic effects of, and powerful mycobactericidal properties of, INH.


Assuntos
Antituberculosos/farmacologia , Proteínas de Bactérias/efeitos dos fármacos , Isoniazida/farmacologia , Mycobacterium tuberculosis/efeitos dos fármacos , Proteoma , Proteínas de Bactérias/metabolismo , Eletroforese em Gel de Poliacrilamida , Mycobacterium tuberculosis/metabolismo
19.
Nat Struct Mol Biol ; 13(5): 408-13, 2006 May.
Artigo em Inglês | MEDLINE | ID: mdl-16648861

RESUMO

Isoniazid is a key drug used in the treatment of tuberculosis. Isoniazid is a pro-drug, which, after activation by the katG-encoded catalase peroxidase, reacts nonenzymatically with NAD(+) and NADP(+) to generate several isonicotinoyl adducts of these pyridine nucleotides. One of these, the acyclic 4S isomer of isoniazid-NAD, targets the inhA-encoded enoyl-ACP reductase, an enzyme essential for mycolic acid biosynthesis in Mycobacterium tuberculosis. Here we show that the acyclic 4R isomer of isoniazid-NADP inhibits the M. tuberculosis dihydrofolate reductase (DHFR), an enzyme essential for nucleic acid synthesis. This biologically relevant form of the isoniazid adduct is a subnanomolar bisubstrate inhibitor of M. tuberculosis DHFR. Expression of M. tuberculosis DHFR in Mycobacterium smegmatis mc(2)155 protects cells against growth inhibition by isoniazid by sequestering the drug. Thus, M. tuberculosis DHFR is the first new target for isoniazid identified in the last decade.


Assuntos
Antagonistas do Ácido Fólico/farmacologia , Isoniazida/farmacologia , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/enzimologia , Tetra-Hidrofolato Desidrogenase/metabolismo , Proliferação de Células , Clonagem Molecular , Cristalografia por Raios X , Antagonistas do Ácido Fólico/química , Antagonistas do Ácido Fólico/metabolismo , Expressão Gênica , Isoniazida/química , Isoniazida/metabolismo , Modelos Moleculares , Mycobacterium tuberculosis/citologia , Mycobacterium tuberculosis/genética , NADP/química , NADP/metabolismo , Pró-Fármacos/química , Pró-Fármacos/metabolismo , Pró-Fármacos/farmacologia , Ligação Proteica , Estrutura Terciária de Proteína , Tetra-Hidrofolato Desidrogenase/genética , Tetra-Hidrofolato Desidrogenase/isolamento & purificação
20.
J Am Chem Soc ; 127(28): 10004-5, 2005 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-16011356

RESUMO

This report describes the first demonstration of slow-onset feedback inhibition of an enzyme that catalyzes the first committed step in a biosynthetic pathway. alpha-Isopropylmalate synthase (IPMS) catalyzes the first committed step of the l-leucine biosynthetic pathway and is feedback-inhibited by l-leucine. Initial velocity experiments on the Mycobacterium tuberculosis IPMS indicate that inhibition by l-leucine is linearly noncompetitive versus alpha-ketoisovalerate. Time-courses displayed a burst of product formation followed by a linear steady-state rate when reactions were initiated by the addition of enzyme. The burst rate showed a hyperbolic dependence on the concentration of l-leucine indicating that inhibition proceeds in two steps, an initial rapid binding step followed by slow isomerization to a more tightly bound complex.


Assuntos
2-Isopropilmalato Sintase/antagonistas & inibidores , 2-Isopropilmalato Sintase/química , Leucina/química , Leucina/farmacologia , Mycobacterium tuberculosis/enzimologia , Ligação Competitiva , Relação Dose-Resposta a Droga , Retroalimentação Fisiológica , Cinética , Fatores de Tempo
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