RESUMO
(1,1-Dioxo-2H-[1,2,4]benzothiadiazin-3-yl) azolo[1,5-a]pyridine and azolo[1,5-a]pyrimidine derivatives have been investigated as potential anti-HCV drugs. Their synthesis, HCV NS5B polymerase inhibition, and replicon activity are discussed.
Assuntos
Antivirais/síntese química , Azóis/síntese química , Benzotiadiazinas/síntese química , Inibidores Enzimáticos/síntese química , Hepatite C/tratamento farmacológico , Piridinas/síntese química , Pirimidinas/síntese química , Proteínas não Estruturais Virais/antagonistas & inibidores , Animais , Antivirais/farmacologia , Azóis/farmacologia , Benzotiadiazinas/farmacologia , Química Farmacêutica/métodos , Desenho de Fármacos , Inibidores Enzimáticos/farmacologia , Hepacivirus/metabolismo , Técnicas In Vitro , Concentração Inibidora 50 , Espectroscopia de Ressonância Magnética , Modelos Químicos , Estrutura Molecular , Piridinas/farmacologia , Pirimidinas/farmacologia , Relação Estrutura-Atividade , Proteínas não Estruturais Virais/químicaRESUMO
Future treatments for chronic hepatitis C virus (HCV) infection are likely to include agents that target viral components directly. Here, the preclinical characteristics of ITMN-191, a peptidomimetic inhibitor of the NS3/4A protease of HCV, are described. ITMN-191 inhibited a reference genotype 1 NS3/4A protein in a time-dependent fashion, a hallmark of an inhibitor with a two-step binding mechanism and a low dissociation rate. Under preequilibrium conditions, 290 pM ITMN-191 half-maximally inhibited the reference NS3/4A protease, but a 35,000-fold-higher concentration did not appreciably inhibit a panel of 79 proteases, ion channels, transporters, and cell surface receptors. Subnanomolar biochemical potency was maintained against NS3/4A derived from HCV genotypes 4, 5, and 6, while single-digit nanomolar potency was observed against NS3/4A from genotypes 2b and 3a. Dilution of a preformed enzyme inhibitor complex indicated ITMN-191 remained bound to and inhibited NS3/4A for more than 5 h after its initial association. In cell-based potency assays, half-maximal reduction of genotype 1b HCV replicon RNA was afforded by 1.8 nM; 45 nM eliminated the HCV replicon from cells. Peginterferon alfa-2a displayed a significant degree of antiviral synergy with ITMN-191 and reduced the concentration of ITMN-191 required for HCV replicon elimination. A 30-mg/kg of body weight oral dose administered to rats or monkeys yielded liver concentrations 12 h after dosing that exceeded the ITMN-191 concentration required to eliminate replicon RNA from cells. These preclinical characteristics compare favorably to those of other inhibitors of NS3/4A in clinical development and therefore support the clinical investigation of ITMN-191 for the treatment of chronic hepatitis C.
Assuntos
Antivirais , Proteínas de Transporte/antagonistas & inibidores , Hepacivirus/efeitos dos fármacos , Hepacivirus/enzimologia , Inibidores de Proteases , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas Virais/antagonistas & inibidores , Animais , Antivirais/química , Antivirais/metabolismo , Antivirais/farmacologia , Linhagem Celular Tumoral , Avaliação Pré-Clínica de Medicamentos , Sinergismo Farmacológico , Hepacivirus/genética , Hepacivirus/fisiologia , Humanos , Interferon alfa-2 , Interferon-alfa/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular , Fígado/metabolismo , Macaca fascicularis , Polietilenoglicóis/farmacologia , Inibidores de Proteases/química , Inibidores de Proteases/metabolismo , Inibidores de Proteases/farmacologia , Ratos , Proteínas Recombinantes , Replicação Viral/efeitos dos fármacosRESUMO
As bacteria continue to develop resistance toward current antibiotics, we find ourselves in a continual battle to identify new antibacterial agents and targets. We report herein a class of boron-containing compounds termed borinic esters that have broad spectrum antibacterial activity with minimum inhibitory concentrations (MIC) in the low microgram/mL range. These compounds were identified by screening for inhibitors against Caulobacter crescentus CcrM, an essential DNA methyltransferase from gram negative alpha-proteobacteria. In addition, we demonstrate that borinic esters inhibit menaquinone methyltransferase in gram positive bacteria using a new biochemical assay for MenH from Bacillus subtilis. Our data demonstrate the potential for further development of borinic esters as antibacterial agents as well as leads to explore more specific inhibitors against two essential bacterial enzymes.
Assuntos
Antibacterianos/síntese química , Ácidos Borínicos/síntese química , Metilases de Modificação do DNA/antagonistas & inibidores , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Antibacterianos/química , Antibacterianos/farmacologia , Bacillus subtilis/efeitos dos fármacos , Bacillus subtilis/enzimologia , Ácidos Borínicos/química , Ácidos Borínicos/farmacologia , Caulobacter crescentus/efeitos dos fármacos , Caulobacter crescentus/enzimologia , Metilases de Modificação do DNA/química , Ésteres/síntese química , Ésteres/química , Ésteres/farmacologia , Bactérias Gram-Negativas/enzimologia , Bactérias Gram-Negativas/crescimento & desenvolvimento , Bactérias Gram-Positivas/enzimologia , Bactérias Gram-Positivas/crescimento & desenvolvimento , Cinética , Testes de Sensibilidade Microbiana , Proteobactérias/enzimologia , Relação Estrutura-AtividadeRESUMO
We have developed a nonradioactive assay method for DNA methyltransferases based on the ability to protect substrate DNA from restriction. DNA immobilized to a microplate well was treated sequentially with methyltransferase and an appropriate endonuclease. The amount of methylated DNA product is reflected by a proportional decrease in endonuclease cleavage, which is in turn reflected by increased retention of the end-labeled affinity probe. A single universal substrate was designed to assay multiple methyltransferases including those that do not have a cognate endonuclease. The methodology developed is suited to screen a large number of compounds for inhibitors of various methyltransferases.
Assuntos
Metilases de Modificação do DNA/análise , Metilases de Modificação do DNA/metabolismo , Sequência de Bases , DNA/síntese química , Enzimas de Restrição-Modificação do DNA/metabolismo , DNA-Citosina Metilases/antagonistas & inibidores , DNA-Citosina Metilases/metabolismo , Avaliação Pré-Clínica de Medicamentos , Reprodutibilidade dos TestesRESUMO
Recently, an alternative has been offered to the concept of transition state (TS) stabilization as an explanation for rate enhancements in enzyme-catalyzed reactions. Instead, most of the rate increase has been ascribed to preorganization of the enzyme active site to bind substrates in a geometry close to that of the TS, which then transit the activation barrier impelled by motions along the reaction coordinate. The question as to how an enzyme achieves such preorganization and concomitant TS stabilization as well as potential coupled motions along the reaction coordinate leads directly to the role of protein dynamic motion. Dihydrofolate reductase (DHFR) is a paradigm in which the role of dynamics in catalysis continues to be unraveled by a wealth of kinetic, structural, and computational studies. DHFR has flexible loop regions adjacent to the active site whose motions modulate passage through the kinetically preferred pathway. The participation of residues distant from the DHFR active site in enhancing the rate of hydride transfer, however, is unanticipated and may signify the importance of long range protein motions. The general significance of protein dynamics in understanding other biological processes is briefly discussed.
Assuntos
Oxirredutases do Álcool/química , Enzimas/química , Oxirredutases do Álcool/metabolismo , Sítios de Ligação , Catálise , Enzimas/metabolismo , Cinética , Modelos Químicos , Movimento (Física) , Conformação ProteicaRESUMO
Recently, the participation in catalysis of residues spatially removed from the enzyme's active site has received considerable attention. The influence of the distal Gly-121 residue on the chemical step of hydride transfer in dihydrofolate reductase (DHFR) catalysis had been demonstrated previously [Cameron, C. E., and Benkovic, S. J. (1997) Biochemistry 36, 15792-15800]. In our continuing effort to identify catalytically important residues that are distal from the active site, we used sequence conservation information, kinetic data on site-directed mutants, dynamic motion information from NMR methods, and correlated motions from MD simulations to identify a subset of residues. Among them, the region spanning positions 41-45 is distal to the active site and was chosen as the focus for the mutagenesis and kinetic studies reported here. Specifically, the highly conserved Met-42 was selected for site-directed mutagenesis. While the reaction kinetics for the M42F mutant enzyme did not deviate from wild-type behavior, a 41-fold reduction in the forward hydride transfer rate was found for the M42W mutant. Given the established role of Gly-121 in the hydride transfer process, double mutant enzymes involving positions 42 and 121 were constructed and characterized. These double mutant enzymes generally showed little changes in substrate and cofactor binding but synergistic decreases in forward hydride transfer rates, while the decreases in reverse rates were additive. Along with supporting information from mixed quantum/classical MD simulations [Agarwal, P. K., Billeter, S. R., Rajagopalan, P. T., Benkovic, S. J., and Hammes-Schiffer, S. (2002) Proc. Natl. Acad. Sci. U.S.A. 99, 2794-2799], the data suggest that a coupled dynamic motion of these distal residues enhances crossing of the chemical reaction barrier and imply an expanded nonstatic role for the protein fold in catalysis.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Hidrogênio/química , Mutagênese Sítio-Dirigida , Tetra-Hidrofolato Desidrogenase/química , Tetra-Hidrofolato Desidrogenase/genética , Sítios de Ligação/genética , Catálise , Biologia Computacional , Sequência Conservada , Evolução Molecular , Transferência Ressonante de Energia de Fluorescência , Glicina/química , Cinética , Ligantes , Metionina/química , NADP/química , Relação Estrutura-Atividade , Especificidade por Substrato/genética , Tetra-Hidrofolato Desidrogenase/isolamento & purificação , TermodinâmicaRESUMO
The interaction of dihydrofolate (H(2)F) and NADPH with a fluorescent derivative of H(2)F reductase (DHFR) was studied by using transient and single-molecule techniques. The fluorescent moiety Alexa 488 was attached to the structural loop that closes over the substrates after they are bound. Fluorescence quenching was found to accompany the binding of both substrates and the hydride transfer reaction. For the binding of H(2)F to DHFR, the simplest mechanism consistent with the data postulates that the enzyme exists as slowly interconverting conformers, with the substrate binding preferentially to one of the conformers. At pH 7.0, the binding reaction has a bimolecular rate constant of 1.8 x 10(7) M(-1).s(-1), and the formation of the initial complex is followed by a conformational change. The binding of NADPH to DHFR is more complex and suggests multiple conformers of the enzyme exist. NADPH binds to a different conformer than H(2)F with a bimolecular rate constant of 2.6-5.7 x 10(6) M(-1).s(-1), with the former value obtained from single-molecule kinetics and the latter from stopped-flow kinetics. Single-molecule studies of DHFR in equilibrium with substrates and products revealed a reaction with ensemble average rate constants of 170 and 470 s(-1) at pH 8.5. The former rate constant has an isotope effect of >2 when NADPD is substituted for NADPH and probably is associated with hydride transfer. The results from stopped-flow and single-molecule methods are complementary and demonstrate that multiple conformations of both the enzyme and enzyme-substrate complexes exist.
Assuntos
Ácido Fólico/análogos & derivados , Ácido Fólico/metabolismo , NADP/metabolismo , Tetra-Hidrofolato Desidrogenase/metabolismo , Escherichia coli/enzimologia , Cinética , Modelos Biológicos , Fatores de TempoRESUMO
A network of coupled promoting motions in the enzyme dihydrofolate reductase is identified and characterized. The present identification is based on genomic analysis for sequence conservation, kinetic measurements of multiple mutations, and mixed quantum/classical molecular dynamics simulations of hydride transfer. The motions in this network span time scales of femtoseconds to milliseconds and are found on the exterior of the enzyme as well as in the active site. This type of network has broad implications for an expanded role of the protein fold in catalysis as well as ancillaries such as the engineering of altered protein function and the action of drugs distal to the active site.
Assuntos
Tetra-Hidrofolato Desidrogenase/química , Animais , Catálise , Humanos , Cinética , Modelos Moleculares , Estrutura Secundária de Proteína , Tetra-Hidrofolato Desidrogenase/genética , Tetra-Hidrofolato Desidrogenase/metabolismoRESUMO
The thermodynamics and kinetics of the interaction of dihydrofolate reductase (DHFR) with methotrexate have been studied by using fluorescence, stopped-flow, and single-molecule methods. DHFR was modified to permit the covalent addition of a fluorescent molecule, Alexa 488, and a biotin at the N terminus of the molecule. The fluorescent molecule was placed on a protein loop that closes over methotrexate when binding occurs, thus causing a quenching of the fluorescence. The biotin was used to attach the enzyme in an active form to a glass surface for single-molecule studies. The equilibrium dissociation constant for the binding of methotrexate to the enzyme is 9.5 nM. The stopped-flow studies revealed that methotrexate binds to two different conformations of the enzyme, and the association and dissociation rate constants were determined. The single-molecule investigation revealed a conformational change in the enzyme-methotrexate complex that was not observed in the stopped-flow studies. The ensemble averaged rate constants for this conformation change in both directions is about 2-4 s(-1) and is attributed to the opening and closing of the enzyme loop over the bound methotrexate. Thus the mechanism of methotrexate binding to DHFR involves multiple steps and protein conformational changes.
Assuntos
Metotrexato/metabolismo , Tetra-Hidrofolato Desidrogenase/metabolismo , Sequência de Bases , Biotina , DNA Bacteriano/genética , Escherichia coli/enzimologia , Escherichia coli/genética , Corantes Fluorescentes , Genes Bacterianos , Cinética , Metotrexato/química , Microscopia de Fluorescência , Modelos Moleculares , Mutação , Conformação Proteica , Tetra-Hidrofolato Desidrogenase/química , Tetra-Hidrofolato Desidrogenase/genética , TermodinâmicaRESUMO
H-transfer was studied in the complex kinetic cascade of dihydrofolate reductase. Intrinsic kinetic isotope effects, their temperature dependence, and other temperature-dependent parameters indicated H-tunneling, but no 1 degrees to 2 degrees coupled motion. The data also suggested environmentally coupled tunneling and commitment to catalysis on pre-steady-state isotope effects.