RESUMO
PYR1/PYL/RCAR proteins (PYLs) are confirmed abscisic acid (ABA) receptors, which inhibit protein phosphatase 2C (PP2C) upon binding to ABA. Arabidopsis thaliana has 14 PYLs, yet their functional distinction remains unclear. Here, we report systematic biochemical characterization of PYLs. A subclass of PYLs, represented by PYL10, inhibited PP2C in the absence of any ligand. Crystal structures of PYL10, both in the free form and in the HAB1 (PP2C)-bound state, revealed the structural basis for its constitutive activity. Structural-guided biochemical analyses revealed that ABA-independent inhibition of PP2C requires the PYLs to exist in a monomeric state. In addition, the residues guarding the entrance to the ligand-binding pocket of these PYLs should be bulky and hydrophobic. Based on these principles, we were able to generate monomeric PYL2 variants that gained constitutive inhibitory effect on PP2Cs. These findings provide an important framework for understanding the complex regulation of ABA signaling by PYL proteins.
Assuntos
Proteínas de Arabidopsis/fisiologia , Arabidopsis/metabolismo , Receptores de Superfície Celular/fisiologia , Ácido Abscísico/metabolismo , Ácido Abscísico/farmacologia , Sequência de Aminoácidos , Arabidopsis/genética , Proteínas de Arabidopsis/antagonistas & inibidores , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Dados de Sequência Molecular , Fosfoproteínas Fosfatases/antagonistas & inibidores , Fosfoproteínas Fosfatases/metabolismo , Estrutura Terciária de Proteína , Receptores de Superfície Celular/química , Receptores de Superfície Celular/genética , Alinhamento de SequênciaRESUMO
Human respiratory syncytial virus (RSV) is a leading cause of lower respiratory tract infections (LRTI) in young children and elderly people worldwide. Recent significant progress in our understanding of the structure and function of RSV proteins has led to the discovery of several clinical candidates targeting RSV fusion and replication. These include both the development of novel small molecule interventions and the isolation of potent monoclonal antibodies. In this review, we summarize the state-of-the-art of RSV drug discovery, with a focus on the characteristics of the candidates that reached the clinical stage of development. We also discuss the lessons learned from failed and discontinued clinical developments and highlight the challenges that remain for development of RSV therapies.
Assuntos
Infecções por Vírus Respiratório Sincicial , Vacinas contra Vírus Sincicial Respiratório , Vírus Sincicial Respiratório Humano , Infecções Respiratórias , Criança , Humanos , Idoso , Pré-Escolar , Anticorpos Monoclonais/uso terapêutico , Vacinas contra Vírus Sincicial Respiratório/uso terapêutico , Proteínas Virais de Fusão , Anticorpos Antivirais , Anticorpos NeutralizantesRESUMO
Ziresovir (RO-0529, AK0529) is reported here for the first time as a promising respiratory syncytial virus (RSV) fusion (F) protein inhibitor that currently is in phase 2 clinical trials. This article describes the process of RO-0529 as a potent, selective, and orally bioavailable RSV F protein inhibitor and highlights the in vitro and in vivo anti-RSV activities and pharmacokinetics in animal species. RO-0529 demonstrates single-digit nM EC50 potency against laboratory strains, as well as clinical isolates of RSV in cellular assays, and more than one log viral load reduction in BALB/c mouse model of RSV viral infection. RO-0529 was proven to be a specific RSV F protein inhibitor by identification of drug resistant mutations of D486N, D489V, and D489Y in RSV F protein and the inhibition of RSV F protein-induced cell-cell fusion in cellular assays.
Assuntos
Antivirais/uso terapêutico , Benzazepinas/uso terapêutico , Quinazolinas/uso terapêutico , Infecções por Vírus Respiratório Sincicial/tratamento farmacológico , Vírus Sincicial Respiratório Humano/efeitos dos fármacos , Tiazepinas/uso terapêutico , Proteínas Virais de Fusão/antagonistas & inibidores , Administração Oral , Animais , Antivirais/administração & dosagem , Antivirais/síntese química , Antivirais/farmacocinética , Benzazepinas/administração & dosagem , Benzazepinas/síntese química , Benzazepinas/farmacocinética , Cães , Descoberta de Drogas , Feminino , Haplorrinos , Masculino , Camundongos Endogâmicos BALB C , Simulação de Acoplamento Molecular , Estrutura Molecular , Quinazolinas/administração & dosagem , Quinazolinas/líquido cefalorraquidiano , Quinazolinas/síntese química , Quinazolinas/farmacocinética , Ratos Wistar , Vírus Sincicial Respiratório Humano/química , Relação Estrutura-Atividade , Sulfonas , Tiazepinas/administração & dosagem , Tiazepinas/líquido cefalorraquidiano , Tiazepinas/farmacocinética , Proteínas Virais de Fusão/químicaRESUMO
VRX0466617 is a novel selective small-molecule inhibitor for Chk2 discovered through a protein kinase screening program. In this study, we provide a detailed biochemical and cellular characterization of VRX0466617. We show that VRX0466617 blocks the enzymatic activity of recombinant Chk2, as well as the ionizing radiation (IR)-induced activation of Chk2 from cells pretreated with the compound, at doses between 5 and 10 micromol/L. These doses of VRX0466617 inhibited, to some extent, the phosphorylation of Chk2 Ser(19) and Ser(33-35), but not of Chk2 Thr(68), which is phosphorylated by the upstream ataxia-telangiectasia mutated (ATM) kinase. Interestingly, VRX0466617 induced the phosphorylation of Chk2 Thr(68) even in the absence of DNA damage, arising from the block of its enzymatic activity. VRX0466617 prevented the IR-induced Chk2-dependent degradation of Hdmx, concordant with the in vivo inhibition of Chk2. Analysis of ATM/ATM and Rad3-related substrates Smc1, p53, and Chk1 excluded a cross-inhibition of these kinases. VRX0466617 did not modify the cell cycle phase distribution, although it caused an increase in multinucleated cells. Whereas VRX0466617 attenuated IR-induced apoptosis, in short-term assays it did not affect the cytotoxicity by the anticancer drugs doxorubicin, Taxol, and cisplatin. These results underscore the specificity of VRX0466617 for Chk2, both in vitro and in vivo, and support the use of this compound as a biological probe to study the Chk2-dependent pathways.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Benzimidazóis/farmacologia , Proliferação de Células/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Tiazóis/farmacologia , Animais , Apoptose/efeitos da radiação , Proteínas Mutadas de Ataxia Telangiectasia , Benzimidazóis/química , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/efeitos da radiação , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Núcleo Celular/efeitos da radiação , Proliferação de Células/efeitos da radiação , Células Cultivadas/efeitos dos fármacos , Células Cultivadas/efeitos da radiação , Quinase 1 do Ponto de Checagem , Quinase do Ponto de Checagem 2 , Cisplatino/farmacologia , Dano ao DNA/efeitos dos fármacos , Dano ao DNA/efeitos da radiação , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Doxorrubicina/farmacologia , Humanos , Imunoprecipitação , Camundongos , Estrutura Molecular , Paclitaxel/farmacologia , Fosforilação/efeitos dos fármacos , Fosforilação/efeitos da radiação , Proteínas Quinases/química , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Radiação Ionizante , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Serina/química , Tiazóis/química , Treonina/química , Proteínas Supressoras de Tumor/antagonistas & inibidores , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismoRESUMO
A novel benzoazepinequnoline (BAQ) series was discovered as RSV fusion inhibitors. BAQ series originated from compound 2, a hit from similarity-based virtual screening. In SAR exploration, benzoazepine allowed modifications in the head moiety. Benzylic sulfonyl on benzoazepine and 6-Me on quinoline were crucial for good anti-RSV activity. Although the basic amine in the head portion was crucial for anti-RSV activity, the attenuated basicity was required to reduce Vss. Introducing oxetane to the head portion led to discovery of compound 1, which demonstrated single-digit nM anti-RSV activity against different RSV strains, reasonable oral exposure in plasma, and 78-fold higher exposure in lung. Compound 1 also displayed 1 log viral reduction in a female BALB/c mice RSV model by b.i.d. oral dosing at 12.5 mg/kg. A single resistant mutant at L138F in fusion protein proved compound 1 to be a RSV fusion inhibitor.
Assuntos
Antivirais/química , Antivirais/farmacologia , Quinolinas/química , Quinolinas/farmacologia , Vírus Sincicial Respiratório Humano/efeitos dos fármacos , Administração Oral , Animais , Antivirais/administração & dosagem , Antivirais/farmacocinética , Disponibilidade Biológica , Feminino , Células Hep G2 , Humanos , Camundongos , Quinolinas/administração & dosagem , Quinolinas/farmacocinética , Relação Estrutura-AtividadeRESUMO
Described herein are the discovery and structure-activity relationship (SAR) studies of the third-generation 4-H heteroaryldihydropyrimidines (4-H HAPs) featuring the introduction of a C6 carboxyl group as novel HBV capsid inhibitors. This new series of 4-H HAPs showed improved anti-HBV activity and better drug-like properties compared to the first- and second-generation 4-H HAPs. X-ray crystallographic study of analogue 12 (HAP_R01) with Cp149 Y132A mutant hexamer clearly elucidated the role of C6 carboxyl group played for the increased binding affinity, which formed strong hydrogen bonding interactions with capsid protein and coordinated waters. The representative analogue 10 (HAP_R10) was extensively characterized in vitro (ADMET) and in vivo (mouse PK and PD) and subsequently selected for further development as oral anti-HBV infection agent.
Assuntos
Capsídeo/efeitos dos fármacos , Vírus da Hepatite B/efeitos dos fármacos , Pirimidinas/farmacologia , Animais , Cristalografia por Raios X , Descoberta de Drogas , Células Hep G2 , Humanos , Espectrometria de Massas , Camundongos , Espectroscopia de Prótons por Ressonância Magnética , Pirimidinas/química , Pirimidinas/farmacocinética , Relação Estrutura-AtividadeRESUMO
Previously we reported that viramidine is a prodrug of ribavirin and that adenosine deaminase catalyses viramidine deamination to ribavirin in vivo. This in vivo study explores this prodrug conversion in rats and inhibition by a potent adenosine deaminase inhibitor, 2'-deoxycoformycin. We found that conversion of viramidine to ribavirin was viramidine dose-dependent in rat plasma. A single intravenous dose of 0.25 mg/kg 2'-deoxycoformycin suppressed orally administered viramidine conversion to ribavirin in plasma by 50%. The inhibition was 2'-deoxycoformycin dose-dependent and a single dose of 2 mg/kg decreased the ribavirin/viramidine area under the concentration-time curve between 0 h and 6 h ratio by 2.5-fold. These findings provide strong evidence that adenosine deaminase plays a major role in converting viramidine to ribavirin in vivo.
Assuntos
Inibidores de Adenosina Desaminase , Adenosina Desaminase/metabolismo , Antivirais/farmacocinética , Pentostatina/farmacologia , Pró-Fármacos/farmacocinética , Ribavirina/análogos & derivados , Ribavirina/farmacocinética , Animais , Área Sob a Curva , Inibidores Enzimáticos/farmacologia , Cromatografia Gasosa-Espectrometria de Massas , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
A series of adenosine 5'-phosphonate analogues were designed to mimic naturally occurring adenosine monophosphate. These compounds (1-5) were synthesized and evaluated in a cellular hepatitis C virus (HCV) replication assay. To improve cellular permeability and enhance the anti-HCV activity of these phosphonates, a bis(S-acyl-2-thioethyl) prodrug for compound 5 was prepared, and its cellular activity was determined. To elucidate the mechanism of action of these novel adenosine phosphonates, their diphosphate derivatives (1a-5a) were synthesized. Further nucleotide incorporation assays by HCV NS5B RNA-dependent RNA polymerase revealed that 2a and 3a can serve as chain terminators, whereas compounds 1a, 4a, and 5a are competitive inhibitors with ATP. Additional steady-state kinetic analysis determined the incorporation efficiency of 2a and 3a as well as the inhibition constants for 1a, 4a, and 5a. The structure-activity relationships among these compounds were analyzed, and the implication for nucleoside phosphonate drug design was discussed.
Assuntos
Antivirais/síntese química , Desoxiadenosinas/síntese química , Hepacivirus/efeitos dos fármacos , Organofosfonatos/síntese química , Antivirais/química , Antivirais/farmacologia , Linhagem Celular , DNA Viral/antagonistas & inibidores , DNA Viral/biossíntese , Desoxiadenosinas/química , Desoxiadenosinas/farmacologia , Desenho de Fármacos , Humanos , Organofosfonatos/química , Organofosfonatos/farmacologia , Pró-Fármacos/síntese química , Pró-Fármacos/química , Pró-Fármacos/farmacologia , Relação Estrutura-Atividade , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas não Estruturais Virais/química , Replicação Viral/efeitos dos fármacosRESUMO
Lack of highly effective and safe therapeutics for hepatitis C virus (HCV) infection provides an opportunity as well as a challenge to discover novel and potent anti-HCV drugs. HCV NS5B RNA-dependent RNA polymerase (RdRp) is responsible for viral genome replication and thus constitutes a valid target for therapeutic intervention. To date, numerous HCV NS5B RdRp inhibitors have been discovered. This review focuses on the recent advances in discovery, mechanism of action studies and biological characterization of several distinct classes of potent inhibitors for NS5B RdRp. The clinical efficacy and developmental status of several promising compounds are also outlined.
Assuntos
Antivirais/farmacologia , Hepacivirus/efeitos dos fármacos , RNA Polimerase Dependente de RNA/antagonistas & inibidores , Proteínas não Estruturais Virais/antagonistas & inibidores , Replicação Viral/efeitos dos fármacos , Animais , Antivirais/uso terapêutico , Farmacorresistência Viral , Genoma Viral , Hepacivirus/fisiologia , Hepatite C/tratamento farmacológico , Humanos , Ligação Proteica , RNA Polimerase Dependente de RNA/metabolismo , Relação Estrutura-Atividade , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/metabolismoRESUMO
The global prevalence of persistent hepatitis C virus (HCV) infection and the lack of a highly effective and well-tolerated antiviral therapy have spurred intensive efforts to discover and develop novel anti-HCV therapy in the pharmaceutical industry. HCV NS5B RNA-dependent RNA polymerase (RdRp), the centerpiece for viral replication, constitutes a valid target for drug discovery. Compared to the host RNA and DNA polymerases, NS5B RdRp has distinct subcellular localization at the interface of the endoplasmic reticulum (ER) membrane and cytoplasm, a novel catalytic mechanism and many unique structural features, all of which make it an attractive target for developing effective anti-HCV therapeutics. High genetic variation among the major HCV genotypes commands that any efficacious NS5B inhibitors have to be broadly active against NS5Bs from various genotypes. Rapid viral replication and its inherent genetic diversity will certainly culminate drug resistance to any NS5B inhibitors. Therefore, iterative drug design and combination therapies of drugs that intervene with different steps in the HCV replicative cycle are needed to combat the viral infection. Many classes of nucleoside and non-nucleoside inhibitors of NS5B RdRp have been identified and appeared in literatures and patent applications. These progresses hold a considerable promise to the development of novel, specific and highly effective therapeutics to achieve sustained response and ultimately the eradication of HCV infection.
Assuntos
Antivirais/farmacologia , RNA Polimerase Dependente de RNA/antagonistas & inibidores , Proteínas não Estruturais Virais/antagonistas & inibidores , Sequência de Aminoácidos , Catálise , Desenho de Fármacos , Genótipo , Hepatite C/tratamento farmacológico , Hepatite C/enzimologia , Hepatite C/genética , Humanos , Dados de Sequência Molecular , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genéticaRESUMO
Nucleoside chain terminators represent one of the most promising classes of antiviral drug for DNA viruses and retroviral infection; however, they have not been fully explored against RNA viral polymerases. In this report, we investigate the notion of employing canonical 3'-deoxyribonucleoside triphosphates (3'-dNTPs) as a chain terminator for hepatitis C virus (HCV) NS5B RNA-dependent RNA polymerase (RdRp). Using a HCV RNA transcript-dependent RNA elongating assay, we found that they inhibit NS5B RdRp with K(i) ranged from 0.7 to 23 microM. Additional structure-activity relationship studies showed that removal of 2'-hydroxyl group, elimination of ribose's 2',3'-carbon-carbon bond, or addition of 5-methyl group to a pyrimidine base is detrimental to 3'-dNTP's potency. Direct evidence was obtained that all four canonical 3'-dNTP are incorporated into elongating RNA chains and the incorporation terminates NS5B RdRp-catalyzed RNA synthesis. The K(i) values for each of 3'-dNTPs were determined in the single nucleotide incorporation experiments. The nucleoside form of 3'-dNTPs was further evaluated in a cell culture-based HCV subgenomic replicon assay. The discrepancy between the potent in vitro activity and the weak cellular activity of these chain terminators was discussed in the context of nucleoside metabolism. This proof of concept study demonstrates that canonical 3'-dNTPs can function as an effective chain terminator for HCV NS5B RdRp with cytidine as the preferred nucleoside scaffold. Our results further sheds light on the potential hurdles that need to be overcome for successful development of active nucleoside chain terminators in vivo for a viral RNA polymerase, especially the HCV NS5B RdRp.
Assuntos
Desoxirribonucleotídeos/farmacologia , Hepacivirus/fisiologia , RNA Viral/biossíntese , RNA Polimerase Dependente de RNA/metabolismo , Proteínas não Estruturais Virais/metabolismo , Citidina/metabolismo , Desoxirribonucleotídeos/química , Desoxirribonucleotídeos/metabolismo , Hepacivirus/genética , Hepacivirus/metabolismo , Humanos , Relação Estrutura-Atividade , Moldes Genéticos , Células Tumorais Cultivadas , Replicação Viral/efeitos dos fármacosRESUMO
Diabetic retinopathy (DR) is of great interest to the drug discovery community owing to the rising worldwide prevalence of diabetes and its associated complications. The complex molecular mechanism associated with DR development and the poor translatability of available animal models to late-stage DR are considered to be major hurdles for drug discovery. Here we will provide an overview of the mechanistic rationale as well as clinical efficacy of drug candidates, and highlight emerging and potential targets for therapeutic intervention at different stages of DR.
Assuntos
Retinopatia Diabética/tratamento farmacológico , Desenho de Fármacos , Terapia de Alvo Molecular , Animais , Retinopatia Diabética/epidemiologia , Retinopatia Diabética/fisiopatologia , Modelos Animais de Doenças , Descoberta de Drogas/métodos , Humanos , PrevalênciaRESUMO
Nasopharyngeal carcinoma is a cancer with its highest prevalence among the southern Chinese and is rare elsewhere in the world. The main treatment modalities include chemotherapy and radiotherapy. However, tumor chemoresistance often limits the efficacy of nasopharyngeal carcinoma treatment and reduces survival rates. Thus, identifying new selective chemotherapeutic drugs for nasopharyngeal carcinoma treatment is needed. In this current study, the antitumor efficacy of a polo-like kinase inhibitor, Ro5203280, was investigated. Ro5203280 induces tumor suppression both in vitro and in vivo. An inhibitory effect was observed with the highly proliferating cancer cell lines tested, but not with the nontumorigenic cell line. Real-time cell proliferation and fluorescence-activated cell sorting (FACS) analysis, together with immunohistochemical (IHC), immunofluorescence, and Annexin V staining assays, were used to evaluate the impact of drug treatment on cell cycle and apoptosis. Ro5203280 induces G2-M cell-cycle arrest and apoptosis. Western blotting shows it inhibits PLK1 phosphorylation and downregulates the downstream signaling molecule, Cdc25c, and upregulates two important mitosis regulators, Wee1 and Securin, as well as the DNA damage-related factor Chk2 in vitro and in vivo. In vivo tumorigenicity assays with Ro5203280 intravenous injection showed its potent ability to inhibit tumor growth in mice, with no observable signs of toxicity. These findings suggest the potential usefulness of Ro5203280 as a chemotherapeutic targeting drug for nasopharyngeal carcinoma treatment.
Assuntos
Antineoplásicos/farmacologia , Benzamidas/farmacologia , Proteínas de Ciclo Celular/antagonistas & inibidores , Compostos Heterocíclicos com 2 Anéis/farmacologia , Neoplasias Nasofaríngeas/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Benzamidas/administração & dosagem , Benzamidas/química , Carcinoma , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Compostos Heterocíclicos com 2 Anéis/administração & dosagem , Compostos Heterocíclicos com 2 Anéis/química , Humanos , Camundongos , Carcinoma Nasofaríngeo , Neoplasias Nasofaríngeas/tratamento farmacológico , Neoplasias Nasofaríngeas/patologia , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Fuso Acromático/efeitos dos fármacos , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto , Quinase 1 Polo-LikeRESUMO
A series of isothiazole carboxamidine compounds were synthesized and discovered as novel and selective inhibitors for Chk2. They are not active against the related Chk1 kinase. The structure-activity relationship studies were performed on the scaffold, and enzymatic kinetic analysis showed they are simple ATP competitive inhibitors with K(i) values as low as 11 nM for Chk2. Computer modeling studies were employed to comprehend the mechanism of action and SAR of these compounds.
Assuntos
Amidinas/química , Inibidores de Proteínas Quinases/química , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Tiazóis/química , Trifosfato de Adenosina/química , Amidinas/síntese química , Ligação Competitiva , Quinase do Ponto de Checagem 2 , Humanos , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/química , Relação Estrutura-Atividade , Tiazóis/síntese química , Tiazóis/farmacologiaRESUMO
Many nucleoside analog drugs, such as ribavirin and viramidine, are activated or metabolized in vivo through 5'-phosphorylation. In this report, we determined the steady-state kinetic parameters for 5'-monophosphorylation of ribavirin and viramidine by adenosine kinase. The apparent Km for ribavirin is 540 microM, and k(cat) is 1.8 min-1. Its catalytic efficiency of 3.3 x 10(-3) min-1 . microM-1 is 1,200-fold lower than that of adenosine. In contrast to the common belief that ribavirin is exclusively phosphorylated by adenosine kinase, cytosolic 5'-nucleotidase II was found to catalyze ribavirin phosphorylation in vitro. The reaction is optimally stimulated by the physiological concentration of ATP or 2,3-bisphosphoglycerate. In phosphate-buffered saline plus ATP and 2,3-bisphosphoglycerate, the apparent Km for ribavirin is 88 microM, and k(cat) is 4.0 min-1. These findings suggest that cytosolic 5'-nucleotidase II may be involved in ribavirin phosphorylation in vivo. Like ribavirin, viramidine was found to be phosphorylated by either adenosine kinase or cytosolic 5'-nucleotidase II, albeit with a much lower activity. The catalytic efficiency for viramidine phosphorylation is 10- to 330-fold lower than that of ribavirin, suggesting that other nucleoside kinase(s) may be involved in viramidine phosphorylation in vivo. Both ribavirin and viramidine are not phosphorylated by deoxycytidine kinase and uridine-cytidine kinase. The coincidence of presence of high concentrated 2,3-bisphosphoglycerate in erythrocytes suggests that cytosolic 5'-nucleotidase II could play an important role in phosphorylating ribavirin and contribute to anabolism of ribavirin triphosphate in erythrocytes. Elucidation of ribavirin and viramidine phosphorylation mechanism should shed light on their in vivo metabolism, especially the ribavirin-induced hemolytic anemia in erythrocytes.
Assuntos
5'-Nucleotidase/metabolismo , Adenosina Quinase/metabolismo , Ribavirina/análogos & derivados , Ribavirina/metabolismo , Animais , Antivirais/metabolismo , Cricetinae , Citosol/enzimologia , Eritrócitos/metabolismo , Humanos , Cinética , Fosforilação , Ribavirina/químicaRESUMO
An investigational nucleoside analogue drug, viramidine, has recently emerged as a potentially safer alternative to ribavirin for the treatment of hepatitis C viral infection. We have reported that viramidine mainly functions as a prodrug of ribavirin that is enriched in the liver. This in vitro study further explores viramidine's activity against nucleoside phosphorylase, a host enzyme that is responsible for phosphorolysis of ribavirin in vivo. Our experiments show that viramidine inhibits ribavirin phosphorolysis with a K(i) of 2.5 microM. This result suggests that viramidine may act through a dual-action mechanism by serving as a prodrug of ribavirin and concomitantly as an inhibitor for nucleoside phosphorylase catabolism of ribavirin.
Assuntos
Antivirais/antagonistas & inibidores , Antivirais/farmacologia , Pró-Fármacos/farmacologia , Ribavirina/análogos & derivados , Ribavirina/farmacologia , Antivirais/metabolismo , Cromatografia em Camada Fina , Humanos , Técnicas In Vitro , Cinética , Fosforilases/sangue , Fosforilases/metabolismo , Ribavirina/metabolismoRESUMO
Inosine monophosphate dehydrogenase (IMPDH) catalyzes the rate-limiting step in GMP biosynthesis. The resulting intracellular pool of guanine nucleotides is of great importance to all cells for use in DNA and RNA synthesis, metabolism, and signal transduction. The enzyme binds IMP and the cofactor NAD(+) in random order, IMP is converted to XMP, NAD(+) is reduced to NADH, and finally, NADH and then XMP are released sequentially. XMP is subsequently converted into GMP by GMP synthetase. Drugs that decrease GMP synthesis by inhibiting IMPDH have been shown to have antiproliferative as well as antiviral activity. Several drugs are in use that target the substrate- or cofactor-binding site; however, due to differences between the mammalian and microbial isoforms, most drugs are far less effective against the microbial form of the enzyme than the mammalian form. The high resolution crystal structures of the protozoan parasite Tritrichomonas foetus IMPDH complexed with the inhibitor ribavirin monophosphate as well as monophosphate together with a second inhibitor, mycophenolic acid, are presented here. These structures reveal an active site cation identified previously only in the Chinese hamster IMPDH structure with covalently bound IMP. This cation was not found previously in apo IMPDH, IMPDH in complex with XMP, or covalently bound inhibitor, indicating that the cation-binding site may be catalysis-dependent. A comparison of T. foetus IMPDH with the Chinese hamster and Streptococcus pyogenes structures reveals differences in the active site loop architecture, which contributes to differences in cation binding during the catalytic sequence and the kinetic rates between bacterial, protozoan, and mammalian enzymes. Exploitation of these differences may lead to novel inhibitors, which favor the microbial form of the enzyme.
Assuntos
IMP Desidrogenase/química , Nucleotídeos/química , Tritrichomonas foetus/enzimologia , Sequência de Aminoácidos , Animais , Sítios de Ligação , Catálise , IMP Desidrogenase/antagonistas & inibidores , Modelos Moleculares , Nucleotídeos/farmacologia , Conformação Proteica , Estrutura Secundária de Proteína , Ribavirina/química , Ribavirina/farmacologiaRESUMO
De novo RNA synthesis by hepatitis C virus (HCV) nonstructural protein 5B (NS5B) RNA-dependent RNA polymerase has been investigated using short RNA templates. Various templates including those derived from the HCV genome were evaluated by examining the early steps of de novo RNA synthesis. NS5B was shown to be able to produce an initiation dinucleotide product from templates as short as 4-mer and from the 3'-terminal sequences of both plus and minus strands of the HCV RNA genome. GMP, GDP, and guanosine were able to act as an initiating nucleotide in de novo RNA synthesis, indicating that the triphosphate moiety is not absolutely required by an initiating nucleotide. Significant amounts of the initiation product accumulated in de novo synthesis, and elongation from the dinucleotide was observed when large amounts of dinucleotide were available. This result suggests that NS5B, a template, and incoming nucleotides are able to form an initiation complex that aborts frequently by releasing the dinucleotide product before transition to an elongation complex. The transition is rate limiting. Furthermore, we discovered that the secondary structure of a template was not essential for de novo initiation and that 3'-terminal bases of a template conferred specificity in selection of an initiation site. Initiation can occur at the +1, +2, or +3 position numbered from the 3' end of a template depending on base composition. Pyrimidine bases at any of the three positions are able to serve as an initiation site, while purine bases at the +2 and +3 positions do not support initiation. This result implies that HCV possesses an intrinsic ability to ensure that de novo synthesis is initiated from the +1 position and to maintain the integrity of the 3' end of its genome. This assay system should be an important tool for investigating the detailed mechanism of de novo initiation by HCV NS5B as well as other viral RNA polymerases.
Assuntos
Regiões 3' não Traduzidas/metabolismo , Hepacivirus/enzimologia , Oligonucleotídeos/metabolismo , RNA Viral/biossíntese , RNA Polimerase Dependente de RNA/metabolismo , Proteínas não Estruturais Virais/metabolismo , Regiões 3' não Traduzidas/genética , Hepacivirus/genética , Magnésio/metabolismo , Oligonucleotídeos/genética , RNA Viral/genética , RNA Polimerase Dependente de RNA/genética , Moldes Genéticos , Proteínas não Estruturais Virais/genética , Virologia/métodosRESUMO
Ribavirin is an approved broad-spectrum antiviral drug. A liver-targeting prodrug of ribavirin, viramidine, is in clinical trial in an attempt to provide a better therapeutic index. The conversion of viramidine to ribavirin, and of ribavirin to an inactive metabolite through adenosine deaminase, is reported. Kinetic analysis indicates that adenosine deaminase is likely involved in activation of viramidine in vivo, and the process is highly pH sensitive. The differential activities of two consecutive deamination reactions are kinetically studied and interpreted based on adenosine deaminase structural information. A comprehensive understanding of the viramidine and ribavirin deamination mechanism should help in designing better nucleoside therapeutics in the future.