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1.
Antimicrob Agents Chemother ; 52(2): 675-83, 2008 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-18070954

RESUMO

A novel class of nonnucleoside hepatitis C virus (HCV) polymerase inhibitors characterized by a dihydropyrone core was identified by high-throughput screening. Crystallographic studies of these compounds in complex with the polymerase identified an allosteric binding site close to the junction of the thumb and finger domains, approximately 30 A away from the catalytic center. AG-021541, a representative compound from this series, displayed measurable in vitro antiviral activity against the HCV genotype 1b subgenomic replicon with a mean 50% effective concentration of 2.9 muM. To identify mutations conferring in vitro resistance to AG-021541, resistance selection was carried out using HCV replicon cells either by serial passages in increasing concentrations of AG-021541 or by direct colony formation at fixed concentrations of the compound. We identified several amino acid substitutions in the AG-021541-binding region of the polymerase, including M423(T/V/I), M426T, I482(S/T), and V494A, with M423T as the predominant change observed. These mutants conferred various levels of resistance to AG-021541 and structurally related compounds but remained sensitive to interferon and HCV polymerase inhibitors known to interact with the active site or other allosteric sites of the protein. In addition, dihydropyrone polymerase inhibitors retained activity against replicons that contain signature resistance changes to other polymerase inhibitors, including S282T, C316N, M414T, and P495(S/L), indicating their potential to be used in combination therapies with these polymerase inhibitors. AG-021541-resistant replicon cell lines provide a valuable tool for mechanism-of-action studies of dihydropyrone polymerase inhibitors. The clinical relevance of in vitro resistance to HCV polymerase inhibitors remains to be investigated.


Assuntos
Farmacorresistência Viral , Inibidores Enzimáticos/farmacologia , Hepacivirus/efeitos dos fármacos , Hepacivirus/enzimologia , Pironas/farmacologia , RNA Polimerase Dependente de RNA/antagonistas & inibidores , Sítio Alostérico , Sítios de Ligação , Linhagem Celular Tumoral , Farmacorresistência Viral/genética , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/toxicidade , Hepacivirus/genética , Hepacivirus/crescimento & desenvolvimento , Humanos , Modelos Moleculares , Mutação , Pironas/química , Pironas/metabolismo , Pironas/toxicidade , RNA Polimerase Dependente de RNA/química , RNA Polimerase Dependente de RNA/metabolismo , Replicon , Replicação Viral
2.
J Med Chem ; 50(17): 3969-72, 2007 Aug 23.
Artigo em Inglês | MEDLINE | ID: mdl-17658778

RESUMO

The discovery and optimization of a novel class of carbon-linked dihydropyrones as allosteric HCV NS5B polymerase inhibitors are presented. Replacement of the sulfur linker atom with carbon reduced compound acidity and greatly increased cell permeation. Further structure-activity relationship (SAR) studies led to the identification of compounds, exemplified by 23 and 24, with significantly improved antiviral activities in the cell-based replicon assay and favorable pharmacokinetic profiles.


Assuntos
Antivirais/síntese química , Hepacivirus/enzimologia , Pironas/síntese química , Proteínas não Estruturais Virais/antagonistas & inibidores , Administração Oral , Regulação Alostérica , Animais , Antivirais/química , Antivirais/farmacologia , Disponibilidade Biológica , Células CACO-2 , Linhagem Celular Tumoral , Meia-Vida , Humanos , Permeabilidade , Pironas/química , Pironas/farmacologia , Ratos , Estereoisomerismo , Relação Estrutura-Atividade , Proteínas não Estruturais Virais/genética
3.
Antimicrob Agents Chemother ; 51(1): 95-102, 2007 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-17060518

RESUMO

Hepatitis C virus (HCV) research and drug discovery have been facilitated by the introduction of cell lines with self-replicating subgenomic HCV replicons. Early attempts to carry out robust, high-throughput screens (HTS) using HCV replicons have met with limited success. Specifically, selectable replicons have required laborious reverse transcription-PCR quantitation, and reporter replicons have generated low signal-to-noise ratios. In this study, we constructed a dicistronic single reporter (DSR)-selectable HCV replicon that contained a humanized Renilla luciferase (hRLuc) gene separated from the selectable Neo(r) marker by a short peptide cleavage site. The mutations E1202G, T1280I, and S2197P were introduced to enhance replicative capability. A dicistronic dual-reporter HCV replicon cell line (DDR) was subsequently created by transfection of Huh-7 cells with the DSR replicon to monitor antiviral activity and by the introduction of the firefly luciferase (FLuc) reporter gene into the host cell genome to monitor cytotoxicity. The DDR cell line demonstrated low signal variation within the HTS format, with a calculated Z' value of 0.8. A pilot HTS consisting of 20 96-well plates with a single concentration (10 microM) of 1,760 different compounds was executed. Hits were defined as compounds that reduced hRLuc and FLuc signals > or =50 and < or =40%, respectively, relative to those in a compound-free control. Good reproducibility was demonstrated, with a calculated confirmation rate of >75%. The development of a robust, high-throughput HCV replicon assay where the effects of inhibitors can be monitored for antiviral activity and cytotoxicity should greatly facilitate HCV drug discovery.


Assuntos
Antivirais/farmacologia , Genes Reporter/genética , Hepacivirus/genética , Replicon/genética , Antivirais/química , Linhagem Celular , Diclororribofuranosilbenzimidazol/química , Diclororribofuranosilbenzimidazol/farmacologia , Avaliação Pré-Clínica de Medicamentos/métodos , Farmacorresistência Viral/genética , Hepacivirus/efeitos dos fármacos , Humanos , Luciferases/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas não Estruturais Virais/genética , Replicação Viral/efeitos dos fármacos , Replicação Viral/genética
4.
Bioorg Med Chem Lett ; 16(18): 4834-8, 2006 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-16824756

RESUMO

A novel class of non-nucleoside HCV NS5B polymerase inhibitors has been identified from screening. A co-crystal structure revealed an allosteric binding site in the protein that required a unique conformational change to accommodate inhibitor binding. Herein we report the structure-activity relationships (SARs) of this novel class of dihydropyrone-containing compounds that show potent inhibitory activities against the HCV RNA polymerase in biochemical assays.


Assuntos
RNA Polimerases Dirigidas por DNA/antagonistas & inibidores , Hepacivirus/efeitos dos fármacos , Hepacivirus/enzimologia , Hidrogênio/química , Pironas/química , Pironas/farmacologia , Cristalografia por Raios X , RNA Polimerases Dirigidas por DNA/química , RNA Polimerases Dirigidas por DNA/metabolismo , Concentração Inibidora 50 , Modelos Moleculares , Estrutura Molecular , Pironas/síntese química , RNA Polimerase Dependente de RNA/antagonistas & inibidores , RNA Polimerase Dependente de RNA/química , Relação Estrutura-Atividade , Proteínas não Estruturais Virais/química
5.
Antimicrob Agents Chemother ; 49(6): 2267-75, 2005 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-15917520

RESUMO

(E)-(S)-4-((S)-2-{3-[(5-methyl-isoxazole-3-carbonyl)-amino]-2-oxo-2H-pyridin-1-yl}-pent-4-ynoylamino)-5-((S)-2-oxo-pyrrolidin-3-yl)-pent-2-enoic acid ethyl ester (Compound 1) is a novel, irreversible inhibitor of human rhinovirus (HRV) 3C protease {inactivation rate constant (Kobs/[I]) of 223,000 M-1s-1}. In cell-based assays, Compound 1 was active against all HRV serotypes (35 of 35), HRV clinical isolates (5 of 5), and related picornaviruses (8 of 8) tested with mean 50% effective concentration (EC50) values of 50 nM (range, 14 to 122 nM), 77 nM (range, 72 to 89 nM), and 75 nM (range, 7 to 249 nM), respectively. Compound 1 inhibited HRV 3C-mediated polyprotein processing in infected cells in a concentration-dependent manner, providing direct confirmation that the cell-based antiviral activity is due to inhibition of 3C protease. In vitro and in vivo nonclinical safety studies showed Compound 1 to be without adverse effects at maximum achievable doses. Single oral doses of Compound 1 up to 2,000 mg in healthy volunteers were found to be safe and well tolerated in a phase I-ascending, single-dose study. Compound 1 estimated free observed maximum concentration in plasma (Cmax) for 500-, 1,000-, and 2,000-mg doses were higher than the protein binding-corrected EC50 required to inhibit 80% of the HRV serotypes tested. Treatment of HRV 52-infected cells with one to five 2-h pulses of 150 nM Compound 1 (corresponding to the Cmax at the 500-mg dose) was sufficient to effect a significant reduction in viral replication. These experiments highlight Compound 1 as a potent, orally bioavailable, irreversible inhibitor of HRV 3C protease and provide data that suggest that Cmax rather than the Cmin might be the key variable predicting clinical efficacy.


Assuntos
Antivirais , Inibidores de Cisteína Proteinase , Rhinovirus/efeitos dos fármacos , Proteínas Virais/antagonistas & inibidores , Proteases Virais 3C , Administração Oral , Adolescente , Adulto , Animais , Antivirais/administração & dosagem , Antivirais/química , Antivirais/farmacocinética , Antivirais/farmacologia , Disponibilidade Biológica , Cisteína Endopeptidases , Inibidores de Cisteína Proteinase/administração & dosagem , Inibidores de Cisteína Proteinase/química , Inibidores de Cisteína Proteinase/farmacocinética , Inibidores de Cisteína Proteinase/farmacologia , Cães , Células HeLa , Humanos , Masculino , Pessoa de Meia-Idade , Rhinovirus/classificação , Rhinovirus/enzimologia , Sorotipagem , Resultado do Tratamento
6.
Structure ; 12(8): 1533-44, 2004 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-15296746

RESUMO

Human rhinoviruses (HRV), the predominant members of the Picornaviridae family of positive-strand RNA viruses, are the major causative agents of the common cold. Given the lack of effective treatments for rhinoviral infections, virally encoded proteins have become attractive therapeutic targets. The HRV genome encodes an RNA-dependent RNA polymerase (RdRp) denoted 3Dpol, which is responsible for replicating the viral genome and for synthesizing a protein primer used in the replication. Here the crystal structures for three viral serotypes (1B, 14, and 16) of HRV 3Dpol have been determined. The three structures are very similar to one another, and to the closely related poliovirus (PV) 3Dpol enzyme. Because the reported PV crystal structure shows significant disorder, HRV 3Dpol provides the first complete view of a picornaviral RdRp. The folding topology of HRV 3Dpol also resembles that of RdRps from hepatitis C virus (HCV) and rabbit hemorrhagic disease virus (RHDV) despite very low sequence homology.


Assuntos
Modelos Moleculares , Dobramento de Proteína , RNA Polimerase Dependente de RNA/química , Rhinovirus/enzimologia , Sequência de Aminoácidos , Clonagem Molecular , Cristalografia por Raios X , Humanos , Dados de Sequência Molecular , Homologia de Sequência de Aminoácidos
7.
Antiviral Res ; 61(3): 153-9, 2004 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-15168795

RESUMO

Human rhinoviruses (HRV) are the main cause of the common cold. Viral replication utilizes the activity of the HRV3C protease (3CP) enzyme [Antimicrob. Agents Chemother. 43 (1999) 2444; Antimicrob. Agents Chemother. 44 (2000) 1236]. Therefore, 3CP is an attractive target for antiviral drug development, and a new class of orally bioavailable irreversible 3CP inhibitors has been designed [J. Med. Chem. 45 (2002) 1607]. We have used related inhibitors to develop a rapid test for rhinovirus. The optical immuno assay (OIA) thin film detection technology utilizes an optically coated silicon surface to convert specific molecular binding events into visual color changes by altering the reflective properties of light through molecular thin films. The purpose of this study was to develop a rapid assay for the determination of 3CP combining the Thermo Electron Bio Star OIA technology and the newly designed inhibitor compounds. The advantage of this assay was in its approach, in which therapeutic and diagnostic targets are the same thus allowing patients with detected rhinoviruses to receive optimal treatment. Three different biotinylated inhibitor compounds were synthesized. The length of the spacer between the inhibitor and biotin core was 5, 10, and 15 atoms. These compounds were incorporated into the OIA format for the HRV assay development. A rapid (20 min) OIA test was developed using a 15 atom spacer biotinylated inhibitor (4). Forty different HRV serotypes were studied and thirty three serotypes of these 40 were detected (80%).


Assuntos
Cisteína Endopeptidases/análise , Imunoensaio , Inibidores de Proteases , Rhinovirus/isolamento & purificação , Proteínas Virais/antagonistas & inibidores , Proteínas Virais/análise , Proteases Virais 3C , Anticorpos Antivirais , Proteínas de Bactérias/metabolismo , Biotinilação , Resfriado Comum/diagnóstico , Resfriado Comum/virologia , Cisteína Endopeptidases/imunologia , Células HeLa , Peroxidase do Rábano Silvestre/metabolismo , Humanos , Estrutura Molecular , Proteínas Virais/imunologia
8.
J Med Chem ; 46(21): 4572-85, 2003 Oct 09.
Artigo em Inglês | MEDLINE | ID: mdl-14521419

RESUMO

The optimization of the pharmacokinetic performance of various 2-pyridone-containing human rhinovirus (HRV) 3C protease (3CP) inhibitors following oral administration to either beagle dogs or CM-monkeys is described. The molecules described in this work are composed of a 2-pyridone-containing peptidomimetic binding determinant and an alpha,beta-unsaturated ester Michael acceptor moiety which forms an irreversible covalent adduct with the active site cysteine residue of the 3C enzyme. Modification of the ester contained within these compounds is detailed along with alteration of the P(2) substituent present in the peptidomimetic portion of the inhibitors. The pharmacokinetics of several inhibitors in both dogs and monkeys are described (7 h plasma concentrations after oral administration) along with their human plasma stabilities, stabilities in incubations with human, dog, and monkey microsomes and hepatocytes, Caco-2 permeabilities, and aqueous solubilities. Compounds containing an alpha,beta-unsaturated ethyl ester fragment and either an ethyl or propargyl P(2) moiety displayed the most promising combination of 3C enzyme inhibition (k(obs)/[I] 170 000-223 000 M(-1) s(-1)), antiviral activity (EC(50) = 0.047-0.058 microM, mean vs seven HRV serotypes), and pharmacokinetics following oral administration (7 h dog plasma levels = 0.248-0.682 microM; 7 h CM-monkey plasma levels = 0.057-0.896 microM).


Assuntos
Antivirais/síntese química , Antivirais/farmacologia , Cisteína Endopeptidases/metabolismo , Inibidores de Proteases/síntese química , Inibidores de Proteases/farmacologia , Piridonas/síntese química , Piridonas/farmacologia , Rhinovirus/enzimologia , Proteínas Virais/metabolismo , Proteases Virais 3C , Animais , Antivirais/farmacocinética , Disponibilidade Biológica , Proteínas Sanguíneas/metabolismo , Células CACO-2 , Cães , Desenho de Fármacos , Meia-Vida , Hepatócitos/metabolismo , Humanos , Técnicas In Vitro , Indicadores e Reagentes , Macaca fascicularis , Espectroscopia de Ressonância Magnética , Masculino , Microssomos Hepáticos/metabolismo , Inibidores de Proteases/farmacocinética , Ligação Proteica , Rhinovirus/efeitos dos fármacos , Solubilidade , Relação Estrutura-Atividade
9.
J Virol ; 77(13): 7575-81, 2003 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-12805457

RESUMO

The virus-encoded nonstructural protein 5B (NS5B) of hepatitis C virus (HCV) is an RNA-dependent RNA polymerase and is absolutely required for replication of the virus. NS5B exhibits significant differences from cellular polymerases and therefore has become an attractive target for anti-HCV therapy. Using a high-throughput screen, we discovered a novel NS5B inhibitor that binds to the enzyme noncompetitively with respect to nucleotide substrates. Here we report the crystal structure of NS5B complexed with this small molecule inhibitor. Unexpectedly, the inhibitor is bound within a narrow cleft on the protein's surface in the "thumb" domain, about 30 A from the enzyme's catalytic center. The interaction between this inhibitor and NS5B occurs without dramatic changes to the structure of the protein, and sequence analysis suggests that the binding site is conserved across known HCV genotypes. Possible mechanisms of inhibition include perturbation of protein dynamics, interference with RNA binding, and disruption of enzyme oligomerization.


Assuntos
Proteínas não Estruturais Virais/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Catálise , Cristalografia por Raios X , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas não Estruturais Virais/química
10.
J Med Chem ; 45(10): 2016-23, 2002 May 09.
Artigo em Inglês | MEDLINE | ID: mdl-11985469

RESUMO

Utilizing the tools of parallel synthesis and structure-based design, a new class of Michael acceptor-containing, irreversible inhibitors of human rhinovirus 3C protease (HRV 3CP) was discovered. These inhibitors are shown to inhibit HRV-14 3CP with rates of inactivation ranging from 886 to 31 400 M(-1) sec(-1). These inhibitors exhibit antiviral activity when tested against HRV-14 infected H1-HeLa cells, with EC(50) values ranging from 1.94 to 0.15 microM. No cytotoxicity was observed at the limits of the assay concentration. A crystal structure of one of the more potent inhibitors covalently bound to HRV-2 3CP is detailed. These compounds were also tested against HRV serotypes other than type 14 and were found to have highly variable activities.


Assuntos
Antivirais/síntese química , Inibidores Enzimáticos/síntese química , Rhinovirus/efeitos dos fármacos , Proteínas Virais/antagonistas & inibidores , Proteases Virais 3C , Antivirais/química , Antivirais/farmacologia , Técnicas de Química Combinatória , Cristalografia por Raios X , Cisteína Endopeptidases , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Células HeLa , Humanos , Ligação Proteica , Rhinovirus/química , Relação Estrutura-Atividade
11.
J Med Chem ; 45(8): 1607-23, 2002 Apr 11.
Artigo em Inglês | MEDLINE | ID: mdl-11931615

RESUMO

The structure-based design, chemical synthesis, and biological evaluation of various 2-pyridone-containing human rhinovirus (HRV) 3C protease (3CP) inhibitors are described. These compounds are comprised of a peptidomimetic binding determinant and a Michael acceptor moiety, which forms an irreversible covalent adduct with the active site cysteine residue of the 3C enzyme. The 2-pyridone-containing inhibitors typically display improved 3CP inhibition properties relative to related peptide-derived molecules along with more favorable antiviral properties. The cocrystal structure of one pyridone-derived 3CP inhibitor complexed with HRV-2 3CP is also described along with certain ab initio conformation analyses. Optimization of the 2-pyridone-containing compounds is shown to provide several highly active 3CP inhibitors (k(obs)/[I] > 500,00 M(-1) s(-1)) that function as potent antirhinoviral agents (EC(50) = <0.05 microM) against multiple virus serotypes in cell culture. One 2-pyridone-containing 3CP inhibitor is shown to be bioavailable in the dog after oral dosing (F = 48%).


Assuntos
Antivirais/síntese química , Peptídeos/química , Inibidores de Proteases/síntese química , Piridonas/síntese química , Rhinovirus/enzimologia , Proteínas Virais/antagonistas & inibidores , Proteases Virais 3C , Administração Oral , Animais , Antivirais/química , Antivirais/farmacologia , Disponibilidade Biológica , Cristalografia por Raios X , Cisteína Endopeptidases , Cães , Estabilidade de Medicamentos , Humanos , Técnicas In Vitro , Ligantes , Microssomos Hepáticos/metabolismo , Modelos Moleculares , Mimetismo Molecular , Inibidores de Proteases/química , Inibidores de Proteases/farmacologia , Ligação Proteica , Piridonas/química , Piridonas/farmacologia , Relação Estrutura-Atividade
12.
Bioorg Med Chem Lett ; 12(5): 733-8, 2002 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-11858991
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