RESUMO
Resistance to the 2'-F-2'-C-methylguanosine monophosphate nucleotide hepatitis C virus (HCV) inhibitors PSI-352938 and PSI-353661 was associated with a combination of amino acid changes (changes of S to G at position 15 [S15G], C223H, and V321I) within the genotype 2a nonstructural protein 5B (NS5B), an RNA-dependent RNA polymerase. To understand the role of these residues in viral replication, we examined the effects of single and multiple point mutations on replication fitness and inhibition by a series of nucleotide analog inhibitors. An acidic residue at position 15 reduced replicon fitness, consistent with its proximity to the RNA template. A change of the residue at position 223 to an acidic or large residue reduced replicon fitness, consistent with its proposed proximity to the incoming nucleoside triphosphate (NTP). A change of the residue at position 321 to a charged residue was not tolerated, consistent with its position within a hydrophobic cavity. This triple resistance mutation was specific to both genotype 2a virus and 2'-F-2'-C-methylguanosine inhibitors. A crystal structure of the NS5B S15G/C223H/V321I mutant of the JFH-1 isolate exhibited rearrangement to a conformation potentially consistent with short primer-template RNA binding, which could suggest a mechanism of resistance accomplished through a change in the NS5B conformation, which was better tolerated by genotype 2a virus than by viruses of other genotypes.
Assuntos
Farmacorresistência Viral/genética , Hepacivirus/genética , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/ultraestrutura , Replicação Viral/genética , Antivirais/farmacologia , Cristalografia por Raios X , Óxidos P-Cíclicos/farmacologia , Guanosina Monofosfato/análogos & derivados , Guanosina Monofosfato/farmacologia , Hepacivirus/efeitos dos fármacos , Hepacivirus/crescimento & desenvolvimento , Humanos , Nucleosídeos/farmacologia , Estrutura Terciária de Proteína , RNA Viral/genética , Proteínas de Ligação a RNA/genéticaRESUMO
The 3',5'-cyclic phosphate prodrug 9-[ß-d-2'-deoxy-2'-α-fluoro-2'-ß-C-methylribofuranosyl]-2-amino-6-ethoxypurine, PSI-352938 1, has demonstrated promising anti-HCV efficacy in vitro and in human clinical trials. A structure-activity relationship study of the nucleoside 3',5'-cyclic phosphate series of ß-d-2'-deoxy-2'-α-fluoro-2'-ß-C-methylribofuranosyl nucleoside prodrugs was undertaken and the anti-HCV activity and in vitro safety profile were assessed. Cycloalkyl 3',5'-cyclic phosphate prodrugs were shown to be significantly more potent as inhibitors of HCV replication than branched and straight chain alkyl 3',5'-cyclic phosphate prodrugs. No cytotoxicity and mitochondrial toxicity for prodrugs 12, 13 and 19 were observed at concentrations up to 100 µm in vitro. Cycloalkyl esters of 3',5'-cyclic phosphate nucleotide prodrugs demonstrated the ability to produce high levels of active triphosphate in clone-A cells and primary human hepatocytes. Compounds 12, 13 and 19 also demonstrated the ability to effectively deliver in vivo high levels of active nucleoside phosphates to rat liver.
Assuntos
Antivirais/farmacologia , Hepacivirus/efeitos dos fármacos , Nucleotídeos Cíclicos/farmacologia , Pró-Fármacos/farmacologia , Animais , Antivirais/síntese química , Antivirais/química , Relação Dose-Resposta a Droga , Estabilidade de Medicamentos , Humanos , Fígado/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Modelos Moleculares , Estrutura Molecular , Nucleotídeos Cíclicos/síntese química , Nucleotídeos Cíclicos/química , Pró-Fármacos/síntese química , Pró-Fármacos/química , Ratos , Relação Estrutura-Atividade , Replicação Viral/efeitos dos fármacosRESUMO
The replication of the hepatitis C viral (HCV) genome is accomplished by the NS5B RNA-dependent RNA polymerase (RdRp), for which mechanistic understanding and structure-guided drug design efforts have been hampered by its propensity to crystallize in a closed, polymerization-incompetent state. The removal of an autoinhibitory ß-hairpin loop from genotype 2a HCV NS5B increases de novo RNA synthesis by >100-fold, promotes RNA binding, and facilitated the determination of the first crystallographic structures of HCV polymerase in complex with RNA primer-template pairs. These crystal structures demonstrate the structural realignment required for primer-template recognition and elongation, provide new insights into HCV RNA synthesis at the molecular level, and may prove useful in the structure-based design of novel antiviral compounds. Additionally, our approach for obtaining the RNA primer-template-bound structure of HCV polymerase may be generally applicable to solving RNA-bound complexes for other viral RdRps that contain similar regulatory ß-hairpin loops, including bovine viral diarrhea virus, dengue virus, and West Nile virus.
Assuntos
Hepacivirus/enzimologia , Hepacivirus/genética , RNA/genética , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/metabolismo , Linhagem Celular , Cristalização , Replicação do DNA , Hepacivirus/química , Hepatite C/virologia , Humanos , Modelos Moleculares , Estrutura Secundária de Proteína , Moldes Genéticos , Proteínas não Estruturais Virais/genéticaRESUMO
PSI-352938 is a novel cyclic phosphate prodrug of ß-D-2'-deoxy-2'-α-fluoro-2'-ß-C-methylguanosine-5'-monophosphate with potent anti-HCV activity. In order to inhibit the NS5B RNA-dependent RNA polymerase, PSI-352938 must be metabolized to the active triphosphate form, PSI-352666. During in vitro incubations with PSI-352938, significantly larger amounts of PSI-352666 were formed in primary hepatocytes than in clone A hepatitis C virus (HCV) replicon cells. Metabolism and biochemical assays were performed to define the molecular mechanism of PSI-352938 activation. The first step, removal of the isopropyl group on the 3',5'-cyclic phosphate moiety, was found to be cytochrome P450 (CYP) 3A4 dependent, with other CYP isoforms unable to catalyze the reaction. The second step, opening of the cyclic phosphate ring, was catalyzed by phosphodiesterases (PDEs) 2A1, 5A, 9A, and 11A4, all known to be expressed in the liver. The role of these enzymes in the activation of PSI-352938 was confirmed in primary human hepatocytes, where prodrug activation was reduced by inhibitors of CYP3A4 and PDEs. The third step, removal of the O(6)-ethyl group on the nucleobase, was shown to be catalyzed by adenosine deaminase-like protein 1. The resulting monophosphate was consecutively phosphorylated to the diphosphate and to the triphosphate PSI-352666 by guanylate kinase 1 and nucleoside diphosphate kinase, respectively. In addition, formation of nucleoside metabolites was observed in primary hepatocytes, and ecto-5'-nucleotidase was able to dephosphorylate the monophosphate metabolites. Since CYP3A4 is highly expressed in the liver, the CYP3A4-dependent metabolism of PSI-352938 makes it an effective liver-targeted prodrug, in part accounting for the potent antiviral activity observed clinically.
Assuntos
Antivirais/metabolismo , Óxidos P-Cíclicos/metabolismo , Hepacivirus/efeitos dos fármacos , Nucleosídeos/metabolismo , Células Cultivadas , Citocromo P-450 CYP3A/metabolismo , Guanilato Quinases/metabolismo , Hepatócitos/metabolismo , Humanos , Núcleosídeo-Difosfato Quinase/metabolismo , Diester Fosfórico Hidrolases/metabolismoRESUMO
The HCV non-structural protein NS5A has been established as a viable target for the development of direct acting antiviral therapy. From computational modeling studies strong intra-molecular hydrogen bonds were found to be a common structural moiety within known NS5A inhibitors that have low pico-molar replicon potency. Efforts to reproduce these γ-turn-like substructures provided a novel NS5A inhibitor based on a fluoro-olefin isostere. This fluoro-olefin containing inhibitor exhibited picomolar activity (EC(50)=79 pM) against HCV genotype 1b replicon without measurable cytotoxicity. This level of activity is comparable to the natural peptide-based inhibitors currently under clinic evaluation, and demonstrates that a peptidomimetic approach can serve as a useful strategy to produce potent and structurally unique inhibitors of HCV NS5A.
Assuntos
Alcenos/química , Flúor/química , Hepacivirus/efeitos dos fármacos , Peptidomiméticos/química , Peptidomiméticos/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Alcenos/farmacologia , Flúor/farmacologia , Humanos , Ligação de Hidrogênio , Modelos MolecularesRESUMO
PSI-7977, a prodrug of 2'-F-2'-C-methyluridine monophosphate, is the purified diastereoisomer of PSI-7851 and is currently being investigated in phase 3 clinical trials for the treatment of hepatitis C. In this study, we profiled the activity of PSI-7977 and its ability to select for resistance using a number of different replicon cells. Results showed that PSI-7977 was active against genotype (GT) 1a, 1b, and 2a (strain JFH-1) replicons and chimeric replicons containing GT 2a (strain J6), 2b, and 3a NS5B polymerase. Cross-resistance studies using GT 1b replicons confirmed that the S282T change conferred resistance to PSI-7977. Subsequently, we evaluated the ability of PSI-7977 to select for resistance using GT 1a, 1b, and 2a (JFH-1) replicon cells. S282T was the common mutation selected among all three genotypes, but while it conferred resistance to PSI-7977 in GT 1a and 1b, JFH-1 GT 2a S282T showed only a very modest shift in 50% effective concentration (EC(50)) for PSI-7977. Sequence analysis of the JFH-1 NS5B region indicated that additional amino acid changes were selected both prior to and after the emergence of S282T. These include T179A, M289L, I293L, M434T, and H479P. Residues 179, 289, and 293 are located within the finger and palm domains, while 434 and 479 are located on the surface of the thumb domain. Data from the JFH-1 replicon variants showed that amino acid changes within the finger and palm domains together with S282T were required to confer resistance to PSI-7977, while the mutations on the thumb domain serve to enhance the replication capacity of the S282T replicons.
Assuntos
Antivirais/farmacologia , Hepacivirus/efeitos dos fármacos , Hepacivirus/genética , Uridina Monofosfato/análogos & derivados , Linhagem Celular , Genótipo , Humanos , Replicon/efeitos dos fármacos , Replicon/genética , Sofosbuvir , Uridina Monofosfato/farmacologia , Replicação Viral/efeitos dos fármacosRESUMO
BACKGROUND: Nucleoside reverse transcriptase inhibitors (NRTIs) are an effective class of agents that has played a vital role in the treatment of HIV infections. (-)-ß-D-(2R,4R)-dioxolane-thymine (DOT) is a thymidine analogue that is active against wild-type and NRTI-resistant HIV-1 mutants. It has been shown that the anti-HIV activity of DOT is limited due to poor monophosphorylation. METHODS: To further enhance the anti-HIV activity of DOT, an extensive structure-activity relationship analysis of phosphoramidate prodrugs of DOT monophosphate was undertaken. These prodrugs were evaluated for anti-HIV activity using Hela CD4 ß-gal reporter cells (P4-CCR5 luc cells). RESULTS: Among the synthesized prodrugs, the 4-bromophenyl benzyloxy l-alanyl phosphate derivative of DOT was the most potent, with a 50% effective concentration of 0.089 µM corresponding to a 75-fold increase in activity relative to the parent nucleoside DOT with no increased cytotoxicity. The metabolic stability of a selected number of potent DOT phosphoramidates was also evaluated in simulated gastric fluid, simulated intestinal fluid, human plasma and liver S9 fractions. CONCLUSIONS: A series of new phosphoramidate prodrugs of DOT were prepared and evaluated as inhibitors of HIV replication in vitro. Metabolic stability studies indicated that these DOT phosphoramidate derivatives have the potential to show acceptable stability in the gastrointestinal tract, but they metabolize rapidly in the liver.
Assuntos
Amidas/farmacologia , Fármacos Anti-HIV/farmacologia , Dioxolanos/farmacologia , Ácidos Fosfóricos/farmacologia , Pró-Fármacos/farmacologia , Timina/análogos & derivados , Cromatografia Líquida de Alta Pressão , Dioxolanos/química , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Espectrofotometria Ultravioleta , Timina/química , Timina/farmacologiaRESUMO
PSI-352938, a cyclic phosphate nucleotide, and PSI-353661, a phosphoramidate nucleotide, are prodrugs of ß-D-2'-deoxy-2'-α-fluoro-2'-ß-C-methylguanosine-5'-monophosphate. Both compounds are metabolized to the same active 5'-triphosphate, PSI-352666, which serves as an alternative substrate inhibitor of the NS5B RNA-dependent RNA polymerase during HCV replication. PSI-352938 and PSI-353661 retained full activity against replicons containing the S282T substitution, which confers resistance to certain 2'-substituted nucleoside/nucleotide analogs. PSI-352666 was also similarly active against both wild-type and S282T NS5B polymerases. In order to identify mutations that confer resistance to these compounds, in vitro selection studies were performed using HCV replicon cells. While no resistant genotype 1a or 1b replicons could be selected, cells containing genotype 2a JFH-1 replicons cultured in the presence of PSI-352938 or PSI-353661 developed resistance to both compounds. Sequencing of the NS5B region identified a number of amino acid changes, including S15G, R222Q, C223Y/H, L320I, and V321I. Phenotypic evaluation of these mutations indicated that single amino acid changes were not sufficient to significantly reduce the activity of PSI-352938 and PSI-353661. Instead, a combination of three amino acid changes, S15G/C223H/V321I, was required to confer a high level of resistance. No cross-resistance exists between the 2'-F-2'-C-methylguanosine prodrugs and other classes of HCV inhibitors, including 2'-modified nucleoside/-tide analogs such as PSI-6130, PSI-7977, INX-08189, and IDX-184. Finally, we determined that in genotype 1b replicons, the C223Y/H mutation failed to support replication, and although the A15G/C223H/V321I triple mutation did confer resistance to PSI-352938 and PSI-353661, this mutant replicated at only about 10% efficiency compared to the wild type.
Assuntos
Óxidos P-Cíclicos/farmacologia , Farmacorresistência Viral , Guanosina Monofosfato/análogos & derivados , Hepacivirus/efeitos dos fármacos , Hepacivirus/genética , Mutação/genética , Nucleosídeos/farmacologia , RNA Viral/genética , Replicon/efeitos dos fármacos , Antivirais/farmacologia , Guanosina Monofosfato/farmacologia , Hepatite C/tratamento farmacológico , Hepatite C/genética , Hepatite C/virologia , Humanos , Fenótipo , Pró-Fármacos/farmacologia , Conformação Proteica , RNA Polimerase Dependente de RNA/antagonistas & inibidores , RNA Polimerase Dependente de RNA/metabolismo , Replicon/genética , Replicação Viral/efeitos dos fármacos , Replicação Viral/genéticaRESUMO
Phenylpropenamides have been reported to be a class of non-nucleoside inhibitors of the hepatitis B virus (HBV). This class of compounds was explored with the objective of developing potent anti-HBV agents, with a novel mechanism of action, that could be combined with nucleos(t)ide analogs currently used to treat HBV infection. To accomplish this objective a series of substituted arylpropenamide derivatives were prepared and the E and Z geometrical isomers were separated. The structural identity of each of the E and Z isomers was determined by single crystal X-ray crystallography. Contrary to previous reports, the activity of this class of molecules resides in the Z isomer. Further structure-activity relationship studies around the active Z isomer identified compounds that displayed potent antiviral activity against HBV with EC(90) value of approximately 0.5 µM in vitro. Attempts to develop ring constrained analogs did not lead to active HBV inhibitors.
Assuntos
Amidas/química , Antivirais/química , Vírus da Hepatite B/efeitos dos fármacos , Amidas/síntese química , Amidas/farmacologia , Antivirais/síntese química , Antivirais/farmacologia , Linhagem Celular , Cristalografia por Raios X , Humanos , Isomerismo , Conformação Molecular , Relação Estrutura-AtividadeRESUMO
PSI-353661, a phosphoramidate prodrug of 2'-deoxy-2'-fluoro-2'-C-methylguanosine-5'-monophosphate, is a highly active inhibitor of genotype 1a, 1b, and 2a HCV RNA replication in the replicon assay and of genotype 1a and 2a infectious virus replication. PSI-353661 is active against replicons harboring the NS5B S282T or S96T/N142T amino acid alterations that confer decreased susceptibility to nucleoside/tide analogs as well as mutations that confer resistance to non-nucleoside inhibitors of NS5B. Replicon clearance studies show that PSI-353661 was able to clear cells of HCV replicon RNA and prevent a rebound in replicon RNA. PSI-353661 showed no toxicity toward bone marrow stem cells or mitochondrial toxicity. The metabolism to the active 5'-triphosphate involves hydrolysis of the carboxyl ester by cathepsin A (Cat A) and carboxylesterase 1 (CES1) followed by a putative nucleophilic attack on the phosphorus by the carboxyl group resulting in the elimination of phenol and the alaninyl phosphate metabolite, PSI-353131. Histidine triad nucleotide-binding protein 1 (Hint 1) then removes the amino acid moiety, which is followed by hydrolysis of the methoxyl group at the O(6)-position of the guanine base by adenosine deaminase-like protein 1 (ADAL1) to give 2'-deoxy-2'-fluoro-2'-C-methylguanosine-5'-monophosphate. The monophosphate is phosphorylated to the diphosphate by guanylate kinase. Nucleoside diphosphate kinase is the primary enzyme involved in phosphorylation of the diphosphate to the active triphosphate, PSI-352666. PSI-352666 is equally active against wild-type NS5B and NS5B containing the S282T amino acid alteration.
Assuntos
Antivirais/farmacologia , Guanosina Monofosfato/análogos & derivados , Hepacivirus/efeitos dos fármacos , Pró-Fármacos/farmacologia , Replicação Viral/efeitos dos fármacos , Biotransformação , Catepsina A/metabolismo , Cromatografia Líquida de Alta Pressão , Clonagem Molecular , Avaliação Pré-Clínica de Medicamentos , Guanosina Monofosfato/antagonistas & inibidores , Guanosina Monofosfato/farmacologia , Guanilato Quinases/metabolismo , Células Hep G2 , Hepacivirus/genética , Hepacivirus/fisiologia , Hepatócitos/efeitos dos fármacos , Humanos , Ácido Láctico/metabolismo , Luciferases/metabolismo , Testes de Sensibilidade Microbiana , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Mutação , Proteínas do Tecido Nervoso/metabolismo , Fenol/metabolismo , Fosforilação , Pró-Fármacos/química , Replicon , Proteínas não Estruturais Virais/antagonistas & inibidoresRESUMO
PSI-352938 is a novel cyclic phosphate prodrug of ß-D-2'-deoxy-2'-α-fluoro-2'-ß-C-methylguanosine 5'-monophosphate that has potent activity against hepatitis C virus (HCV) in vitro. The studies described here characterize the in vitro anti-HCV activity of PSI-352938, alone and in combination with other inhibitors of HCV, and the cross-resistance profile of PSI-352938. The effective concentration required to achieve 50% inhibition for PSI-352938, determined using genotype 1a-, 1b-, and 2a-derived replicons stably expressed in the Lunet cell line, were 0.20, 0.13, and 0.14 µM, respectively. The active 5'-triphosphate metabolite, PSI-352666, inhibited recombinant NS5B polymerase from genotypes 1 to 4 with comparable 50% inhibitory concentrations. In contrast, PSI-352938 did not inhibit the replication of hepatitis B virus or human immunodeficiency virus in vitro. PSI-352666 did not significantly affect the activity of human DNA and RNA polymerases. PSI-352938 and its cyclic phosphate metabolites did not affect the cyclic GMP-mediated activation of protein kinase G. Clearance studies using replicon cells demonstrated that PSI-352938 cleared cells of HCV replicon RNA and prevented replicon rebound. An additive to synergistic effect was observed when PSI-352938 was combined with other classes of HCV inhibitors, including alpha interferon, ribavirin, NS3/4A inhibitors, an NS5A inhibitor, and nucleoside/nucleotide and nonnucleoside inhibitors. Cross-resistance studies showed that PSI-352938 remained fully active against replicons containing the S282T or the S96T/N142T amino acid alteration. Replicons that contain mutations conferring resistance to various classes of nonnucleoside inhibitors also remained sensitive to inhibition by PSI-352938. PSI-352938 is currently being evaluated in a phase I clinical study in genotype 1-infected individuals.
Assuntos
Antivirais/farmacologia , Óxidos P-Cíclicos/farmacologia , Desoxiguanosina/análogos & derivados , Hepacivirus/efeitos dos fármacos , Nucleosídeos/farmacologia , Pró-Fármacos/farmacologia , RNA Viral/biossíntese , Replicon/efeitos dos fármacos , Desoxiguanosina/farmacologia , Farmacorresistência Viral , HumanosRESUMO
Hepatitis C virus afflicts approximately 180 million people worldwide, and the development of direct acting antivirals may offer substantial benefit compared to the current standard of care. Accordingly, prodrugs of 2'-deoxy-2'-fluoro-2'-C-methylguanosine monophosphate analogues were prepared and evaluated for their anti-HCV efficacy and tolerability. These prodrugs demonstrated >1000 fold greater potency than the parent nucleoside in a cell-based replicon assay as a result of higher intracellular triphosphate levels. Further optimization led to the discovery of the clinical candidate PSI-353661, which has demonstrated strong in vitro inhibition against HCV without cytotoxicity and equipotent activity against both the wild type and the known S282T nucleoside/tide resistant replicon. PSI-353661 is currently in preclinical development for the treatment of HCV.
RESUMO
A series of novel 2'-deoxy-2'-α-fluoro-2'-ß-C-methyl 3',5'-cyclic phosphate nucleotide prodrug analogs were synthesized and evaluated for their in vitro anti-HCV activity and safety. These prodrugs demonstrated a 10-100-fold greater potency than the parent nucleoside in a cell-based replicon assay due to higher cellular triphosphate levels. Our structure-activity relationship (SAR) studies provided compounds that gave high levels of active triphosphate in rat liver when administered orally to rats. These studies ultimately led to the selection of the clinical development candidate 24a (PSI-352938).
Assuntos
Antivirais/química , Óxidos P-Cíclicos/química , Inibidores Enzimáticos/química , Hepacivirus/enzimologia , Nucleosídeos/química , Nucleotídeos Cíclicos/química , Pró-Fármacos/química , Proteínas não Estruturais Virais/antagonistas & inibidores , Administração Oral , Animais , Antivirais/farmacocinética , Antivirais/toxicidade , Linhagem Celular Tumoral , Cristalografia por Raios X , Óxidos P-Cíclicos/farmacocinética , Óxidos P-Cíclicos/toxicidade , Inibidores Enzimáticos/farmacocinética , Inibidores Enzimáticos/toxicidade , Humanos , Conformação Molecular , Nucleosídeos/farmacocinética , Nucleosídeos/toxicidade , Nucleotídeos Cíclicos/síntese química , Nucleotídeos Cíclicos/toxicidade , Pró-Fármacos/farmacocinética , Pró-Fármacos/farmacologia , Ratos , Relação Estrutura-Atividade , Proteínas não Estruturais Virais/metabolismoRESUMO
Hepatitis C virus (HCV) is a global health problem requiring novel approaches for effective treatment of this disease. The HCV NS5B polymerase has been demonstrated to be a viable target for the development of HCV therapies. ß-d-2'-Deoxy-2'-α-fluoro-2'-ß-C-methyl nucleosides are selective inhibitors of the HCV NS5B polymerase and have demonstrated potent activity in the clinic. Phosphoramidate prodrugs of the 5'-phosphate derivative of the ß-d-2'-deoxy-2'-α-fluoro-2'-ß-C-methyluridine nucleoside were prepared and showed significant potency in the HCV subgenomic replicon assay (<1 µM) and produced high levels of triphosphate 6 in primary hepatocytes and in the livers of rats, dogs, and monkeys when administered in vivo. The single diastereomer 51 of diastereomeric mixture 14 was crystallized, and an X-ray structure was determined establishing the phosphoramidate stereochemistry as Sp, thus correlating for the first time the stereochemistry of a phosphoramidate prodrug with biological activity. 51 (PSI-7977) was selected as a clinical development candidate.
Assuntos
Antivirais/síntese química , Hepacivirus/efeitos dos fármacos , Pró-Fármacos/síntese química , Uridina Monofosfato/análogos & derivados , Proteínas não Estruturais Virais/antagonistas & inibidores , Animais , Antivirais/farmacocinética , Antivirais/farmacologia , Linhagem Celular , Cristalografia por Raios X , Cães , Farmacorresistência Viral , Ésteres , Hepacivirus/genética , Hepatócitos/metabolismo , Humanos , Técnicas In Vitro , Fígado/metabolismo , Macaca fascicularis , Mutação , Pró-Fármacos/farmacocinética , Pró-Fármacos/farmacologia , Ratos , Replicon , Sofosbuvir , Estereoisomerismo , Relação Estrutura-Atividade , Uridina Monofosfato/síntese química , Uridina Monofosfato/farmacocinética , Uridina Monofosfato/farmacologia , Proteínas não Estruturais Virais/genéticaRESUMO
A phosphoramidate prodrug of 2'-deoxy-2'-α-fluoro-ß-C-methyluridine-5'-monophosphate, PSI-7851, demonstrates potent anti-hepatitis C virus (HCV) activity both in vitro and in vivo. PSI-7851 is a mixture of two diastereoisomers, PSI-7976 and PSI-7977, with PSI-7977 being the more active inhibitor of HCV RNA replication in the HCV replicon assay. To inhibit the HCV NS5B RNA-dependent RNA polymerase, PSI-7851 must be metabolized to the active triphosphate form. The first step, hydrolysis of the carboxyl ester by human cathepsin A (CatA) and/or carboxylesterase 1 (CES1), is a stereospecific reaction. Western blot analysis showed that CatA and CES1 are both expressed in primary human hepatocytes. However, expression of CES1 is undetectable in clone A replicon cells. Studies with inhibitors of CatA and/or CES1 indicated that CatA is primarily responsible for hydrolysis of the carboxyl ester in clone A cells, although in primary human hepatocytes, both CatA and CES1 contribute to the hydrolysis. Hydrolysis of the ester is followed by a putative nucleophilic attack on the phosphorus by the carboxyl group resulting in the spontaneous elimination of phenol and the production of an alaninyl phosphate metabolite, PSI-352707, which is common to both isomers. The removal of the amino acid moiety of PSI-352707 is catalyzed by histidine triad nucleotide-binding protein 1 (Hint1) to give the 5'-monophosphate form, PSI-7411. siRNA-mediated Hint1 knockdown studies further indicate that Hint1 is, at least in part, responsible for converting PSI-352707 to PSI-7411. PSI-7411 is then consecutively phosphorylated to the diphosphate, PSI-7410, and to the active triphosphate metabolite, PSI-7409, by UMP-CMP kinase and nucleoside diphosphate kinase, respectively.
Assuntos
Antivirais/farmacocinética , Hepacivirus/fisiologia , Pró-Fármacos/farmacocinética , Uridina Monofosfato/análogos & derivados , Proteínas não Estruturais Virais/antagonistas & inibidores , Replicação Viral/efeitos dos fármacos , Antivirais/farmacologia , Hidrolases de Éster Carboxílico/metabolismo , Catepsina A/metabolismo , Linhagem Celular , Hepatócitos/metabolismo , Hepatócitos/virologia , Humanos , Hidrólise , Núcleosídeo-Fosfato Quinase/metabolismo , Pró-Fármacos/farmacologia , RNA Viral/metabolismo , Sofosbuvir , Estereoisomerismo , Uridina Monofosfato/farmacocinética , Uridina Monofosfato/farmacologia , Proteínas não Estruturais Virais/metabolismo , Replicação Viral/fisiologiaRESUMO
Hepatitis C virus afflicts approximately 180 million people worldwide and currently there are no direct acting antiviral agents available to treat this disease. Our first generation nucleoside HCV inhibitor, RG7128 has already established proof-of-concept in the clinic and is currently in phase IIb clinical trials. As part of our continuing efforts to discover novel anti-HCV agents, 3',4'-oxetane cytidine and adenosine nucleosides were prepared as inhibitors of HCV RNA replication. These nucleosides were shown not to be inhibitors of HCV as determined in a whole cell subgenomic replicon assay. However, 2'-mono/diflouro analogs, 4, 5, and 6 were readily phosphorylated to their monophosphate metabolites by deoxycytidine kinase and their triphosphate derivatives were shown to be inhibitors of HCV NS5B polymerase in vitro. Lack of anti-HCV activity in the replicon assay may be due to the inability of the monophosphates to be converted to their corresponding diphosphates.
Assuntos
Antivirais/síntese química , Éteres Cíclicos/química , Hepacivirus/efeitos dos fármacos , Nucleosídeos/química , Antivirais/química , Antivirais/farmacologia , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Hepacivirus/enzimologia , Nucleosídeos/síntese química , Nucleosídeos/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas não Estruturais Virais/metabolismoRESUMO
The hepatitis C virus (HCV) NS5B RNA polymerase facilitates the RNA synthesis step during the HCV replication cycle. Nucleoside analogs targeting the NS5B provide an attractive approach to treating HCV infections because of their high barrier to resistance and pan-genotype activity. PSI-7851, a pronucleotide of beta-D-2'-deoxy-2'-fluoro-2'-C-methyluridine-5'-monophosphate, is a highly active nucleotide analog inhibitor of HCV for which a phase 1b multiple ascending dose study of genotype 1-infected individuals was recently completed (M. Rodriguez-Torres, E. Lawitz, S. Flach, J. M. Denning, E. Albanis, W. T. Symonds, and M. M. Berry, Abstr. 60th Annu. Meet. Am. Assoc. Study Liver Dis., abstr. LB17, 2009). The studies described here characterize the in vitro antiviral activity and cytotoxicity profile of PSI-7851. The 50% effective concentration for PSI-7851 against the genotype 1b replicon was determined to be 0.075+/-0.050 microM (mean+/-standard deviation). PSI-7851 was similarly effective against replicons derived from genotypes 1a, 1b, and 2a and the genotype 1a and 2a infectious virus systems. The active triphosphate, PSI-7409, inhibited recombinant NS5B polymerases from genotypes 1 to 4 with comparable 50% inhibitory concentrations. PSI-7851 is a specific HCV inhibitor, as it lacks antiviral activity against other closely related and unrelated viruses. PSI-7409 also lacked any significant activity against cellular DNA and RNA polymerases. No cytotoxicity, mitochondrial toxicity, or bone marrow toxicity was associated with PSI-7851 at the highest concentration tested (100 microM). Cross-resistance studies using replicon mutants conferring resistance to modified nucleoside analogs showed that PSI-7851 was less active against the S282T replicon mutant, whereas cells expressing a replicon containing the S96T/N142T mutation remained fully susceptible to PSI-7851. Clearance studies using replicon cells demonstrated that PSI-7851 was able to clear cells of HCV replicon RNA and prevent viral rebound.
Assuntos
Antivirais/farmacologia , Nucleotídeos de Desoxiuracil/farmacologia , Inibidores Enzimáticos/farmacologia , Hepacivirus/efeitos dos fármacos , Pró-Fármacos/farmacologia , Replicação Viral/efeitos dos fármacos , Amidas/química , Amidas/farmacologia , Antivirais/química , Linhagem Celular Tumoral , Nucleotídeos de Desoxiuracil/química , Inibidores Enzimáticos/química , Genótipo , Hepacivirus/classificação , Hepacivirus/enzimologia , Humanos , Ácidos Fosfóricos/química , Ácidos Fosfóricos/farmacologia , Pró-Fármacos/química , RNA Viral/genética , RNA Viral/metabolismo , RNA Polimerase Dependente de RNA/antagonistas & inibidores , Replicon/efeitos dos fármacos , Proteínas não Estruturais Virais/antagonistas & inibidoresRESUMO
beta-D-2'-Deoxy-2'-fluoro-2'-C-methylcytidine (PSI-6130) is a potent inhibitor of hepatitis C virus (HCV) RNA replication in an HCV replicon assay. The 5'-triphosphate of PSI-6130 is a competitive inhibitor of the HCV RNA-dependent RNA polymerase (RdRp) and acts as a nonobligate chain terminator. Recently, it has been shown that the metabolism of PSI-6130 also results in the formation of the 5'-triphosphate of the uridine congener, beta-D-2'-deoxy-2'-fluoro-2'-C-methyluridine (PSI-6206; RO2433). Here we show that the formation of the 5'-triphosphate of RO2433 (RO2433-TP) requires the deamination of PSI-6130 monophosphate and that RO2433 monophosphate is subsequently phosphorylated to the corresponding di- and triphosphates by cellular UMP-CMP kinase and nucleoside diphosphate kinase, respectively. RO2433-TP is a potent inhibitor of the HCV RdRp; however, both enzymatic and cell-based assays show that PSI-6130 triphosphate is a more potent inhibitor of the HCV RdRp than RO2433-TP.
Assuntos
Desoxicitidina/análogos & derivados , Hepacivirus/efeitos dos fármacos , RNA Polimerase Dependente de RNA/antagonistas & inibidores , Uridina Trifosfato/metabolismo , Uridina Trifosfato/farmacologia , Antivirais/química , Antivirais/metabolismo , Antivirais/farmacologia , Linhagem Celular , Citidina/análogos & derivados , Citidina/farmacologia , Desoxicitidina/química , Desoxicitidina/metabolismo , Desoxicitidina/farmacologia , Hepacivirus/enzimologia , Humanos , Testes de Sensibilidade Microbiana , Fosforilação , Replicon/efeitos dos fármacos , Relação Estrutura-Atividade , Uridina Trifosfato/análogos & derivados , Uridina Trifosfato/químicaRESUMO
(-)-(2R,4R)-1-(2-Hydroxymethyl-1,3-dioxolan-4yl)thymine (DOT) is a thymidine analogue that has potent in vitro activity against wild-type and nucleoside reverse transcriptase inhibitor (NRTI)-resistant HIV. For nucleoside analogues to inhibit viral replication, they must be metabolized to the active triphosphate, which inhibits the viral reverse transcriptase (RT). Using purified enzymes, the kinetics of DOT phosphorylation, inhibition of wild-type and drug-resistant HIV-1 reverse transcriptase activity, and excision of DOT-5'-monophosphate (DOT-MP) from a chain-terminated primer were examined. DOT was phosphorylated by human thymidine kinase-1 (TK-1) but not by other pyrimidine nucleoside kinases, including the mitochondrial thymidine kinase (TK-2). Resistance to NRTIs involves decreased binding/incorporation and/or increased excision of the chain-terminating NRTI. RTs containing the D67N/K70R/T215Y/K219Q or T695-SS/T215Y mutations show enhanced removal of DOT-MP from terminated primer as well as approximately four-fold decreased binding/incorporation. The Q151M and K65R mutations appear to cause decreased inhibition by DOT-TP. However, both the K65R and Q151M mutations show decreased excision, which would confer greater stability on the terminated primer. These opposing mechanisms could offset the overall resistance profile and susceptibility. Little or no resistance was observed with the enzymes harbouring mutations resistant to lamivudine (M184V) and non-nucleoside RT inhibitors (K103N).
Assuntos
Fármacos Anti-HIV/farmacologia , Transcriptase Reversa do HIV/antagonistas & inibidores , Inibidores da Transcriptase Reversa/farmacologia , Timina/análogos & derivados , Trifosfato de Adenosina/farmacologia , Farmacorresistência Viral , Fosforilação , Timina/farmacologiaRESUMO
Beta-D-2'-deoxy-2'-fluoro-2'-C-methylcytidine (PSI-6130) is a potent specific inhibitor of hepatitis C virus (HCV) RNA synthesis in Huh-7 replicon cells. To inhibit the HCV NS5B RNA polymerase, PSI-6130 must be phosphorylated to the 5'-triphosphate form. The phosphorylation of PSI-6130 and inhibition of HCV NS5B were investigated. The phosphorylation of PSI-6130 by recombinant human 2'-deoxycytidine kinase (dCK) and uridine-cytidine kinase 1 (UCK-1) was measured by using a coupled spectrophotometric reaction. PSI-6130 was shown to be a substrate for purified dCK, with a Km of 81 microM and a kcat of 0.007 s-1, but was not a substrate for UCK-1. PSI-6130 monophosphate (PSI-6130-MP) was efficiently phosphorylated to the diphosphate and subsequently to the triphosphate by recombinant human UMP-CMP kinase and nucleoside diphosphate kinase, respectively. The inhibition of wild-type and mutated (S282T) HCV NS5B RNA polymerases was studied. The steady-state inhibition constant (Ki) for PSI-6130 triphosphate (PSI-6130-TP) with the wild-type enzyme was 4.3 microM. Similar results were obtained with 2'-C-methyladenosine triphosphate (Ki=1.5 microM) and 2'-C-methylcytidine triphosphate (Ki=1.6 microM). NS5B with the S282T mutation, which is known to confer resistance to 2'-C-methyladenosine, was inhibited by PSI-6130-TP as efficiently as the wild type. Incorporation of PSI-6130-MP into RNA catalyzed by purified NS5B RNA polymerase resulted in chain termination.