RESUMO
Numerous interventions are in clinical development for respiratory syncytial virus (RSV) infection, including small molecules that target viral transcription and replication. These processes are catalyzed by a complex comprising the RNA-dependent RNA polymerase (L) and the tetrameric phosphoprotein (P). RSV P recruits multiple proteins to the polymerase complex and, with the exception of its oligomerization domain, is thought to be intrinsically disordered. Despite their critical roles in RSV transcription and replication, structures of L and P have remained elusive. Here, we describe the 3.2-Å cryo-EM structure of RSV L bound to tetrameric P. The structure reveals a striking tentacular arrangement of P, with each of the four monomers adopting a distinct conformation. The structure also rationalizes inhibitor escape mutants and mutations observed in live-attenuated vaccine candidates. These results provide a framework for determining the molecular underpinnings of RSV replication and transcription and should facilitate the design of effective RSV inhibitors.
Assuntos
Fosfoproteínas/ultraestrutura , RNA Polimerase Dependente de RNA/ultraestrutura , Infecções por Vírus Respiratório Sincicial/virologia , Vírus Sincicial Respiratório Humano/enzimologia , Proteínas Virais/ultraestrutura , Acetatos/química , Animais , Antivirais/química , Antivirais/uso terapêutico , Domínio Catalítico , Microscopia Crioeletrônica , Desoxicitidina/análogos & derivados , Desoxicitidina/química , Desoxicitidina/farmacologia , Desoxicitidina/uso terapêutico , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Conformação Proteica em alfa-Hélice , Domínios e Motivos de Interação entre Proteínas , Quinolinas/química , RNA Polimerase Dependente de RNA/antagonistas & inibidores , RNA Polimerase Dependente de RNA/química , RNA Polimerase Dependente de RNA/metabolismo , Infecções por Vírus Respiratório Sincicial/tratamento farmacológico , Vacinas contra Vírus Sincicial Respiratório/química , Células Sf9 , Spodoptera , Proteínas Virais/química , Proteínas Virais/metabolismo , Replicação Viral/efeitos dos fármacosRESUMO
Respiratory syncytial virus (RSV) causes severe lower respiratory tract infections, yet no vaccines or effective therapeutics are available. ALS-8176 is a first-in-class nucleoside analog prodrug effective in RSV-infected adult volunteers, and currently under evaluation in hospitalized infants. Here, we report the mechanism of inhibition and selectivity of ALS-8176 and its parent ALS-8112. ALS-8176 inhibited RSV replication in non-human primates, while ALS-8112 inhibited all strains of RSV in vitro and was specific for paramyxoviruses and rhabdoviruses. The antiviral effect of ALS-8112 was mediated by the intracellular formation of its 5'-triphosphate metabolite (ALS-8112-TP) inhibiting the viral RNA polymerase. ALS-8112 selected for resistance-associated mutations within the region of the L gene of RSV encoding the RNA polymerase. In biochemical assays, ALS-8112-TP was efficiently recognized by the recombinant RSV polymerase complex, causing chain termination of RNA synthesis. ALS-8112-TP did not inhibit polymerases from host or viruses unrelated to RSV such as hepatitis C virus (HCV), whereas structurally related molecules displayed dual RSV/HCV inhibition. The combination of molecular modeling and enzymatic analysis showed that both the 2'F and the 4'ClCH2 groups contributed to the selectivity of ALS-8112-TP. The lack of antiviral effect of ALS-8112-TP against HCV polymerase was caused by Asn291 that is well-conserved within positive-strand RNA viruses. This represents the first comparative study employing recombinant RSV and HCV polymerases to define the selectivity of clinically relevant nucleotide analogs. Understanding nucleotide selectivity towards distant viral RNA polymerases could not only be used to repurpose existing drugs against new viral infections, but also to design novel molecules.
Assuntos
Antivirais/farmacologia , Citidina Trifosfato/análogos & derivados , Citidina Trifosfato/farmacologia , RNA Polimerases Dirigidas por DNA/metabolismo , Infecções por Vírus Respiratório Sincicial/tratamento farmacológico , Vírus Sincicial Respiratório Humano/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Animais , Chlorocebus aethiops , Humanos , RNA Viral/genética , Infecções por Vírus Respiratório Sincicial/virologia , Vírus Sincicial Respiratório Humano/genética , Proteínas Virais/genéticaRESUMO
Ribonucleotide analog inhibitors of the RNA-dependent RNA polymerase of hepatitis C virus (HCV) represent one of the most exciting recent developments in HCV antiviral therapy. Although it is well established that these molecules cause chain termination by competing at the triphosphate level with natural nucleotides for incorporation into elongating RNA, strategies to rationally optimize antiviral potency based on enzyme kinetics remain elusive. In this study, we used the isolated HCV polymerase elongation complex to determine the pre-steady-state kinetics of incorporation of 2'F-2'C-Me-UTP, the active metabolite of the anti-HCV drug sofosbuvir. 2'F-2'C-Me-UTP was efficiently incorporated by HCV polymerase with apparent Kd (equilibrium constant) and kpol (rate of nucleotide incorporation at saturating nucleotide concentration) values of 113 ± 28 µM and 0.67 ± 0.05 s(-1), respectively, giving an overall substrate efficiency (kpol/Kd) of 0.0059 ± 0.0015 µM(-1) s(-1). We also measured the substrate efficiency of other UTP analogs and found that substitutions at the 2' position on the ribose can greatly affect their level of incorporation, with a rank order of OH > F > NH2 > F-C-Me > C-Me > N3 > ara. However, the efficiency of chain termination following the incorporation of UMP analogs followed a different order, with only 2'F-2'C-Me-, 2'C-Me-, and 2'ara-UTP causing complete and immediate chain termination. The chain termination profile of the 2'-modified nucleotides explains the apparent lack of correlation observed across all molecules between substrate efficiency at the single-nucleotide level and their overall inhibition potency. To our knowledge, these results provide the first attempt to use pre-steady-state kinetics to uncover the mechanism of action of 2'-modified NTP analogs against HCV polymerase.
Assuntos
Antivirais/farmacologia , Inibidores Enzimáticos/farmacologia , Hepacivirus/efeitos dos fármacos , Hepacivirus/enzimologia , RNA Polimerase Dependente de RNA/antagonistas & inibidores , Uridina Trifosfato/análogos & derivados , Uridina Trifosfato/farmacologia , Algoritmos , Guanosina Trifosfato/metabolismo , Humanos , CinéticaRESUMO
Respiratory syncytial virus (RSV) can cause pulmonary complications in infants, elderly and immunocompromised patients. While two vaccines and two prophylactic monoclonal antibodies are now available, treatment options are still needed. JNJ-7184 is a non-nucleoside inhibitor of the RSV-Large (L) polymerase, displaying potent inhibition of both RSV-A and -B strains. Resistance selection and hydrogen-deuterium exchange experiments suggest JNJ-7184 binds RSV-L in the connector domain. JNJ-7184 prevents RSV replication and transcription by inhibiting initiation or early elongation. JNJ-7184 is effective in air-liquid interface cultures and therapeutically in neonatal lambs, acting to drastically reverse the appearance of lung pathology.
Assuntos
Antivirais , Infecções por Vírus Respiratório Sincicial , Vírus Sincicial Respiratório Humano , Replicação Viral , Antivirais/farmacologia , Antivirais/química , Infecções por Vírus Respiratório Sincicial/tratamento farmacológico , Infecções por Vírus Respiratório Sincicial/virologia , Animais , Humanos , Replicação Viral/efeitos dos fármacos , Vírus Sincicial Respiratório Humano/efeitos dos fármacos , Ovinos , Farmacorresistência Viral , Proteínas Virais/antagonistas & inibidores , Proteínas Virais/metabolismo , Proteínas Virais/genética , Pulmão/virologiaRESUMO
The respiratory syncytial virus polymerase complex, consisting of the polymerase (L) and phosphoprotein (P), catalyzes nucleotide polymerization, cap addition, and cap methylation via the RNA dependent RNA polymerase, capping, and Methyltransferase domains on L. Several nucleoside and non-nucleoside inhibitors have been reported to inhibit this polymerase complex, but the structural details of the exact inhibitor-polymerase interactions have been lacking. Here, we report a non-nucleoside inhibitor JNJ-8003 with sub-nanomolar inhibition potency in both antiviral and polymerase assays. Our 2.9 Å resolution cryo-EM structure revealed that JNJ-8003 binds to an induced-fit pocket on the capping domain, with multiple interactions consistent with its tight binding and resistance mutation profile. The minigenome and gel-based de novo RNA synthesis and primer extension assays demonstrated that JNJ-8003 inhibited nucleotide polymerization at the early stages of RNA transcription and replication. Our results support that JNJ-8003 binding modulates a functional interplay between the capping and RdRp domains, and this molecular insight could accelerate the design of broad-spectrum antiviral drugs.
Assuntos
Vírus Sincicial Respiratório Humano , RNA Polimerase Dependente de RNA/química , Ligação Proteica , RNA/metabolismo , Nucleotídeos/metabolismoRESUMO
Conformational modeling has been successfully applied to the design of cyclic bioisosteres used to replace a conformationally rigid amide bond in a series of thiophene carboxylate inhibitors of HCV NS5B polymerase. Select compounds were equipotent with the original amide series. Single-point mutant binding studies, in combination with inhibition structure-activity relationships, suggest this new series interacts at the Thumb-II domain of NS5B. Inhibitor binding at the Thumb-II site was ultimately confirmed by solving a crystal structure of 8b complexed with NS5B.
Assuntos
Amidas/química , Antivirais/síntese química , Inibidores Enzimáticos/síntese química , Hepacivirus/efeitos dos fármacos , Tiofenos/síntese química , Proteínas não Estruturais Virais/antagonistas & inibidores , Amidas/síntese química , Amidas/farmacologia , Antivirais/química , Antivirais/farmacologia , Sítios de Ligação , Cristalografia por Raios X , Desenho de Fármacos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Estrutura Terciária de Proteína , Relação Estrutura-Atividade , Tiofenos/química , Tiofenos/farmacologia , Proteínas não Estruturais Virais/metabolismoRESUMO
Chronic hepatitis C (CHC) is a major liver disease caused by the hepatitis C virus. The current standard of care for CHC can achieve cure rates above 95%; however, the drugs in current use are administered for a period of 8-16 weeks. A combination of safe and effective drugs with a shorter treatment period is highly desirable. We report synthesis and biological evaluation of a series of 2',3'- and 2',4'-substituted guanosine nucleotide analogues. Their triphosphates exhibited potent inhibition of the HCV NS5B polymerase with IC50 as low as 0.13 µM. In the HCV replicon assay, the phosphoramidate prodrugs of these analogues demonstrated excellent activity with EC50 values as low as 5 nM. A lead compound AL-611 showed high levels of the nucleoside 5'-triphosphate in vitro in primary human hepatocytes and in vivo in dog liver following oral administration.
Assuntos
Antivirais/farmacologia , RNA Polimerases Dirigidas por DNA/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Nucleotídeos de Guanina/farmacologia , Hepacivirus/efeitos dos fármacos , Pró-Fármacos/farmacologia , Animais , Antivirais/síntese química , Antivirais/toxicidade , Cães , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/toxicidade , Feminino , Nucleotídeos de Guanina/síntese química , Nucleotídeos de Guanina/toxicidade , Humanos , Masculino , Pró-Fármacos/síntese química , Pró-Fármacos/toxicidade , Proteínas não Estruturais Virais/antagonistas & inibidores , Replicação Viral/efeitos dos fármacosRESUMO
Compound 1 (SNS-314) is a potent and selective Aurora kinase inhibitor that is currently in clinical trials in patients with advanced solid tumors. This communication describes the synthesis of prodrug derivatives of 1 with improved aqueous solubility profiles. In particular, phosphonooxymethyl-derived prodrug 2g has significantly enhanced solubility and is converted to the biologically active parent (1) following iv as well as po administration to rodents.
Assuntos
Compostos de Fenilureia/química , Pró-Fármacos/química , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Tiazóis/química , Água/química , Animais , Antineoplásicos/química , Antineoplásicos/farmacocinética , Antineoplásicos/farmacologia , Aurora Quinases , Masculino , Camundongos , Compostos de Fenilureia/farmacocinética , Compostos de Fenilureia/farmacologia , Pró-Fármacos/farmacocinética , Pró-Fármacos/farmacologia , Proteínas Serina-Treonina Quinases/metabolismo , Ratos , Ratos Sprague-Dawley , Solubilidade , Tiazóis/farmacocinética , Tiazóis/farmacologiaRESUMO
This Letter describes the discovery and key structure-activity relationship (SAR) of a series of 2-aminobenzimidazoles as potent Aurora kinase inhibitors. 2-Aminobenzimidazole serves as a bioisostere of the biaryl urea residue of SNS-314 (1c), which is a potent Aurora kinase inhibitor and entered clinical testing in patients with solid tumors. Compared to SNS-314, this series of compounds offers better aqueous solubility while retaining comparable in vitro potency in biochemical and cell-based assays; in particular, 6m has also demonstrated a comparable mouse iv PK profile to SNS-314.
Assuntos
Antineoplásicos/química , Benzimidazóis/química , Inibidores de Proteínas Quinases/química , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Animais , Antineoplásicos/síntese química , Antineoplásicos/farmacocinética , Aurora Quinases , Benzimidazóis/síntese química , Benzimidazóis/farmacocinética , Linhagem Celular Tumoral , Humanos , Camundongos , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/farmacocinética , Proteínas Serina-Treonina Quinases/metabolismo , Relação Estrutura-AtividadeRESUMO
We report the synthesis and biological evaluation of a series of 4'-fluoro-2'- C-substituted uridines. Triphosphates of the uridine analogues exhibited a potent inhibition of hepatitis C virus (HCV) NS5B polymerase with IC50 values as low as 27 nM. In an HCV subgenomic replicon assay, the phosphoramidate prodrugs of these uridine analogues demonstrated a very potent activity with EC50 values as low as 20 nM. A lead compound AL-335 (53) demonstrated high levels of the nucleoside triphosphate in vitro in primary human hepatocytes and Huh-7 cells as well as in dog liver following a single oral dose. Compound 53 was selected for the clinical development where it showed promising results in phase 1 and 2 trials.
Assuntos
Alanina/análogos & derivados , Antivirais/farmacologia , Hepacivirus/efeitos dos fármacos , Pró-Fármacos/farmacologia , Nucleotídeos de Uracila/farmacologia , Uridina/análogos & derivados , Alanina/síntese química , Alanina/farmacologia , Animais , Antivirais/síntese química , Linhagem Celular Tumoral , Cães , Hepacivirus/enzimologia , Hepatite C/tratamento farmacológico , Humanos , Inibidores da Síntese de Ácido Nucleico/síntese química , Inibidores da Síntese de Ácido Nucleico/farmacologia , Fosforamidas , Pró-Fármacos/síntese química , Replicon/efeitos dos fármacos , Nucleotídeos de Uracila/síntese química , Nucleotídeos de Uracila/metabolismo , Uridina/síntese química , Uridina/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidoresRESUMO
We demonstrate a fragment-based lead discovery method that combines site-directed ligand discovery with dynamic combinatorial chemistry. Our technique targets dynamic combinatorial screening to a specified region of a protein by using reversible disulfide chemistry. We have used this technology to rapidly identify inhibitors of the drug target Aurora A that span the purine-binding site and the adaptive pocket of the kinase. The binding mode of a noncovalent inhibitor has been further characterized through crystallography.
Assuntos
Química Farmacêutica/métodos , Técnicas de Química Combinatória/métodos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/síntese química , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Aurora Quinases , Sítios de Ligação/efeitos dos fármacos , Cristalografia por Raios X , Desenho de Fármacos , Ligantes , Espectrometria de Massas/métodos , Modelos Químicos , Estrutura Molecular , Purinas/química , Relação Estrutura-AtividadeRESUMO
This communication describes the discovery of a novel series of Aurora kinase inhibitors. Key SAR and critical binding elements are discussed. Some of the more advanced analogues potently inhibit cellular proliferation and induce phenotypes consistent with Aurora kinase inhibition. In particular, compound 21 (SNS-314) is a potent and selective Aurora kinase inhibitor that exhibits significant activity in pre-clinical in vivo tumor models.
Assuntos
Neoplasias Experimentais/tratamento farmacológico , Compostos de Fenilureia/química , Compostos de Fenilureia/farmacologia , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Quinazolinas/farmacologia , Tiazóis/química , Tiazóis/farmacologia , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Aurora Quinases , Ensaios de Seleção de Medicamentos Antitumorais , Células HCT116 , Humanos , Camundongos , Transplante de Neoplasias , Neoplasias Experimentais/enzimologia , Quinazolinas/química , Relação Estrutura-AtividadeRESUMO
OBJECTIVE: To examine the relation between perceived overprotection and the psychological states of cerebral palsy patients and their primary caretakers in Hong Kong. DESIGN: Cross-sectional survey, in which data of 14 pairs of cerebral palsy patients and their caretakers were analysed. SETTING: Duchess of Kent Children's Hospital, Hong Kong. PARTICIPANTS: Cerebral palsy patients and their primary caretakers in Hong Kong. MAIN OUTCOME MEASURES: Perceived overprotection and psychological states. RESULTS: Nearly two thirds of the 14 patients (mean age of 15 years) and 86% of the 14 primary caretakers (mean age of 47 years) perceived various levels of overprotection. For both patients and caretakers, perceived overprotection was positively associated with anxiety and unhappiness. The patients' and caretakers' psychological states and perception of overprotection were not related to the actual motor ability of the patients. Perceived overprotection of the patients was not related to that of the caretakers. CONCLUSION: Caretakers should be mindful that a well-meaning move may have undesirable consequences. More support and child-rearing education should be considered for caretakers.
Assuntos
Cuidadores/psicologia , Paralisia Cerebral/psicologia , Pessoas com Deficiência/psicologia , Relações Interpessoais , Qualidade de Vida , Atividades Cotidianas , Adaptação Fisiológica , Adaptação Psicológica , Adolescente , Adulto , Ansiedade/epidemiologia , Ansiedade/psicologia , Paralisia Cerebral/diagnóstico , Paralisia Cerebral/reabilitação , Distribuição de Qui-Quadrado , Estudos Transversais , Transtorno Depressivo/epidemiologia , Transtorno Depressivo/psicologia , Avaliação da Deficiência , Feminino , Hong Kong , Humanos , Masculino , Pessoa de Meia-Idade , Probabilidade , Perfil de Impacto da Doença , Apoio Social , Inquéritos e Questionários , Adulto JovemRESUMO
The fission yeast Dbf4 homologue Dfp1 has a well-characterized role in regulating the initiation of DNA replication. Sequence analysis of Dfp1 homologues reveals three highly conserved regions, referred to as motifs N, M, and C. To determine the roles of these conserved regions in Dfp1 function, we have generated dfp1 alleles with mutations in these regions. Mutations in motif N render cells sensitive to a broad range of DNA-damaging agents and replication inhibitors, yet these mutant proteins are efficient activators of Hsk1 kinase in vitro. In contrast, mutations in motif C confer sensitivity to the alkylating agent methyl methanesulfonate (MMS) but, surprisingly, not to UV, ionizing radiation, or hydroxyurea. Motif C mutants are poor activators of Hsk1 in vitro but can fulfill the essential function(s) of Dfp1 in vivo. Strains carrying dfp1 motif C mutants have an intact mitotic and intra-S-phase checkpoint, and epistasis analysis indicates that dfp1 motif C mutants function outside of the known MMS damage repair pathways, suggesting that the observed MMS sensitivity is due to defects in recovery from DNA damage. The motif C mutants are most sensitive to MMS during S phase and are partially suppressed by deletion of the S-phase checkpoint kinase cds1. Following treatment with MMS, dfp1 motif C mutants exhibit nuclear fragmentation, chromosome instability, precocious recombination, and persistent checkpoint activation. We propose that Dfp1 plays at least two genetically separable roles in the DNA damage response in addition to its well-characterized role in the initiation of DNA replication and that motif C plays a critical role in the response to alkylation damage, perhaps by restarting or stabilizing stalled replication forks.
Assuntos
Proteínas de Ciclo Celular , Cromossomos Fúngicos , Proteínas Fúngicas/genética , Proteínas Serina-Treonina Quinases , Fase S/genética , Proteínas de Saccharomyces cerevisiae , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces/genética , Alquilação , Sequência de Aminoácidos , Antineoplásicos Alquilantes/farmacologia , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/genética , Núcleo Celular/ultraestrutura , Quinase 1 do Ponto de Checagem , Sequência Conservada , Dano ao DNA/efeitos dos fármacos , Dano ao DNA/genética , Replicação do DNA/efeitos dos fármacos , Proteínas Fúngicas/metabolismo , Metanossulfonato de Metila/farmacologia , Mitose , Mutação , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Recombinação Genética , Schizosaccharomyces/citologia , Schizosaccharomyces/efeitos dos fármacosRESUMO
PURPOSE: To evaluate the effectiveness of psychological intervention in the care of cancer patients and to determine whether routine use of individual psychological therapies is indicated. PATIENTS AND METHODS: Patients with newly diagnosed gynecologic malignancies from August 1999 to November 2000 were recruited and randomly assigned to either a control group receiving routine medical care or to an intervention group receiving individual psychotherapy. A set of fixed-choice, self-report questionnaires assessing the patients' psychological status, quality of life, and their perceptions related to the medical consultations was completed at recruitment and then every 3 months for 18 months. Data analysis was performed according to the intention-to-treat principle by fitting the data into a linear mixed-effects model. Multivariable analyses were performed to examine the effects of confounding factors. RESULTS: One hundred fifty-five patients participated in the trial. There were no statistically significant differences between the two groups at baseline. There was a trend toward better quality of life and functional status and also improvement of the symptoms over time for both groups. No differences were found between the groups in the scores measured by any of the instruments at baseline and at any time points after the cancer diagnosis. Psychological intervention had no significant effects on the psychosocial parameters. CONCLUSION: Routine use of psychological therapies as given in our format has no significant effect on the patients' quality of life and psychological status.
Assuntos
Neoplasias dos Genitais Femininos/psicologia , Neoplasias dos Genitais Femininos/reabilitação , Psicoterapia , Atividades Cotidianas , Adulto , China/etnologia , Feminino , Neoplasias dos Genitais Femininos/etnologia , Humanos , Pessoa de Meia-Idade , Qualidade de Vida , Resultado do TratamentoRESUMO
ALS-8112 is the parent molecule of ALS-8176, a first-in-class nucleoside analog prodrug effective in the clinic against respiratory syncytial virus (RSV) infection. The antiviral activity of ALS-8112 is mediated by its 5'-triphosphate metabolite (ALS-8112-TP, or 2'F-4'ClCH2-cytidine triphosphate) inhibiting the RNA polymerase activity of the RSV L-P protein complex through RNA chain termination. Four amino acid mutations in the RNA-dependent RNA polymerase (RdRp) domain of L (QUAD: M628L, A789V, L795I, and I796V) confer in vitro resistance to ALS-8112-TP by increasing its discrimination relative to natural CTP. In this study, we show that the QUAD mutations specifically recognize the ClCH2 group of ALS-8112-TP. Among the four mutations, A789V conferred the greatest resistance phenotype, which was consistent with its putative position in the active site of the RdRp domain. AZ-27, a non-nucleoside inhibitor of RSV, also inhibited the RdRp activity, with decreased inhibition potency in the presence of the Y1631H mutation. The QUAD mutations had no effect on the antiviral activity of AZ-27, and the Y1631H mutation did not significantly increase the discrimination of ALS-8112-TP. Combining ALS-8112 with AZ-27 in vitro resulted in significant synergistic inhibition of RSV replication. Overall, this is the first mechanistic study showing a lack of cross-resistance between mutations selected by different classes of RSV polymerase inhibitors acting in synergy, opening the door to future potential combination therapies targeting different regions of the L protein.
Assuntos
Antivirais/farmacologia , Benzazepinas/farmacologia , Citidina Trifosfato/análogos & derivados , RNA Polimerases Dirigidas por DNA/antagonistas & inibidores , Farmacorresistência Viral/genética , Niacinamida/análogos & derivados , Mutação Puntual , Proteínas Virais/antagonistas & inibidores , Linhagem Celular Tumoral , Citidina Trifosfato/farmacologia , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Combinação de Medicamentos , Sinergismo Farmacológico , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/virologia , Expressão Gênica , Humanos , Niacinamida/farmacologia , RNA Viral/antagonistas & inibidores , RNA Viral/biossíntese , RNA Viral/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
Respiratory syncytial virus (RSV) is a leading pathogen of childhood and is associated with significant morbidity and mortality. To date, ribavirin is the only approved small molecule drug, which has limited use. The only other RSV drug is palivizumab, a monoclonal antibody, which is used for RSV prophylaxis. Clearly, there is an urgent need for small molecule RSV drugs. This article reports the design, synthesis, anti-RSV activity, metabolism, and pharmacokinetics of a series of 4'-substituted cytidine nucleosides. Among tested compounds 4'-chloromethyl-2'-deoxy-2'-fluorocytidine (2c) exhibited the most promising activity in the RSV replicon assay with an EC50 of 0.15 µM. The 5'-triphosphate of 2c (2c-TP) inhibited RSV polymerase with an IC50 of 0.02 µM without appreciable inhibition of human DNA and RNA polymerases at 100 µM. ALS-8176 (71), the 3',5'-di-O-isobutyryl prodrug of 2c, demonstrated good oral bioavailability and a high level of 2c-TP in vivo. Compound 71 is a first-in-class nucleoside RSV polymerase inhibitor that demonstrated excellent anti-RSV efficacy and safety in a phase 2 clinical RSV challenge study.
Assuntos
Antivirais/farmacologia , Desoxicitidina/análogos & derivados , Inibidores Enzimáticos/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases , Pró-Fármacos/farmacologia , Infecções por Vírus Respiratório Sincicial/tratamento farmacológico , Vírus Sinciciais Respiratórios/efeitos dos fármacos , Vírus Sinciciais Respiratórios/enzimologia , Animais , Antivirais/administração & dosagem , Antivirais/química , Cricetinae , DNA Polimerase Dirigida por DNA/metabolismo , RNA Polimerases Dirigidas por DNA/antagonistas & inibidores , RNA Polimerases Dirigidas por DNA/metabolismo , Desoxicitidina/síntese química , Desoxicitidina/química , Desoxicitidina/farmacologia , Relação Dose-Resposta a Droga , Descoberta de Drogas , Inibidores Enzimáticos/administração & dosagem , Inibidores Enzimáticos/química , Haplorrinos , Humanos , Masculino , Conformação Molecular , Poli(ADP-Ribose) Polimerases/metabolismo , Pró-Fármacos/administração & dosagem , Pró-Fármacos/química , Ratos , Ratos Sprague-Dawley , Infecções por Vírus Respiratório Sincicial/virologia , Relação Estrutura-Atividade , Replicação Viral/efeitos dos fármacosRESUMO
In the past few years, there have been many advances in the efforts to cure patients with hepatitis C virus (HCV). The ultimate goal of these efforts is to develop a combination therapy consisting of only direct-antiviral agents (DAAs). In this paper, we discuss our efforts that led to the identification of a bicyclic template with potent activity against the NS5B polymerase, a critical enzyme on the life cycle of HCV. In continuation of our exploration to improve the stilbene series, the 3,5,6,8-tetrasubstituted quinoline core was identified as replacement of the stilbene moiety. 6-Methoxy-2(1H)-pyridone was identified among several heterocyclic headgroups to have the best potency. Solubility of the template was improved by replacing a planar aryl linker with a saturated pyrrolidine. Profiling of the most promising compounds led to the identification of quinoline 41 (RG7109), which was selected for advancement to clinical development.
Assuntos
Antivirais/farmacologia , Inibidores Enzimáticos/farmacologia , Hepacivirus/efeitos dos fármacos , Quinolinas/farmacologia , Sulfonamidas/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Animais , Antivirais/química , Antivirais/farmacocinética , Cães , Descoberta de Drogas , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacocinética , Hepacivirus/enzimologia , Humanos , Modelos Moleculares , Quinolinas/química , Quinolinas/farmacocinética , Ratos , Sulfonamidas/química , Sulfonamidas/farmacocinéticaRESUMO
Hepatitis C virus (HCV) is a major global public health problem. While the current standard of care, a direct-acting antiviral (DAA) protease inhibitor taken in combination with pegylated interferon and ribavirin, represents a major advancement in recent years, an unmet medical need still exists for treatment modalities that improve upon both efficacy and tolerability. Toward those ends, much effort has continued to focus on the discovery of new DAAs, with the ultimate goal to provide interferon-free combinations. The RNA-dependent RNA polymerase enzyme NS5B represents one such DAA therapeutic target for inhibition that has attracted much interest over the past decade. Herein, we report the discovery and optimization of a novel series of inhibitors of HCV NS5B, through the use of structure-based design applied to a fragment-derived starting point. Issues of potency, pharmacokinetics, and early safety were addressed in order to provide a clinical candidate in fluoropyridone 19.
Assuntos
Antivirais/farmacologia , Inibidores Enzimáticos/farmacologia , RNA Polimerase Dependente de RNA/antagonistas & inibidores , Proteínas não Estruturais Virais/antagonistas & inibidores , Animais , Antivirais/química , Antivirais/farmacocinética , Área Sob a Curva , Linhagem Celular Tumoral , Cães , Descoberta de Drogas/métodos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacocinética , Hepacivirus/efeitos dos fármacos , Hepacivirus/enzimologia , Hepacivirus/fisiologia , Hepatite C/prevenção & controle , Hepatite C/virologia , Interações Hospedeiro-Patógeno/genética , Humanos , Modelos Moleculares , Terapia de Alvo Molecular/métodos , Ligação Proteica , Estrutura Terciária de Proteína , Piridonas/síntese química , Piridonas/farmacocinética , Piridonas/farmacologia , RNA Polimerase Dependente de RNA/química , RNA Polimerase Dependente de RNA/metabolismo , Ratos , Relação Estrutura-Atividade , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/metabolismoRESUMO
BIO8898 is one of several synthetic organic molecules that have recently been reported to inhibit receptor binding and function of the constitutively trimeric tumor necrosis factor (TNF) family cytokine CD40 ligand (CD40L, aka CD154). Small molecule inhibitors of protein-protein interfaces are relatively rare, and their discovery is often very challenging. Therefore, to understand how BIO8898 achieves this feat, we characterized its mechanism of action using biochemical assays and X-ray crystallography. BIO8898 inhibited soluble CD40L binding to CD40-Ig with a potency of IC(50) = 25 µM and inhibited CD40L-dependent apoptosis in a cellular assay. A co-crystal structure of BIO8898 with CD40L revealed that one inhibitor molecule binds per protein trimer. Surprisingly, the compound binds not at the surface of the protein but by intercalating deeply between two subunits of the homotrimeric cytokine, disrupting a constitutive protein-protein interface and breaking the protein's 3-fold symmetry. The compound forms several hydrogen bonds with the protein, within an otherwise hydrophobic binding pocket. In addition to the translational splitting of the trimer, binding of BIO8898 was accompanied by additional local and longer-range conformational perturbations of the protein, both in the core and in a surface loop. Binding of BIO8898 is reversible, and the resulting complex is stable and does not lead to detectable dissociation of the protein trimer. Our results suggest that a set of core aromatic residues that are conserved across a subset of TNF family cytokines might represent a generic hot-spot for the induced-fit binding of trimer-disrupting small molecules.