RESUMO
Hepatitis C virus (HCV) infection is a major global health burden and is associated with an increased risk of liver cirrhosis and hepatocellular carcinoma. There remains an unmet medical need for efficacious and safe direct antivirals with complementary modes of action for combination in treatment regimens to deliver a high cure rate with a short duration of treatment for HCV patients. Here we report the in vitro inhibitory activity, mode of action, binding kinetics, and resistance profile of TMC647055, a novel and potent nonnucleoside inhibitor of the HCV NS5B RNA-dependent RNA polymerase. In vitro combination studies with an HCV NS3/4A protease inhibitor demonstrated potent suppression of HCV RNA replication, confirming the potential for combination of these two classes in the treatment of chronic HCV infection. TMC647055 is a potent nonnucleoside NS5B polymerase inhibitor of HCV replication with a promising in vitro biochemical, kinetic, and virological profile that is currently undergoing clinical evaluation.
Assuntos
Antivirais/farmacologia , Inibidores Enzimáticos/farmacologia , Hepacivirus/efeitos dos fármacos , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , RNA Polimerase Dependente de RNA/antagonistas & inibidores , Sulfonamidas/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Linhagem Celular , Clonagem Molecular , Combinação de Medicamentos , Sinergismo Farmacológico , Escherichia coli/genética , Genes Reporter , Hepacivirus/enzimologia , Hepacivirus/genética , Hepacivirus/crescimento & desenvolvimento , Hepatite C Crônica/tratamento farmacológico , Hepatite C Crônica/virologia , Humanos , Plasmídeos , RNA Polimerase Dependente de RNA/metabolismo , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/metabolismo , Transfecção , Proteínas não Estruturais Virais/metabolismo , Replicação Viral/efeitos dos fármacosRESUMO
Novel conformationaly constrained 1,6- and 2,6-macrocyclic HCV NS5b polymerase inhibitors, in which either the nitrogen or the phenyl ring in the C2 position of the central indole core is tethered to an acylsulfamide acid bioisostere, have been designed and tested for their anti-HCV potency. This transformational route toward non-zwitterionic finger loop-directed inhibitors led to the discovery of derivatives with improved cell potency and pharmacokinetic profile.
Assuntos
Antivirais/química , Inibidores Enzimáticos/química , Hepacivirus/enzimologia , Indóis/química , Proteínas não Estruturais Virais/antagonistas & inibidores , Regulação Alostérica , Animais , Antivirais/síntese química , Antivirais/farmacocinética , Linhagem Celular Tumoral , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacocinética , Humanos , Indóis/síntese química , Indóis/farmacocinética , Microssomos Hepáticos/metabolismo , Ratos , Ratos Sprague-Dawley , Relação Estrutura-Atividade , Proteínas não Estruturais Virais/metabolismo , Replicação Viral/efeitos dos fármacosRESUMO
Optimization of a novel series of macrocyclic indole-based inhibitors of the HCV NS5b polymerase targeting the finger loop domain led to the discovery of lead compounds exhibiting improved potency in cellular assays and superior pharmacokinetic profile. Further lead optimization performed on the most promising unsaturated-bridged subseries provided the clinical candidate 27-cyclohexyl-12,13,16,17-tetrahydro-22-methoxy-11,17-dimethyl-10,10-dioxide-2,19-methano-3,7:4,1-dimetheno-1H,11H-14,10,2,9,11,17-benzoxathiatetraazacyclo docosine-8,18(9H,15H)-dione, TMC647055 (compound 18a). This non-zwitterionic 17-membered ring macrocycle combines nanomolar cellular potency (EC(50) of 82 nM) with minimal associated cell toxicity (CC(50)>20 µM) and promising pharmacokinetic profiles in rats and dogs. TMC647055 is currently being evaluated in the clinic.
Assuntos
Antivirais/química , Inibidores Enzimáticos/química , Hepacivirus/enzimologia , Compostos Heterocíclicos de 4 ou mais Anéis/síntese química , Indóis/química , Sulfonamidas/síntese química , Proteínas não Estruturais Virais/antagonistas & inibidores , Regulação Alostérica , Animais , Antivirais/síntese química , Antivirais/farmacocinética , Linhagem Celular Tumoral , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacocinética , Compostos Heterocíclicos de 4 ou mais Anéis/química , Compostos Heterocíclicos de 4 ou mais Anéis/farmacocinética , Humanos , Fígado/metabolismo , Compostos Macrocíclicos/síntese química , Compostos Macrocíclicos/química , Compostos Macrocíclicos/farmacocinética , Ratos , Ratos Sprague-Dawley , Relação Estrutura-Atividade , Sulfonamidas/química , Sulfonamidas/farmacocinética , Proteínas não Estruturais Virais/metabolismo , Replicação Viral/efeitos dos fármacosRESUMO
OBJECTIVES: In this study, the influence of several key elements of the cytologic sample workflow on DNA and RNA content was evaluated. METHODS: The A549 cell line, patient-derived organoids, and pleural effusions were used to investigate the effect of (1) several collection media and delayed time to processing; (2) cytology specimens; (3) cytologic staining; and (4) formalin-fixed, paraffin-embedded (FFPE) cell block processing on nucleic acid quality and quantity as determined by fragment analyzer, Qubit analysis (Thermo Fisher Scientific), and quantitative polymerase chain reaction-based analysis on the Idylla platform (Biocartis). RESULTS: Alcohol-based collection media (CytoRich Red [Thermo Fisher Scientific] and EtOH95%) displayed high DNA and RNA preservation capacity, while phosphate-buffered saline and, to a lesser extent, formalin were associated with high RNA quality. Cytospin and smear cytology specimens were subject to DNA and RNA loss. Cytologic staining had no further impact on sample quality, hence destaining is not necessary. Both H&E-stained and unstained FFPE sections are compatible with nucleic acid analysis, despite a strong decrease in DNA and RNA quality. CONCLUSIONS: Although several key elements of the cytologic sample workflow have an influence on DNA and RNA quality and quantity, the selection of these elements is also dependent on the downstream (ancillary) testing methods.
Assuntos
Formaldeído , Ácidos Nucleicos , Humanos , Inclusão em Parafina/métodos , RNA/genética , Manejo de Espécimes/métodos , Fixação de Tecidos/métodosRESUMO
Chronic infection with hepatitis C virus (HCV) is a major global health burden and is associated with an increased risk of liver cirrhosis and hepatocellular carcinoma. Current therapy for HCV infection has limited efficacy, particularly against genotype 1 virus, and is hampered by a range of adverse effects. Therefore, there is a clear unmet medical need for efficacious and safe direct antiviral drugs for use in combination with current treatments to increase cure rates and shorten treatment times. The broad genotypic coverage achievable with nucleosides or nucleotides and the high genetic barrier to resistance of these compounds observed in vitro and in vivo suggest that this class of inhibitors could be a valuable component of future therapeutic regimens. Here, we report the in vitro inhibitory activity and mode of action of 2'-deoxy-2'-spirocyclopropylcytidine (TMC647078), a novel and potent nucleoside inhibitor of the HCV NS5B RNA-dependent RNA polymerase that causes chain termination of the nascent HCV RNA chain. In vitro combination studies with a protease inhibitor resulted in additive efficacy in the suppression of HCV RNA replication, highlighting the potential for the combination of these two classes in the treatment of chronic HCV infection. No cytotoxic effects were observed in various cell lines. Biochemical studies indicated that TMC647078 is phosphorylated mainly by deoxycytidine kinase (dCK) without inhibiting the phosphorylation of the natural substrate, and high levels of triphosphate were observed in Huh7 cells and in primary hepatocytes in vitro. TMC647078 is a potent novel nucleoside inhibitor of HCV replication with a promising in vitro virology and biology profile.
Assuntos
Antivirais/farmacologia , Citidina/análogos & derivados , Hepacivirus/efeitos dos fármacos , Compostos de Espiro/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Antivirais/metabolismo , Linhagem Celular , Citidina/metabolismo , Citidina/farmacologia , Desoxicitidina Quinase/metabolismo , Humanos , Mitocôndrias/efeitos dos fármacos , Fenótipo , Fosforilação , Inibidores de Proteases/metabolismo , Inibidores de Proteases/farmacologia , RNA Viral/genética , RNA Viral/metabolismo , Compostos de Espiro/metabolismo , Proteínas não Estruturais Virais/genéticaRESUMO
TMC435 is a small-molecule inhibitor of the NS3/4A serine protease of hepatitis C virus (HCV) currently in phase 2 development. The in vitro resistance profile of TMC435 was characterized by selection experiments with HCV genotype 1 replicon cells and the genotype 2a JFH-1 system. In 80% (86/109) of the sequences from genotype 1 replicon cells analyzed, a mutation at NS3 residue D168 was observed, with changes to V or A being the most frequent. Mutations at NS3 positions 43, 80, 155, and 156, alone or in combination, were also identified. A transient replicon assay confirmed the relevance of these positions for TMC435 inhibitory activity. The change in the 50% effective concentrations (EC(50)s) observed for replicons with mutations at position 168 ranged from <10-fold for those with the D168G or D168N mutation to approximately 2,000-fold for those with the D168V or D168I mutation, compared to the EC(50) for the wild type. Of the positions identified, mutations at residue Q80 had the least impact on the activity of TMC435 (<10-fold change in EC(50)s), while greater effects were observed for some replicons with mutations at positions 43, 155, and 156. TMC435 remained active against replicons with the specific mutations observed after in vitro or in vivo exposure to telaprevir or boceprevir, including most replicons with changes at positions 36, 54, and 170 (<3-fold change in EC(50)s). Replicons carrying mutations affecting the activity of TMC435 remained fully susceptible to alpha interferon and NS5A and NS5B inhibitors. Finally, combinations of TMC435 with alpha interferon and NS5B polymerase inhibitors prevented the formation of drug-resistant replicon colonies.
Assuntos
Hepacivirus/efeitos dos fármacos , Hepatite C/tratamento farmacológico , Compostos Heterocíclicos com 3 Anéis/farmacologia , Inibidores de Proteases/farmacologia , Sulfonamidas/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Antivirais/farmacologia , Linhagem Celular , Farmacorresistência Viral/efeitos dos fármacos , Farmacorresistência Viral/genética , Sinergismo Farmacológico , Genótipo , Hepacivirus/enzimologia , Hepacivirus/genética , Hepatite C/virologia , Humanos , Técnicas In Vitro , Interferon-alfa/farmacologia , Mutagênese , Simeprevir , Proteínas não Estruturais Virais/genética , Replicação Viral/efeitos dos fármacosAssuntos
Inibidores Enzimáticos/farmacocinética , Hepacivirus/metabolismo , Proteínas não Estruturais Virais/antagonistas & inibidores , Administração Oral , Animais , Sítios de Ligação , Cristalografia por Raios X , Ciclização , Cães , Inibidores Enzimáticos/química , Indóis/química , Masculino , Estrutura Terciária de Proteína , Ratos , Ratos Sprague-Dawley , Proteínas não Estruturais Virais/metabolismoRESUMO
Structure-based macrocyclization of a 6-carboxylic acid indole chemotype has yielded potent and selective finger-loop inhibitors of the hepatitis C virus (HCV) NS5B polymerase. Lead optimization in conjunction with in vivo evaluation in rats identified several compounds showing (i) nanomolar potency in HCV replicon cells, (ii) limited toxicity and off-target activities, and (iii) encouraging preclinical pharmacokinetic profiles characterized by high liver distribution. This effort culminated in the identification of TMC647055 (10a), a nonzwitterionic 17-membered-ring macrocycle characterized by high affinity, long polymerase residence time, and broad genotypic coverage. In vitro results of the combination of 10a with the HCV protease inhibitor TMC435 (simeprevir) supported an evaluation of this combination in patients with regard to virus suppression and resistance emergence. In a phase 1b trial with HCV genotype 1-infected patients, 10a was considered to be safe and well-tolerated and demonstrated potent antiviral activity, which was further enhanced in a combination study with TMC435.
Assuntos
Antivirais/farmacologia , Inibidores Enzimáticos/farmacologia , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Sulfonamidas/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Animais , Antivirais/química , Antivirais/farmacocinética , Cristalografia por Raios X , Descoberta de Drogas , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacocinética , Compostos Heterocíclicos de 4 ou mais Anéis/química , Compostos Heterocíclicos de 4 ou mais Anéis/farmacocinética , Humanos , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Modelos Moleculares , Ratos , Relação Estrutura-Atividade , Sulfonamidas/química , Sulfonamidas/farmacocinéticaRESUMO
TMC125 is a potent new investigational nonnucleoside reverse transcriptase inhibitor (NNRTI) that is active against human immunodeficiency virus type 1 (HIV-1) with resistance to currently licensed NNRTIs. Sequential passage experiments with both wild-type virus and NNRTI-resistant virus were performed to identify mutations selected by TMC125 in vitro. In addition to "classic" selection experiments at a low multiplicity of infection (MOI) with increasing concentrations of inhibitors, experiments at a high MOI with fixed concentrations of inhibitors were performed to ensure a standardized comparison between TMC125 and current NNRTIs. Both low- and high-MOI experiments demonstrated that the development of resistance to TMC125 required multiple mutations which frequently conferred cross-resistance to efavirenz and nevirapine. In high-MOI experiments, 1 muM TMC125 completely inhibited the breakthrough of resistant virus from wild-type and NNRTI-resistant HIV-1, in contrast to efavirenz and nevirapine. Furthermore, breakthrough of virus from site-directed mutant (SDM) SDM-K103N/Y181C occurred at the same time or later with TMC125 as breakthrough from wild-type HIV-1 with efavirenz or nevirapine. The selection experiments identified mutations selected by TMC125 that included known NNRTI-associated mutations L100I, Y181C, G190E, M230L, and Y318F and the novel mutations V179I and V179F. Testing the antiviral activity of TMC125 against a panel of SDMs indicated that the impact of these individual mutations on resistance was highly dependent upon the presence and identity of coexisting mutations. These results demonstrate that TMC125 has a unique profile of activity against NNRTI-resistant virus and possesses a high genetic barrier to the development of resistance in vitro.