RESUMO
The most recent Ebola virus outbreak in West Africa, which was unprecedented in the number of cases and fatalities, geographic distribution, and number of nations affected, highlights the need for safe, effective, and readily available antiviral agents for treatment and prevention of acute Ebola virus (EBOV) disease (EVD) or sequelae. No antiviral therapeutics have yet received regulatory approval or demonstrated clinical efficacy. Here we report the discovery of a novel small molecule GS-5734, a monophosphoramidate prodrug of an adenosine analogue, with antiviral activity against EBOV. GS-5734 exhibits antiviral activity against multiple variants of EBOV and other filoviruses in cell-based assays. The pharmacologically active nucleoside triphosphate (NTP) is efficiently formed in multiple human cell types incubated with GS-5734 in vitro, and the NTP acts as an alternative substrate and RNA-chain terminator in primer-extension assays using a surrogate respiratory syncytial virus RNA polymerase. Intravenous administration of GS-5734 to nonhuman primates resulted in persistent NTP levels in peripheral blood mononuclear cells (half-life, 14 h) and distribution to sanctuary sites for viral replication including testes, eyes, and brain. In a rhesus monkey model of EVD, once-daily intravenous administration of 10 mg kg(-1) GS-5734 for 12 days resulted in profound suppression of EBOV replication and protected 100% of EBOV-infected animals against lethal disease, ameliorating clinical disease signs and pathophysiological markers, even when treatments were initiated three days after virus exposure when systemic viral RNA was detected in two out of six treated animals. These results show the first substantive post-exposure protection by a small-molecule antiviral compound against EBOV in nonhuman primates. The broad-spectrum antiviral activity of GS-5734 in vitro against other pathogenic RNA viruses, including filoviruses, arenaviruses, and coronaviruses, suggests the potential for wider medical use. GS-5734 is amenable to large-scale manufacturing, and clinical studies investigating the drug safety and pharmacokinetics are ongoing.
Assuntos
Alanina/análogos & derivados , Antivirais/uso terapêutico , Doença pelo Vírus Ebola/tratamento farmacológico , Macaca mulatta/virologia , Ribonucleotídeos/uso terapêutico , Monofosfato de Adenosina/análogos & derivados , Alanina/farmacocinética , Alanina/farmacologia , Alanina/uso terapêutico , Sequência de Aminoácidos , Animais , Antivirais/farmacocinética , Antivirais/farmacologia , Linhagem Celular Tumoral , Ebolavirus/efeitos dos fármacos , Feminino , Células HeLa , Doença pelo Vírus Ebola/prevenção & controle , Humanos , Masculino , Dados de Sequência Molecular , Especificidade de Órgãos , Pró-Fármacos/farmacocinética , Pró-Fármacos/farmacologia , Pró-Fármacos/uso terapêutico , Ribonucleotídeos/farmacocinética , Ribonucleotídeos/farmacologiaRESUMO
A series of 2'-fluorinated C-nucleosides were prepared and tested for anti-HCV activity. Among them, the triphosphate of 2'-fluoro-2'-C-methyl adenosine C-nucleoside (15) was a potent and selective inhibitor of the NS5B polymerase and maintained activity against the S282T resistance mutant. A number of phosphoramidate prodrugs were then prepared and evaluated leading to the identification of the 1-aminocyclobutane-1-carboxylic acid isopropyl ester variant (53) with favorable pharmacokinetic properties including efficient liver delivery in animals.
Assuntos
Antivirais/química , Antivirais/farmacologia , Hepacivirus/efeitos dos fármacos , Nucleosídeos/química , Nucleosídeos/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Amidas/química , Amidas/farmacocinética , Amidas/farmacologia , Animais , Antivirais/farmacocinética , Células CACO-2 , Linhagem Celular , Cricetinae , Descoberta de Drogas , Farmacorresistência Viral , Halogenação , Hepacivirus/genética , Hepacivirus/fisiologia , Hepatite C/tratamento farmacológico , Humanos , Metilação , Simulação de Acoplamento Molecular , Nucleosídeos/farmacocinética , Ácidos Fosfóricos/química , Ácidos Fosfóricos/farmacocinética , Ácidos Fosfóricos/farmacologia , Mutação Puntual , Pró-Fármacos/química , Pró-Fármacos/farmacocinética , Pró-Fármacos/farmacologia , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo , Replicação Viral/efeitos dos fármacosRESUMO
Novel 4'-substituted ß-d-2'-deoxy-2'-α-fluoro (2'd2'F) nucleoside inhibitors of respiratory syncytial virus (RSV) are reported. The introduction of 4'-substitution onto 2'd2'F nucleoside analogs resulted in compounds demonstrating potent cell based RSV inhibition, improved inhibition of the RSV polymerase by the nucleoside triphosphate metabolites, and enhanced selectivity over incorporation by mitochondrial RNA and DNA polymerases. Selectivity over the mitochondrial polymerases was found to be extremely sensitive to the specific 4'-substitution and not readily predictable. Combining the most potent and selective 4'-groups from N-nucleoside analogs onto a 2'd2'F C-nucleoside analog resulted in the identification of ß-D-2'-deoxy-2'-α-fluoro-4'-α-cyano-5-aza-7,9-dideaza adenosine as a promising nucleoside lead for RSV.
Assuntos
Adenosina/química , Antivirais/química , DNA Polimerase Dirigida por DNA/química , Inibidores da Síntese de Ácido Nucleico/química , RNA Polimerase Dependente de RNA/antagonistas & inibidores , RNA/química , Vírus Sinciciais Respiratórios/enzimologia , Vírus Sinciciais Respiratórios/fisiologia , Adenosina/síntese química , Adenosina/farmacologia , Antivirais/síntese química , Antivirais/farmacologia , Compostos Aza/química , DNA Polimerase Dirigida por DNA/metabolismo , Avaliação Pré-Clínica de Medicamentos , Inibidores da Síntese de Ácido Nucleico/síntese química , Inibidores da Síntese de Ácido Nucleico/farmacologia , RNA/metabolismo , RNA Mitocondrial , RNA Polimerase Dependente de RNA/metabolismo , Vírus Sinciciais Respiratórios/efeitos dos fármacos , Relação Estrutura-Atividade , Replicação Viral/efeitos dos fármacosRESUMO
Acute respiratory viral infections, such as pneumovirus and respiratory picornavirus infections, exacerbate disease in COPD and asthma patients. A research program targeting respiratory syncytial virus (RSV) led to the discovery of GS-7682 (1), a novel phosphoramidate prodrug of a 4'-CN-4-aza-7,9-dideazaadenosine C-nucleoside GS-646089 (2) with broad antiviral activity against RSV (EC50 = 3-46 nM), human metapneumovirus (EC50 = 210 nM), human rhinovirus (EC50 = 54-61 nM), and enterovirus (EC50 = 83-90 nM). Prodrug optimization for cellular potency and lung cell metabolism identified 5'-methyl [(S)-hydroxy(phenoxy)phosphoryl]-l-alaninate in combination with 2',3'-diisobutyrate promoieties as being optimal for high levels of intracellular triphosphate formation in vitro and in vivo. 1 demonstrated significant reductions of viral loads in the lower respiratory tract of RSV-infected African green monkeys when administered once daily via intratracheal nebulized aerosol. Together, these findings support additional evaluation of 1 and its analogues as potential therapeutics for pneumo- and picornaviruses.
Assuntos
Antivirais , Picornaviridae , Pró-Fármacos , Infecções por Vírus Respiratório Sincicial , Animais , Antivirais/farmacologia , Antivirais/química , Pró-Fármacos/farmacologia , Pró-Fármacos/química , Pró-Fármacos/síntese química , Chlorocebus aethiops , Infecções por Vírus Respiratório Sincicial/tratamento farmacológico , Infecções por Vírus Respiratório Sincicial/virologia , Humanos , Picornaviridae/efeitos dos fármacos , Relação Estrutura-Atividade , Vírus Sinciciais Respiratórios/efeitos dos fármacos , Descoberta de Drogas , Nucleosídeos/química , Nucleosídeos/farmacologia , Infecções por Picornaviridae/tratamento farmacológico , Infecções por Picornaviridae/virologiaRESUMO
GS-9669 is a highly optimized thumb site II nonnucleoside inhibitor of the hepatitis C virus (HCV) RNA polymerase, with a binding affinity of 1.35 nM for the genotype (GT) 1b protein. It is a selective inhibitor of HCV RNA replication, with a mean 50% effective concentration (EC(50)) of ≤ 11 nM in genotype 1 and 5 replicon assays, but lacks useful activity against genotypes 2 to 4. The M423T mutation is readily generated clinically upon monotherapy with the thumb site II inhibitors filibuvir and lomibuvir, and it is notable that GS-9669 exhibited only a 3-fold loss in potency against this variant in the genotype 1b replicon. Rather than M423T, resistance predominantly tracks to residues R422K and L419M and residue I482L in GT 1b and 1a replicons, respectively. GS-9669 exhibited at least additive activity in combination with agents encompassing four other direct modes of action (NS3 protease, NS5A, NS5B via an alternative allosteric binding site, and NS5B nucleotide) as well as with alpha interferon or ribavirin in replicon assays. It exhibited high metabolic stability in in vitro human liver microsomal assays, which, in combination with its pharmacokinetic profiles in rat, dog, and two monkey species, is predictive of good human pharmacokinetics. GS-9669 is well suited for combination with other orally active, direct-acting antiviral agents in the treatment of genotype 1 chronic HCV infection. (This study has been registered at ClinicalTrials.gov under registration number NCT01431898.).
Assuntos
Antivirais/farmacologia , Furanos/farmacologia , Hepacivirus/efeitos dos fármacos , RNA Polimerase Dependente de RNA/antagonistas & inibidores , Tiofenos/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Animais , Antivirais/química , Linhagem Celular Tumoral , Cães , Farmacorresistência Viral , Furanos/química , Humanos , Interferon-alfa/farmacologia , Masculino , Mutação , Polimorfismo de Nucleotídeo Único , Pironas/farmacologia , Ratos , Ratos Sprague-Dawley , Ribavirina/farmacologia , Tiofenos/química , Triazóis/farmacologiaRESUMO
A novel, potent, and orally bioavailable class of product-like inhibitors of the HCV NS3 protease was discovered by constraining the P2-P3 amide bond and the P3 hydrocarbon substituent to the protease-bound conformation. This preorganization was accomplished by incorporation of the P2-P3 amide into a six-membered ring attached to the P2-proline 5-position. Isothermal calorimetric characterization of the role of hydrocarbon substitution of this six-membered ring, upon binding the HCV NS3 protease, was found to be exclusively entropic in nature. The synthesis, preliminary SAR and pharmacokinetic profiling of this compact, indolizidinone-derived scaffold are described.
Assuntos
Inibidores Enzimáticos/farmacologia , Indolizinas/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Administração Oral , Disponibilidade Biológica , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/administração & dosagem , Inibidores Enzimáticos/química , Indolizinas/administração & dosagem , Indolizinas/química , Modelos Moleculares , Estrutura Molecular , Relação Estrutura-Atividade , Proteínas não Estruturais Virais/metabolismoRESUMO
The discovery of GS-9451 is reported. Modification of the P3 cap and P2 quinoline with a series of solubilizing groups led to the identification of potent HCV NS3 protease inhibitors with greatly improved pharmacokinetic properties in rats, dogs and monkeys.
Assuntos
Antivirais/síntese química , Ácidos Carboxílicos/síntese química , Hepacivirus/efeitos dos fármacos , Quinolinas/síntese química , Inibidores de Serina Proteinase/síntese química , Proteínas não Estruturais Virais/antagonistas & inibidores , Animais , Antivirais/farmacocinética , Antivirais/farmacologia , Disponibilidade Biológica , Células CACO-2 , Ácidos Carboxílicos/farmacocinética , Ácidos Carboxílicos/farmacologia , Cristalografia por Raios X , Cães , Hepacivirus/química , Hepacivirus/enzimologia , Hepatite C/tratamento farmacológico , Hepatite C/virologia , Humanos , Macaca fascicularis , Modelos Moleculares , Quinolinas/farmacocinética , Quinolinas/farmacologia , Ratos , Inibidores de Serina Proteinase/farmacocinética , Inibidores de Serina Proteinase/farmacologia , Relação Estrutura-Atividade , Proteínas não Estruturais Virais/químicaRESUMO
A potent and novel class of phosphinic acid derived product-like inhibitors of the HCV NS3/4A protease was discovered previously. Modification of the phosphinic acid and quinoline heterocycle led to GS-9256 with potent cell-based activity and favorable pharmacokinetic parameters. Based on these attributes, GS-9256 was advanced to human clinical trial as a treatment for chronic infection with genotype 1 HCV.
Assuntos
Proteínas de Transporte/antagonistas & inibidores , Descoberta de Drogas , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Hepacivirus/efeitos dos fármacos , Peptídeos Cíclicos/química , Ácidos Fosfínicos/química , Proteínas não Estruturais Virais/antagonistas & inibidores , Animais , Cães , Inibidores Enzimáticos/síntese química , Hepacivirus/enzimologia , Humanos , Concentração Inibidora 50 , Peptídeos e Proteínas de Sinalização Intracelular , Estrutura Molecular , Peptídeos Cíclicos/síntese química , Peptídeos Cíclicos/farmacologia , Ácidos Fosfínicos/síntese química , Ácidos Fosfínicos/farmacologia , SuínosRESUMO
A potent and novel class of product-like inhibitors of the HCV NS3 protease was discovered by employing a phosphinic acid as a carboxylate isostere. The replicon activity and pharmacokinetic profile of this series of compounds was optimized by exploring the substitution of the phosphinic acid, as well as conformationally constraining these compounds through macrocyclization. The syntheses and preliminary biological evaluation of these phosphinic acids is described.
Assuntos
Hepacivirus/efeitos dos fármacos , Hepacivirus/enzimologia , Ácidos Fosfínicos/síntese química , Ácidos Fosfínicos/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Administração Oral , Ciclização , Humanos , Concentração Inibidora 50 , Estrutura Molecular , Ácidos Fosfínicos/químicaRESUMO
A novel class of phosphonate derivatives was designed to mimic the interaction of product-like carboxylate based inhibitors of HCV NS3 protease. A phosphonic acid (compound 2) was demonstrated to be a potent HCV NS3 protease inhibitor, and a potential candidate for treating HCV infection. The syntheses and preliminary biological evaluation of this phosphonate class of inhibitor are described.
Assuntos
Antivirais/síntese química , Hepacivirus/efeitos dos fármacos , Organofosfonatos/síntese química , Inibidores de Serina Proteinase/síntese química , Proteínas não Estruturais Virais/antagonistas & inibidores , Animais , Antivirais/química , Antivirais/farmacocinética , Linhagem Celular Tumoral , Simulação por Computador , Cães , Descoberta de Drogas , Humanos , Organofosfonatos/química , Organofosfonatos/farmacocinética , Inibidores de Serina Proteinase/química , Inibidores de Serina Proteinase/farmacocinética , Proteínas não Estruturais Virais/metabolismoRESUMO
The recent Ebola virus (EBOV) outbreak in West Africa was the largest recorded in history with over 28,000 cases, resulting in >11,000 deaths including >500 healthcare workers. A focused screening and lead optimization effort identified 4b (GS-5734) with anti-EBOV EC50 = 86 nM in macrophages as the clinical candidate. Structure activity relationships established that the 1'-CN group and C-linked nucleobase were critical for optimal anti-EBOV potency and selectivity against host polymerases. A robust diastereoselective synthesis provided sufficient quantities of 4b to enable preclinical efficacy in a non-human-primate EBOV challenge model. Once-daily 10 mg/kg iv treatment on days 3-14 postinfection had a significant effect on viremia and mortality, resulting in 100% survival of infected treated animals [ Nature 2016 , 531 , 381 - 385 ]. A phase 2 study (PREVAIL IV) is currently enrolling and will evaluate the effect of 4b on viral shedding from sanctuary sites in EBOV survivors.
Assuntos
Alanina/análogos & derivados , Amidas/química , Doença pelo Vírus Ebola/tratamento farmacológico , Ácidos Fosfóricos/química , Pró-Fármacos/química , Pró-Fármacos/farmacologia , Ribonucleotídeos/química , Viroses/tratamento farmacológico , Monofosfato de Adenosina/análogos & derivados , Alanina/química , Linhagem Celular , Descoberta de Drogas , Humanos , Testes de Sensibilidade Microbiana , Pró-Fármacos/síntese química , Relação Estrutura-AtividadeRESUMO
Hepatitis C virus (HCV) infection presents an unmet medical need requiring more effective treatment options. Nucleoside inhibitors (NI) of HCV polymerase (NS5B) have demonstrated pan-genotypic activity and durable antiviral response in the clinic, and they are likely to become a key component of future treatment regimens. NI candidates that have entered clinical development thus far have all been N-nucleoside derivatives. Herein, we report the discovery of a C-nucleoside class of NS5B inhibitors. Exploration of adenosine analogs in this class identified 1'-cyano-2'-C-methyl 4-aza-7,9-dideaza adenosine as a potent and selective inhibitor of NS5B. A monophosphate prodrug approach afforded a series of compounds showing submicromolar activity in HCV replicon assays. Further pharmacokinetic optimization for sufficient oral absorption and liver triphosphate loading led to identification of a clinical development candidate GS-6620. In a phase I clinical study, the potential for potent activity was demonstrated but with high intra- and interpatient pharmacokinetic and pharmacodynamic variability.
Assuntos
Antivirais/uso terapêutico , Inibidores Enzimáticos/uso terapêutico , Hepacivirus/enzimologia , Hepatite C/tratamento farmacológico , Nucleosídeos/farmacologia , Compostos Organofosforados/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Animais , Antivirais/farmacocinética , Antivirais/farmacologia , Cães , Inibidores Enzimáticos/farmacocinética , Inibidores Enzimáticos/farmacologia , Hepatite C/enzimologia , Hepatite C/virologia , Humanos , Nucleosídeos/química , Compostos Organofosforados/química , Ratos , Carga ViralRESUMO
Investigation of thiophene-2-carboxylic acid HCV NS5B site II inhibitors, guided by measurement of cell culture medium binding, revealed the structure-activity relationships for intrinsic cellular potency. The pharmacokinetic profile was enhanced through incorporation of heterocyclic ethers on the N-alkyl substituent. Hydroxyl groups were incorporated to modulate protein binding. Intrinsic potency was further improved through enantiospecific introduction of an olefin in the N-acyl motif, resulting in the discovery of the phase 2 clinical candidate GS-9669. The unexpected activity of this compound against the clinically relevant NS5B M423T mutant, relative to the wild type, was shown to arise from both the N-alkyl substituent and the N-acyl group.
Assuntos
Antivirais/farmacologia , Inibidores Enzimáticos/farmacologia , Furanos/farmacologia , Tiofenos/farmacologia , Proteínas não Estruturais Virais/farmacologia , Antivirais/química , Inibidores Enzimáticos/química , Furanos/química , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Tiofenos/química , Proteínas não Estruturais Virais/químicaRESUMO
A novel series of HCV replication inhibitors based on a pyrido[3,2-d]pyrimidine core were optimized for pharmacokinetics (PK) in rats. Several associations between physicochemical properties and PK were identified and exploited to guide the design of compounds. In addition, a simple new metric that may aid in the prediction of bioavailability for compounds with higher polar surface area is described (3*HBD-cLogP).