RESUMO
IMPORTANCE: Chronic hepatitis B is the most important cause of liver cancer worldwide and affects more than 290 million people. Current treatments are mostly suppressive and rarely lead to a cure. Therefore, there is a need for novel and curative drugs that target the host or the causative agent, hepatitis B virus itself. Capsid assembly modulators are an interesting class of antiviral molecules that may one day become part of curative treatment regimens for chronic hepatitis B. Here we explore the characteristics of a particularly interesting subclass of capsid assembly modulators. These so-called non-HAP CAM-As have intriguing properties in cell culture but also clear virus-infected cells from the mouse liver in a gradual and sustained way. We believe they represent a considerable improvement over previously reported molecules and may one day be part of curative treatment combinations for chronic hepatitis B.
Assuntos
Antivirais , Capsídeo , Vírus da Hepatite B , Hepatite B Crônica , Montagem de Vírus , Animais , Humanos , Camundongos , Antivirais/classificação , Antivirais/farmacologia , Antivirais/uso terapêutico , Capsídeo/química , Capsídeo/efeitos dos fármacos , Capsídeo/metabolismo , Proteínas do Capsídeo/química , Proteínas do Capsídeo/efeitos dos fármacos , Proteínas do Capsídeo/metabolismo , Células Cultivadas , Vírus da Hepatite B/química , Vírus da Hepatite B/efeitos dos fármacos , Vírus da Hepatite B/crescimento & desenvolvimento , Vírus da Hepatite B/metabolismo , Hepatite B Crônica/tratamento farmacológico , Hepatite B Crônica/virologia , Técnicas In Vitro , Montagem de Vírus/efeitos dos fármacos , Modelos Animais de DoençasRESUMO
BACKGROUND AND AIMS: Effective therapies leading to a functional cure for chronic hepatitis B are still lacking. Class A capsid assembly modulators (CAM-As) are an attractive modality to address this unmet medical need. CAM-As induce aggregation of the HBV core protein (HBc) and lead to sustained HBsAg reductions in a chronic hepatitis B mouse model. Here, we investigate the underlying mechanism of action for CAM-A compound RG7907. APPROACH AND RESULTS: RG7907 induced extensive HBc aggregation in vitro , in hepatoma cells, and in primary hepatocytes. In the adeno-associated virus (AAV)-HBV mouse model, the RG7907 treatment led to a pronounced reduction in serum HBsAg and HBeAg, concomitant with clearance of HBsAg, HBc, and AAV-HBV episome from the liver. Transient increases in alanine transaminase, hepatocyte apoptosis, and proliferation markers were observed. These processes were confirmed by RNA sequencing, which also uncovered a role for interferon alpha and gamma signaling, including the interferon-stimulated gene 15 (ISG15) pathway. Finally, the in vitro observation of CAM-A-induced HBc-dependent cell death through apoptosis established the link of HBc aggregation to in vivo loss of infected hepatocytes. CONCLUSIONS: Our study unravels a previously unknown mechanism of action for CAM-As such as RG7907 in which HBc aggregation induces cell death, resulting in hepatocyte proliferation and loss of covalently closed circular DNA or its equivalent, possibly assisted by an induced innate immune response. This represents a promising approach to attain a functional cure for chronic hepatitis B.
Assuntos
Hepatite B Crônica , Hepatite B , Camundongos , Animais , Vírus da Hepatite B , Antígenos de Superfície da Hepatite B/metabolismo , Capsídeo/metabolismo , Hepatócitos/metabolismo , Interferon-alfa/farmacologia , Hepatite B/metabolismo , DNA Viral/genéticaRESUMO
There is an urgent need for antivirals targeting the SARS-CoV-2 virus to fight the current COVID-19 pandemic. The SARS-CoV-2 main protease (3CLpro) represents a promising target for antiviral therapy. The lack of selectivity for some of the reported 3CLpro inhibitors, specifically versus cathepsin L, raises potential safety and efficacy concerns. ALG-097111 potently inhibited SARS-CoV-2 3CLpro (IC50 = 7 nM) without affecting the activity of human cathepsin L (IC50 > 10 µM). When ALG-097111 was dosed in hamsters challenged with SARS-CoV-2, a robust and significant 3.5 log10 (RNA copies/mg) reduction of the viral RNA copies and 3.7 log10 (TCID50/mg) reduction in the infectious virus titers in the lungs was observed. These results provide the first in vivo validation for the SARS-CoV-2 3CLpro as a promising therapeutic target for selective small molecule inhibitors.
Assuntos
Amidas/farmacologia , Tratamento Farmacológico da COVID-19 , Proteases 3C de Coronavírus/antagonistas & inibidores , Inibidores de Cisteína Proteinase/farmacologia , Modelos Animais de Doenças , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/enzimologia , Amidas/farmacocinética , Animais , COVID-19/virologia , Catepsina L/antagonistas & inibidores , Linhagem Celular , Cricetinae , Inibidores de Cisteína Proteinase/farmacocinética , Feminino , Humanos , Concentração Inibidora 50 , Masculino , Mesocricetus/virologia , Reprodutibilidade dos Testes , SARS-CoV-2/crescimento & desenvolvimento , Serina Endopeptidases , Especificidade por Substrato , Replicação Viral/efeitos dos fármacosRESUMO
The 3-chymotrypsin-like cysteine protease (3CLpro) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is considered a major target for the discovery of direct antiviral agents. We previously reported the evaluation of SARS-CoV-2 3CLpro inhibitors in a novel self-assembled monolayer desorption ionization mass spectrometry (SAMDI-MS) enzymatic assay (Gurard-Levin et al., 2020). The assay was further improved by adding the rhinovirus HRV3C protease to the same well as the SARS-CoV-2 3CLpro enzyme. High substrate specificity for each enzyme allowed the proteases to be combined in a single assay reaction without interfering with their individual activities. This novel duplex assay was used to profile a diverse set of reference protease inhibitors. The protease inhibitors were grouped into three categories based on their relative potency against 3CLpro and HRV3C including those that are: equipotent against 3CLpro and HRV3C (GC376 and calpain inhibitor II), selective for 3CLpro (PF-00835231, calpain inhibitor XII, boceprevir), and selective for HRV3C (rupintrivir). Structural analysis showed that the combination of minimal interactions, conformational flexibility, and limited bulk allows GC376 and calpain inhibitor II to potently inhibit both enzymes. In contrast, bulkier compounds interacting more tightly with pockets P2, P3, and P4 due to optimization for a specific target display a more selective inhibition profile. Consistently, the most selective viral protease inhibitors were relatively weak inhibitors of human cathepsin L. Taken together, these results can guide the design of cysteine protease inhibitors that are either virus-specific or retain a broad antiviral spectrum against coronaviruses and rhinoviruses.
Assuntos
Antivirais/farmacologia , Proteases 3C de Coronavírus/antagonistas & inibidores , Inibidores de Proteases/farmacologia , Rhinovirus/efeitos dos fármacos , SARS-CoV-2/efeitos dos fármacos , Antivirais/química , Sítios de Ligação , Catepsina L/metabolismo , Descoberta de Drogas , Glicoproteínas/farmacologia , Humanos , Cinética , Modelos Moleculares , Inibidores de Proteases/química , Pirrolidinas/farmacologia , Ácidos SulfônicosRESUMO
Thyroid hormones are important modulators of metabolic activity in mammals and alter cholesterol and fatty acid levels through activation of the nuclear thyroid hormone receptor (THR). Currently, there are several THRß agonists in clinical trials for the treatment of non-alcoholic steatohepatitis (NASH) that have demonstrated the potential to reduce liver fat and restore liver function. In this study, we tested three THRß-agonism-based NASH treatment candidates, GC-1 (sobetirome), MGL-3196 (resmetirom), and VK2809, and compared their selectivity for THRß and their ability to modulate the expression of genes specific to cholesterol and fatty acid biosynthesis and metabolism in vitro using human hepatic cells and in vivo using a rat model. Treatment with GC-1 upregulated the transcription of CPT1A in the human hepatocyte-derived Huh-7 cell line with a dose-response comparable to that of the native THR ligand, triiodothyronine (T3). VK2809A (active parent of VK2809), MGL-3196, and VK2809 were approximately 30-fold, 1,000-fold, and 2,000-fold less potent than T3, respectively. Additionally, these relative potencies were confirmed by quantification of other direct gene targets of THR, namely, ANGPTL4 and DIO1. In primary human hepatocytes, potencies were conserved for every compound except for VK2809, which showed significantly increased potency that was comparable to that of its active counterpart, VK2809A. In high-fat diet fed rats, a single dose of T3 significantly reduced total cholesterol levels and concurrently increased liver Dio1 and Me1 RNA expression. MGL-3196 treatment resulted in concentration-dependent decreases in total and low-density lipoprotein cholesterol with corresponding increases in liver gene expression, but the compound was significantly less potent than T3. In conclusion, we have implemented a strategy to rank the efficacy of THRß agonists by quantifying changes in the transcription of genes that lead to metabolic alterations, an effect that is directly downstream of THR binding and activation.
Assuntos
Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Receptores beta dos Hormônios Tireóideos/agonistas , Transcrição Gênica/efeitos dos fármacos , Acetatos/farmacologia , Acetatos/uso terapêutico , Proteína 4 Semelhante a Angiopoietina/metabolismo , Animais , Linhagem Celular Tumoral , LDL-Colesterol/sangue , LDL-Colesterol/metabolismo , Dieta Hiperlipídica/efeitos adversos , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Hepatócitos , Humanos , Iodeto Peroxidase/genética , Iodeto Peroxidase/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Malato Desidrogenase/genética , Malato Desidrogenase/metabolismo , Masculino , Hepatopatia Gordurosa não Alcoólica/sangue , Hepatopatia Gordurosa não Alcoólica/etiologia , Hepatopatia Gordurosa não Alcoólica/patologia , Organofosfonatos/farmacologia , Organofosfonatos/uso terapêutico , Fenóis/farmacologia , Fenóis/uso terapêutico , Cultura Primária de Células , Piridazinas/farmacologia , Piridazinas/uso terapêutico , Ratos , Uracila/análogos & derivados , Uracila/farmacologia , Uracila/uso terapêuticoRESUMO
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of the COVID-19 pandemic that began in 2019. The coronavirus 3-chymotrypsin-like cysteine protease (3CLpro) controls replication and is therefore considered a major target for antiviral discovery. This study describes the evaluation of SARS-CoV-2 3CLpro inhibitors in a novel self-assembled monolayer desorption ionization mass spectrometry (SAMDI-MS) enzymatic assay. Compared with a traditional FRET readout, the label-free SAMDI-MS assay offers greater sensitivity and eliminates false positive inhibition from compound interference with the optical signal. The SAMDI-MS assay was optimized and validated with known inhibitors of coronavirus 3CLpro such as GC376 (IC50 = 0.060 µM), calpain inhibitors II and XII (IC50 ~20-25 µM). The FDA-approved drugs shikonin, disulfiram, and ebselen did not inhibit SARS-CoV-2 3CLpro activity in the SAMDI-MS assay under physiologically relevant reducing conditions. The three drugs did not directly inhibit human ß-coronavirus OC-43 or SARS-CoV-2 in vitro, but instead induced cell death. In conclusion, the SAMDI-MS 3CLpro assay, combined with antiviral and cytotoxic assessment, provides a robust platform to evaluate antiviral agents directed against SARS-CoV-2.
Assuntos
Antivirais/farmacologia , Betacoronavirus/efeitos dos fármacos , Betacoronavirus/enzimologia , Inibidores de Cisteína Proteinase/química , Inibidores de Cisteína Proteinase/farmacologia , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Proteínas não Estruturais Virais/antagonistas & inibidores , COVID-19 , Proteases 3C de Coronavírus , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/virologia , Cisteína Endopeptidases/química , Cisteína Endopeptidases/metabolismo , Glicoproteínas/farmacologia , Células HeLa , Humanos , Pandemias , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/virologia , SARS-CoV-2 , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/metabolismo , Tratamento Farmacológico da COVID-19RESUMO
Dengue is the most important mosquito-transmitted viral disease and a major global health concern. Over the last decade, dengue virus (DENV) drug discovery and development has intensified, however, this has not resulted in approved DENV-specific antiviral treatments yet. DENV and hepatitis C virus (HCV) belong to the same Flaviviridae family and, in contrast to DENV, antiviral treatments for HCV have been licensed. Therefore, applying the knowledge gained on anti-HCV drugs may foster the discovery and development of dengue antiviral drugs. Here, we screened a library of compounds with established anti-HCV activity in a DENV-2 sub-genomic replicon inhibition assay and selected compounds with single-digit micromolar activity. These compounds were advanced into a hit-to-lead medicinal chemistry program resulting in lead compound JNJ-1A, which inhibited the DENV-2 sub-genomic replicon at 0.7 µM, in the absence of cytotoxicity. In addition, JNJ-1A showed equipotent antiviral activity against DENV serotypes 1, 2, and 4. In vitro resistance selection experiments with JNJ-1A induced mutation T108I in non-structural protein 4B (NS4B), pointing towards a mechanism of action linked to this protein. Collectively, we described the discovery and characterization of a novel DENV inhibitor potentially targeting NS4B.
Assuntos
Antivirais/farmacologia , Vírus da Dengue/efeitos dos fármacos , Farmacorresistência Viral/genética , Proteínas não Estruturais Virais/genética , Replicação Viral/efeitos dos fármacos , Animais , Antivirais/química , Antivirais/farmacocinética , Antivirais/toxicidade , Linhagem Celular Tumoral , Chlorocebus aethiops , Dengue , Vírus da Dengue/genética , Vírus da Dengue/fisiologia , Descoberta de Drogas , Farmacorresistência Viral/efeitos dos fármacos , Hepacivirus/genética , Humanos , Mutação , RNA Viral/genética , Replicon/efeitos dos fármacos , Análise de Sequência de RNA , Bibliotecas de Moléculas Pequenas , Células VeroRESUMO
Respiratory syncytial virus is a major cause of acute lower respiratory tract infection in young children, immunocompromised adults, and the elderly. Intervention with small-molecule antivirals specific for respiratory syncytial virus presents an important therapeutic opportunity, but no such compounds are approved today. Here we report the structure of JNJ-53718678 bound to respiratory syncytial virus fusion (F) protein in its prefusion conformation, and we show that the potent nanomolar activity of JNJ-53718678, as well as the preliminary structure-activity relationship and the pharmaceutical optimization strategy of the series, are consistent with the binding mode of JNJ-53718678 and other respiratory syncytial virus fusion inhibitors. Oral treatment of neonatal lambs with JNJ-53718678, or with an equally active close analog, efficiently inhibits established acute lower respiratory tract infection in the animals, even when treatment is delayed until external signs of respiratory syncytial virus illness have become visible. Together, these data suggest that JNJ-53718678 is a promising candidate for further development as a potential therapeutic in patients at risk to develop respiratory syncytial virus acute lower respiratory tract infection.Respiratory syncytial virus causes lung infections in children, immunocompromised adults, and in the elderly. Here the authors show that a chemical inhibitor to a viral fusion protein is effective in reducing viral titre and ameliorating infection in rodents and neonatal lambs.
Assuntos
Imidazolidinas/metabolismo , Indóis/metabolismo , Vírus Sincicial Respiratório Humano/metabolismo , Inibidores de Proteínas Virais de Fusão/metabolismo , Proteínas Virais de Fusão/metabolismo , Animais , Animais Recém-Nascidos , Linhagem Celular Tumoral , Chlorocebus aethiops , Células Epiteliais , Humanos , Imidazolidinas/farmacologia , Imidazolidinas/uso terapêutico , Indóis/farmacologia , Indóis/uso terapêutico , Estrutura Molecular , Pneumonia Viral/tratamento farmacológico , Ratos , Mucosa Respiratória/citologia , Infecções por Vírus Respiratório Sincicial/tratamento farmacológico , Vírus Sincicial Respiratório Humano/efeitos dos fármacos , Vírus Sinciciais Respiratórios/efeitos dos fármacos , Vírus Sinciciais Respiratórios/metabolismo , Ovinos , Relação Estrutura-Atividade , Células Vero , Inibidores de Proteínas Virais de Fusão/farmacologia , Inibidores de Proteínas Virais de Fusão/uso terapêuticoRESUMO
Hepatitis C virus is a blood-borne infection and the leading cause of chronic liver disease (including cirrhosis and cancer) and liver transplantation. Since the identification of HCV in 1989, there has been an extensive effort to identify and improve treatment options. An important milestone was reached in 2011 with the approval of the first-generation HCV NS3/4A protease inhibitors. However, new therapies are needed to improve cure rates, shorten treatment duration, and improve tolerability. Here we summarize the extensive medicinal chemistry effort to develop novel P2 cyclopentane macrocyclic inhibitors guided by HCV NS3 protease assays, the cellular replicon system, structure-based design, and a panel of DMPK assays. The selection of compound 29 (simeprevir, TMC435) as clinical candidate was based on its excellent biological, PK, and safety pharmacology profile. Compound 29 has recently been approved for treatment of chronic HCV infection in combination with pegylated interferon-α and ribavirin in Japan, Canada, and USA.
Assuntos
Antivirais/química , Descoberta de Drogas , Compostos Heterocíclicos com 3 Anéis/química , Sulfonamidas/química , Proteínas não Estruturais Virais/antagonistas & inibidores , Antivirais/farmacologia , Compostos Heterocíclicos com 3 Anéis/farmacologia , Simeprevir , Sulfonamidas/farmacologiaRESUMO
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
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
The exogenous control of hepatitis C virus (HCV) replication can be mediated through the inhibition of the RNA-dependent RNA polymerase (RdRp) activity of NS5B. Small-molecule inhibitors of NS5B include nucleoside and nonnucleoside analogs. Here, we report the discovery of a novel class of HCV polymerase nonnucleoside inhibitors, 1,5-benzodiazepines (1,5-BZDs), identified by high-throughput screening of a library of small molecules. A fluorescence-quenching assay and X-ray crystallography revealed that 1,5-BZD 4a bound stereospecifically to NS5B next to the catalytic site. When introduced into replicons, mutations known to confer resistance against chemotypes that bind at this site were detrimental to inhibition by 1,5-BZD 7a. Using a panel of enzyme isolates that covered genotypes 1 to 6, we showed that compound 4a inhibited genotype 1 only. In mechanistic studies, 4a was found to inhibit the RdRp activity of NS5B noncompetitively with GTP and to inhibit the formation of the first phosphodiester bond during the polymerization cycle. The specificity for the HCV target was evaluated by profiling the 1,5-BZDs against other viral and human polymerases, as well as BZD receptors.
Assuntos
Benzodiazepinas/farmacologia , Inibidores Enzimáticos/farmacologia , Hepacivirus/efeitos dos fármacos , RNA Polimerase Dependente de RNA/antagonistas & inibidores , Proteínas não Estruturais Virais/antagonistas & inibidores , Antivirais/metabolismo , Antivirais/farmacologia , Benzodiazepinas/química , Benzodiazepinas/metabolismo , Sítios de Ligação , Linhagem Celular Tumoral , Cristalografia por Raios X , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Genótipo , Hepacivirus/enzimologia , Hepacivirus/genética , Hepacivirus/fisiologia , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Mutação , RNA Polimerase Dependente de RNA/química , RNA Polimerase Dependente de RNA/genética , RNA Polimerase Dependente de RNA/metabolismo , Relação Estrutura-Atividade , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo , Replicação Viral/efeitos dos fármacosRESUMO
A series of 8-substituted pyrazolo[1,5-a]-1,3,5-triazines were considered as a bioisosteric replacement for the 9-substituted adenine derivatives resulting in the discovery of 8-(2-methoxybenzyl)-4-(N-methylamino)-2-n-propylpyrazolo[1,5-a]-1,3,5-triazine (14d) and 2-trifluoromethyl-8-(2-methoxybenzyl)-4-(N-methylamino)pyrazolo[1,5-a]-1,3,5-triazine (14e) as a new structural class of potent phosphodiesterase type 4 inhibitors (IC(50)=13 nM and 11 nM, respectively) with high isoenzyme selectivity. An original tandem of reactions involving a palladium-mediated cross-coupling reaction (PMCCR) of the readily available 8-iodo-2-methyl-4-(N-methyl-N-phenylamino)pyrazolo[1,5-a]-1,3,5-triazine (11a) and arylboronic acids or alkynes followed by the displacement of the N-methyl-N-phenylamino group constitute the key steps in a novel synthetic approach developed herein. The treatment of 11a-c with n-BuLi and selected aldehydes represents an interesting alternative to the PMCCR for the synthesis of benzylic derivatives 14a-i. Preliminary biological testing has shown that compounds 14d and 14e strongly inhibit LPS-induced TNFalpha release from human mononuclear cells from healthy subjects. These two compounds were selected for further biological evaluation.
Assuntos
Adenina/química , Inibidores Enzimáticos/farmacologia , Hidrocarbonetos Fluorados/farmacologia , Metilaminas/farmacologia , Inibidores da Fosfodiesterase 4 , Triazinas/farmacologia , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/metabolismo , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Humanos , Hidrocarbonetos Fluorados/síntese química , Hidrocarbonetos Fluorados/química , Lipopolissacarídeos/farmacologia , Metilaminas/síntese química , Metilaminas/química , Modelos Moleculares , Estrutura Molecular , Paládio/química , Relação Estrutura-Atividade , Triazinas/síntese química , Triazinas/química , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Screening of a focused library of TGF beta kinase inhibitors in the cellular HCV replicon model with luciferase read out yielded a number of low micromolar HCV inhibitors. Medicinal chemistry driven optimization resulted in the discovery of 4-[2-(5-bromo-2-fluoro-phenyl)pteridin-4-ylamino]-N-[3-(2- oxopyrrolidin-1-yl)propyl]nicotinamide 36 with a replicon EC(50) of 64nM, associated with a selective kinase inhibitory profile for human JNK kinases 2 and 3 as well as VEGFR-1, 2, and 3 kinases. Moreover, 36 showed an advantageous PK profile in mice. Experiments performed using different replicon constructs suggest that this series of kinase inhibitors might mediate their effect through the HCV non-structural protein 5A (NS5A).
Assuntos
Antivirais/síntese química , Antivirais/farmacologia , Química Farmacêutica/métodos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Hepacivirus/metabolismo , MAP Quinase Quinase 4/antagonistas & inibidores , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Proteínas não Estruturais Virais/farmacologia , Animais , Área Sob a Curva , Linhagem Celular , Desenho de Fármacos , Estudos de Avaliação como Assunto , Humanos , Concentração Inibidora 50 , Masculino , Camundongos , Modelos Químicos , Conformação Molecular , Proteínas não Estruturais Virais/químicaRESUMO
The vitronectin receptor alpha(v)beta(3) has been identified as a promising potential target for the treatment of osteoporosis, diabetic retinopathy and cancer. We have recently reported 5-substituted indoles 3-[5-[2-(5,6,7,8-tetrahydro[1,8]naphthyridin-2-yl)ethoxy]indol-1-yl]-3-(3-pyridyl)propionic acid 3 and 3-[5-[2-(5,6,7,8-tetrahydro[1,8]naphthyridin-2-yl)ethoxy]indol-1-yl]-3-(3,4-methylenedioxyphenyl)propionic acid 4, as an original series of potent alpha(v)beta(3) antagonists with subnanomolar activity. Ligand-protein docking analyses have been performed to generate binding models of three different chemical classes of known alpha(v)beta(3) antagonists with alpha(v)beta(3). Results of this docking study suggested that indoles bearing the basic tetrahydronaphthyridine group at position 4 can easily adopt the correct binding conformation and should be as potent as our current 5-substituted indole leads 3 and 4. This hypothesis was nicely demonstrated by the synthesis of a series of 1,4-disubstituted indoles through a tandem of reactions involving: (i) the N-alkylation of indoles 15 and 22 with propargyl esters and cesium fluoride, and (ii) a Heck coupling reaction between 4-bromoindole and 7-vinyl-3,4-dihydro-2H-[1,8]naphthyridine-1-carboxylic acid tert-butyl ester 12, or (iii) a reductive amination involving the N-substituted-4-aminoindole 23 and the BOC-protected tetrahydro[1,8]naphthyridine aldehyde 13. Among the compounds assayed, 3-(3-pyridyl)-3-[4-[2-(5,6,7,8-tetrahydro[1,8]naphthyridin-2-yl)ethyl]indol-1-yl]propionic acid 21 showed the most promising activity on alpha(v)beta(3) (IC(50)=0.5 nM), and was found to have the same potency as our current leads 3 and 4, while maintaining selectivity over alpha(IIb)beta(IIIa). Moreover, based on the reasonable apparent permeability coefficient in an in vitro CACO-2 cell monolayer assay (P(app) apical/basolateral=2.2 x 10(-6)cm/s, P(app) basolateral/apical=2.5 x 10(-6)cm/s), compound 21 is expected to be absorbed through the intestine in human. Thus, 1,4-disubstituted indole 21 represents a new lead for this novel class of conformationally restricted alpha(v)beta(3) antagonists. Additionally, this study validates the pharmacophore model previously postulated and provides an improved basis for further structure-based drug design in the field of alpha(v)beta(3).
Assuntos
Indóis/síntese química , Indóis/farmacologia , Integrina alfaVbeta3/antagonistas & inibidores , Desenho de Fármacos , Ensaio de Imunoadsorção Enzimática , Humanos , Indicadores e Reagentes , Indóis/química , Integrina alfa5beta1/antagonistas & inibidores , Integrinas/antagonistas & inibidores , Ligantes , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Glicoproteína IIb da Membrana de Plaquetas/efeitos dos fármacos , Receptores de Vitronectina/antagonistas & inibidores , Relação Estrutura-Atividade , Difração de Raios XRESUMO
The binding of lead compounds and drugs to human serum albumin (HSA) is a ubiquitous problem in drug discovery since it modulates the availability of the leads and drugs to their intended target, which is linked to biological efficacy. In our continuing efforts to identify small molecule alpha(V)beta(3) and alpha(V)beta(5) dual antagonists, we recently reported indoles 2-4 as potent and selective alpha(V)beta(3)/alpha(V)beta(5) antagonists with good oral bioavailability profile. In spite of subnanomolar binding affinity of these compounds to human alpha(V)beta(3) and alpha(V)beta(5) integrins, high HSA binding (96.5-97.3%) emerged as a limiting feature for these leads. Structure-activity HSA binding data of organic acids reported in the literature have demonstrated that the incorporation of polar groups into a given molecule can dramatically decrease the affinity toward HSA. We sought to apply this strategy by examining the effects of such modifications in both the central core constrain and the substituent beta to the carboxylate. Most of these derivatives were prepared in good yields through a cesium fluoride-catalyzed coupling reaction. This reaction was successful with a variety of nitrogen-containing scaffolds (20, 33, and 43) and selected acetylenic derivatives (16, 19, and 34). Among the compounds synthesized, the 3-[5-[2-(5,6,7,8-tetrahydro [1,8]naphthyridin-2-yl)ethoxy]indol-1-yl]-3-[5-(N,N-dimethylaminomethyl)-3-pyridyl]propionic acid (25) was found to be the most promising derivative within this novel series with a subnanomolar affinity for both alpha(v)beta(3) and alpha(v)beta(5) (IC(50) = 0.29 and 0.16 nM, respectively), similar to our initial lead receptor antagonists 2-4, and exhibiting a low HSA protein binding (40% bound, K(d) = 1.1+/-0.4 x 10(3) microM) and an improved in vitro stability profile toward human and mouse microsomes (99.9% and 98.7% remaining after 10 min). Moreover, the selectivity of 25 toward alpha(5)beta(1) and IIbIIIa integrins was perfectly maintained when compared to the parent leads 2-4. Thus, compound 25 was selected as a new lead with improved drug-like properties for further evaluations in the field of oncology and osteoporosis.
Assuntos
Integrina alfaVbeta3/antagonistas & inibidores , Integrinas/antagonistas & inibidores , Receptores de Vitronectina/antagonistas & inibidores , Albumina Sérica/metabolismo , Alcinos/síntese química , Alcinos/química , Benzimidazóis/química , Esterificação , Humanos , Hipoxantinas/síntese química , Hipoxantinas/química , Indóis/síntese química , Indóis/química , Estrutura Molecular , Propionatos/síntese química , Propionatos/química , Ligação Proteica , Relação Estrutura-AtividadeRESUMO
The disruption of the p53-Hdm2 protein-protein interaction induces cell growth arrest and apoptosis. We have identified the 1,4-benzodiazepine-2,5-dione scaffold as a suitable template for inhibiting this interaction by binding to the Hdm2 protein. Several compounds have been made with improved potency, solubility, and cell-based activities.
Assuntos
Benzodiazepinas/farmacologia , Proteínas Proto-Oncogênicas c-mdm2/antagonistas & inibidores , Apoptose/efeitos dos fármacos , Benzodiazepinas/síntese química , Benzodiazepinas/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Estrutura Molecular , Ligação Proteica/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Estereoisomerismo , Relação Estrutura-Atividade , Proteína Supressora de Tumor p53/antagonistas & inibidores , Proteína Supressora de Tumor p53/metabolismoRESUMO
Guided by structure-based drug design, modification of the 1,4-benzodiazepin-2,5-dione lead compound 1 resulted in the discovery of 19, a potent and orally bioavailable antagonist of the HDM2-p53 protein-protein interaction (FP IC50 = 0.7 microM, F approximately 100%).