RESUMEN
The majority of FDA-approved HCV therapeutics target the viral replicative machinery. An automated high-throughput phenotypic screen identified several small molecules as potent inhibitors of hepatitis C virus replication. Here, we disclose the discovery and optimization of a 4-aminopiperidine (4AP) scaffold targeting the assembly stages of the HCV life cycle. The original screening hit (1) demonstrates efficacy in the HCVcc assay but does not show potency prior to or during viral replication. Colocalization and infectivity studies indicate that the 4AP chemotype inhibits the assembly and release of infectious HCV. Compound 1 acts synergistically with FDA-approved direct-acting antiviral compounds Telaprevir and Daclatasvir, as well as broad spectrum antivirals Ribavirin and cyclosporin A. Following an SAR campaign, several derivatives of the 4AP series have been identified with increased potency against HCV, reduced in vitro toxicity, as well as improved in vitro and in vivo ADME properties.
Asunto(s)
Antivirales/farmacología , Descubrimiento de Drogas , Hepacivirus/efectos de los fármacos , Piperidinas/farmacología , Animales , Antivirales/síntesis química , Antivirales/química , Células Cultivadas , Relación Dosis-Respuesta a Droga , Humanos , Masculino , Ratones , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Piperidinas/síntesis química , Piperidinas/química , Ratas , Relación Estructura-Actividad , Replicación Viral/efectos de los fármacosRESUMEN
Fluoxazolevir is an aryloxazole-based entry inhibitor of hepatitis C virus (HCV). We show that fluoxazolevir inhibits fusion of HCV with hepatic cells by binding HCV envelope protein 1 to prevent fusion. Nine of ten fluoxazolevir resistance-associated substitutions are in envelope protein 1, and four are in a putative fusion peptide. Pharmacokinetic studies in mice, rats and dogs revealed that fluoxazolevir localizes to the liver. A 4-week intraperitoneal regimen of fluoxazolevir in humanized chimeric mice infected with HCV genotypes 1b, 2a or 3 resulted in a 2-log reduction in viraemia, without evidence of drug resistance. In comparison, daclatasvir, an approved HCV drug, suppressed more than 3 log of viraemia but is associated with the emergence of resistance-associated substitutions in mice. Combination therapy using fluoxazolevir and daclatasvir cleared HCV genotypes 1b and 3 in mice. Fluoxazolevir combined with glecaprevir and pibrentasvir was also effective in clearing multidrug-resistant HCV replication in mice. Fluoxazolevir may be promising as the next generation of combination drug cocktails for HCV treatment.
Asunto(s)
Antivirales/administración & dosificación , Hepacivirus/efectos de los fármacos , Hepatitis C/tratamiento farmacológico , Internalización del Virus/efectos de los fármacos , Animales , Carbamatos/administración & dosificación , Modelos Animales de Enfermedad , Perros , Quimioterapia Combinada , Genotipo , Hepacivirus/clasificación , Hepacivirus/genética , Hepacivirus/fisiología , Hepatitis C/virología , Humanos , Imidazoles/administración & dosificación , Masculino , Ratones , Pirrolidinas/administración & dosificación , Ratas , Ratas Sprague-Dawley , Valina/administración & dosificación , Valina/análogos & derivados , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/metabolismoRESUMEN
Oxidative stress (OS) in non-alcoholic fatty liver disease (NAFLD) promotes liver injury and inflammation. Treatment with vitamin E (α-tocopherol, αT), a lipid-soluble antioxidant, improves liver injury but also decreases steatosis, thought to be upstream of OS, through an unknown mechanism. To elucidate the mechanism, we combined a mechanistic human trial interrogating pathways of intrahepatic triglyceride (IHTG) accumulation and in vitro experiments. 50% of NAFLD patients (n = 20) treated with αT (200-800 IU/d) for 24 weeks had a ≥ 25% relative decrease in IHTG by magnetic resonance spectroscopy. Paired liver biopsies at baseline and week 4 of treatment revealed a decrease in markers of hepatic de novo lipogenesis (DNL) that strongly predicted week 24 response. In vitro, using HepG2 cells and primary human hepatocytes, αT inhibited glucose-induced DNL by decreasing SREBP-1 processing and lipogenic gene expression. This mechanism is dependent on the antioxidant capacity of αT, as redox-silenced methoxy-αT is unable to inhibit DNL in vitro. OS by itself was sufficient to increase S2P expression in vitro, and S2P is upregulated in NAFLD livers. In summary, we utilized αT to demonstrate a vicious cycle in which NAFLD generates OS, which feeds back to augment DNL and increases steatosis. Clinicaltrials.gov: NCT01792115.
Asunto(s)
Enfermedad del Hígado Graso no Alcohólico , Humanos , Lipogénesis , Hígado/metabolismo , Enfermedad del Hígado Graso no Alcohólico/tratamiento farmacológico , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Estrés Oxidativo , Regulación hacia Arriba , Vitamina E/metabolismo , Vitamina E/farmacologíaRESUMEN
Ageing, and particularly the onset of age-related diseases, is associated with tissue dysfunction and macromolecular damage, some of which can be attributed to accumulation of oxidative damage. Polyphenolic natural products such as stilbenoids, flavonoids and chalcones have been shown to be effective at ameliorating several age-related phenotypes, including oxidative stress, inflammation, impaired proteostasis and cellular senescence, both in vitro and in vivo. Here we aim to identify the structural basis underlying the pharmacology of polyphenols towards ROS and related biochemical pathways involved in age-related disease. We compile and describe SAR trends across different polyphenol chemotypes including stilbenoids, flavonoids and chalcones, review their different molecular targets and indications, and identify common structural ground between chemotypes and mechanisms of action. In particular, we focus on the structural requirements for the direct scavenging of reactive oxygen/nitrogen species such as radicals as well as coordination of a broader antioxidant response. We further suggest that it is important to consider multiple (rather than single) biological activities when identifying and developing new medicinal chemistry entities with utility in modulating complex biological properties such as cell ageing.
RESUMEN
Chlorcyclizine (CCZ) is a potent hepatitis C virus (HCV) entry inhibitor, but its molecular mechanism is unknown. Here, we show that CCZ directly targets the fusion peptide of HCV E1 and interferes with the fusion process. Generation of CCZ resistance-associated substitutions of HCV in vitro revealed six missense mutations in the HCV E1 protein, five being in the putative fusion peptide. A viral fusion assay demonstrated that CCZ blocked HCV entry at the membrane fusion step and that the mutant viruses acquired resistance to CCZ's action in blocking membrane fusion. UV cross-linking of photoactivatable CCZ-diazirine-biotin in both HCV-infected cells and recombinant HCV E1/E2 protein demonstrated direct binding to HCV E1 glycoprotein. Mass spectrometry analysis revealed that CCZ cross-linked to an E1 sequence adjacent to the putative fusion peptide. Docking simulations demonstrate a putative binding model, wherein CCZ binds to a hydrophobic pocket of HCV E1 and forms extensive interactions with the fusion peptide.
Asunto(s)
Hepacivirus/metabolismo , Piperazinas/química , Proteínas del Envoltorio Viral/antagonistas & inhibidores , Antivirales/síntesis química , Antivirales/química , Antivirales/farmacología , Sitios de Unión , Biotina/química , Diazometano/química , Farmacorresistencia Viral/efectos de los fármacos , Genotipo , Hepacivirus/efectos de los fármacos , Hepacivirus/genética , Humanos , Fusión de Membrana/efectos de los fármacos , Simulación del Acoplamiento Molecular , Piperazinas/metabolismo , Piperazinas/farmacología , Rayos Ultravioleta , Proteínas del Envoltorio Viral/metabolismo , Internalización del Virus/efectos de los fármacosRESUMEN
In the original publication of the article, Fig. 2 was published incorrectly. The corrected Figure is given below. The original article has been corrected.
RESUMEN
Cellular senescence has been shown to be sufficient for the development of multiple age-related pathologies. Senescent cells adopt a secretory phenotype (the SASP) which comprises a large number of pro-inflammatory cytokines, chemokines and proteases. The SASP itself is thought to be causative in many pathologies of age-related diseases, and there is growing interest in developing seno-modifying agents that can suppress the SASP. However, in order to identify new agents, it is necessary to conduct moderate to high throughput screening with robust assays for the required outcome. Here, we describe optimisation and validation of a cell-based biosensor HEK cell line for measurement of IL-6 concentrations within the range secreted into conditioned medium by primary senescent fibroblasts, adapted for a 384 well plate format suitable for library screening applications. We further show that the assay can measure changes in IL-6 secretion dependent on cell population age, and that the assay is responsive to mTOR inhibition in the senescent cells, which reduces the SASP, including IL-6. Hence, we propose that this optimised biosensor, which we term HEK-SASP, may prove of value in studies requiring robust, renewable and relatively inexpensive assays for measuring SASP factors.
Asunto(s)
Senescencia Celular/fisiología , Inflamación/metabolismo , Interleucina-6/metabolismo , Transducción de Señal , Técnicas Biosensibles , Ensayo de Inmunoadsorción Enzimática , Células HEK293 , HumanosRESUMEN
The unusual α-amino, ß-hydroxy acid MeBmt is a key structural feature of cyclosporin A, an important naturally occurring immunosuppressant and antiviral agent. We present a convergent synthesis of MeBmt which relies on new aspects of dynamic kinetic resolution (DKR) to establish simultaneously the chirality at C(2) and C(3). We also show that this route is applicable to the synthesis of other derivatives.
RESUMEN
Hepatitis C virus (HCV) is a small, single-stranded, positive-sense RNA virus that infects more than an estimated 70 million people worldwide. Untreated, persistent HCV infection often results in chronic hepatitis, cirrhosis, or liver failure, with progression to hepatocellular carcinoma. Current anti-HCV regimens comprising direct acting antivirals (DAAs) can provide curative treatment; however, due to high costs there remains a need for effective, shorter-duration, and affordable treatments. Recently, we disclosed anti-HCV activity of the cheap antihistamine chlorcyclizine, targeting viral entry. Following our hit-to-lead optimization campaign, we report evaluation of preclinical in vitro absorption, distribution, metabolism, and excretion properties, and in vivo pharmacokinetic profiles of lead compounds. This led to selection of a new lead compound and evaluation of efficacy in chimeric mice engrafted with primary human hepatocytes infected with HCV. Further development and incorporation of this compound into DAA regimens has the potential to improve treatment efficacy, affordability, and accessibility.
Asunto(s)
Antivirales/farmacología , Hepacivirus/efectos de los fármacos , Hepatitis C Crónica/tratamiento farmacológico , Piperazinas/farmacología , Animales , Carcinoma Hepatocelular/virología , Línea Celular , Genotipo , Hepatocitos/virología , Humanos , Cirrosis Hepática/virología , Neoplasias Hepáticas/virología , Masculino , Ratones , Ratones SCID , Internalización del Virus/efectos de los fármacosRESUMEN
Recently, we reported that chlorcyclizine (CCZ, Rac-2), an over-the-counter antihistamine piperazine drug, possesses in vitro and in vivo activity against hepatitis C virus. Here, we describe structure-activity relationship (SAR) efforts that resulted in the optimization of novel chlorcyclizine derivatives as anti-HCV agents. Several compounds exhibited EC50 values below 10 nM against HCV infection, cytotoxicity selectivity indices above 2000, and showed improved in vivo pharmacokinetic properties. The optimized molecules can serve as lead preclinical candidates for the treatment of hepatitis C virus infection and as probes to study hepatitis C virus pathogenesis and host-virus interaction.