RESUMEN
Antibody-based therapy is emerging as a critical therapeutic countermeasure to treat acute viral infections by offering rapid protection against clinical disease. The advancements in structural biology made it feasible to rationalize monoclonal antibodies (mAbs) by identifying key and, possibly, neutralizing epitopes of viral proteins for therapeutic purposes. A critical component in assessing mAbs during pandemics requires the development of rapid but detailed methods to detect and quantitate the neutralization activity. In this study, we developed and optimized two high-content image (HCI)-based assays: one to detect viral proteins by staining and the second to quantify cytopathic viral effects by a label-free phenotypic assay. These assays were employed to screen for therapeutic antibodies against the monkeypox virus (MPXV) using surrogate poxviruses such as vaccinia virus (VACV). Plaque-based neutralization results confirmed the HCI data. The phenotypic assay found pox virus-induced syncytia formation in various cells, and we were able to quantitate and use this phenotype to screen mAbs. The HCI identified several potent VACV-neutralizing antibodies that showed in vitro efficacy against both clades of MPXV. In addition, a combination study of ST-246/tecovirimat/TPOXX a single neutralizing antibody Ab-40, showed synergistic activity against VACV in an in-vitro neutralization assay. This rapid high-content method utilizing state-of-the-art technologies enabled the evaluation of hundreds of mAbs quickly to identify several potent anti-MPXV neutralizing mAbs for further development.
Asunto(s)
Anticuerpos Antivirales , Monkeypox virus , Anticuerpos Neutralizantes , Virus Vaccinia/genética , Proteínas Virales , Anticuerpos Monoclonales/farmacología , Pruebas de NeutralizaciónRESUMEN
XIAP-associated factor 1(XAF1) is a tumor suppressor with its functional mechanisms not fully understood. The zinc-finger cluster located at the N-terminus is the only domain structure. Four and a half LIM domain protein 2 (FHL2) also contains a tandem zinc finger structure, and its protein functions as an important adaptor and modifier in protein-protein interactions. Both of their structures are relatively simple, while the association between them is still unclear. In this study, we detected the interaction between XAF1 and FHL2 by using the yeast two-hybrid system. We identified FHL2 as a XAF1 binding protein. Furthermore, both XAF1 and FHL2 localized to the cytoplasm, mitochondria, and nucleus of gastric cancer cells. Over-expression of XAF1 excluded FHL2 from the nucleus and suppressed the trans-activity of FHL2 in stimulating the transcriptional activities of ß-catenin and AP-1. In conclusion, our findings unraveled an antagonistic mechanism between a tumor suppressor and an oncoprotein in cancer cells.
Asunto(s)
Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias Gástricas/genética , Transactivadores , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Proteínas Reguladoras de la Apoptosis , Western Blotting , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Técnica del Anticuerpo Fluorescente , Humanos , Inmunoprecipitación , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas con Homeodominio LIM , Luciferasas/metabolismo , Mitocondrias/metabolismo , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/genética , Regiones Promotoras Genéticas , Unión Proteica , ARN Interferente Pequeño/genética , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patología , Factor de Transcripción AP-1/genética , Factor de Transcripción AP-1/metabolismo , Transcripción Genética , Activación Transcripcional , Células Tumorales Cultivadas , Técnicas del Sistema de Dos Híbridos , beta Catenina/genética , beta Catenina/metabolismoRESUMEN
Approximately 257 million people chronically infected with hepatitis B virus (HBV) worldwide are at risk of developing hepatocellular carcinoma (HCC). However, despite the availability of potent nucleoside/tide inhibitors, currently there are no curative therapies for chronic HBV infections. To identify potential new antiviral molecules, a select group of compounds previously evaluated in clinical studies were tested against 12 different viruses. Amongst the compounds tested, SRI-32007 (CYT997) demonstrated antiviral activity against HBV (genotype D) in HepG2.2.2.15 cell-based virus yield assay with 50% effective concentration (EC50) and selectivity index (SI) of 60.1 nM and 7.2, respectively. Anti-HBV activity of SRI-32007 was further confirmed against HBV genotype B in huh7 cells with secreted HBe antigen endpoint (EC50 40 nM and SI 250). To determine the stage of HBV life cycle inhibited by SRI-32007, time of addition experiment was conducted in HepG2-NTCP cell-based HBV infectious assay. Results indicated that SRI-32007 retained anti-HBV activity even when added 72 hours postinfection (72 h). Additional mechanism of action studies demonstrated potent inhibition of HBV core promoter activity by SRI-32007 with an EC50 of 40 nM and SI of >250. This study demonstrates anti-HBV activity of a repurposed compound SRI-32007 through inhibition of HBV core promoter activity. Further evaluation of SRI-32007 in HBV animal models is needed to confirm its activity in vivo. Our experiments illustrate the utility of repurposing strategy to identify novel antiviral chemical leads. HBV core promoter inhibitors such as SRI-32007 might enable the development of novel therapeutic strategies to combat HBV infections.