RESUMEN
Cepharanthine (CEP) is a natural biscoclaurine alkaloid of plant origin and was recently demonstrated to have anti-severe acute respiratory syndrome coronavirus 2 (anti-SARS-CoV-2) activity. In this study, we evaluated whether natural analogues of CEP may act as potential anti-coronavirus disease 2019 drugs. A total of 24 compounds resembling CEP were extracted from the KNApSAcK database, and their binding affinities to target proteins, including the spike protein and main protease of SARS-CoV-2, NPC1 and TPC2 in humans, were predicted via molecular docking simulations. Selected analogues were further evaluated by a cell-based SARS-CoV-2 infection assay. In addition, the efficacies of CEP and its analogue tetrandrine were assessed. A comparison of the docking conformations of these compounds suggested that the diphenyl ester moiety of the molecules was a putative pharmacophore of the CEP analogues.
Asunto(s)
Antivirales/farmacología , Bencilisoquinolinas/farmacología , COVID-19/prevención & control , Preparaciones de Plantas/farmacología , SARS-CoV-2/efectos de los fármacos , Animales , Antivirales/química , Antivirales/metabolismo , Bencilisoquinolinas/química , Bencilisoquinolinas/metabolismo , COVID-19/virología , Chlorocebus aethiops , Proteínas M de Coronavirus/antagonistas & inhibidores , Proteínas M de Coronavirus/química , Proteínas M de Coronavirus/metabolismo , Evaluación Preclínica de Medicamentos/métodos , Humanos , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Preparaciones de Plantas/química , Preparaciones de Plantas/metabolismo , Unión Proteica , Conformación Proteica , SARS-CoV-2/metabolismo , SARS-CoV-2/fisiología , Stephania/química , Células VeroRESUMEN
Chondroitin sulfate is found in a variety of tissues as proteoglycans and consists of repeating disaccharide units of N-acetylgalactosamine and glucuronic acid residues with sulfate residues at various places. We found a novel human gene (GenBank accession number AB086063) that possesses a sequence homologous with the human chondroitin sulfate glucuronyltransferase gene which we recently cloned and characterized. The full-length open reading frame encodes a typical type II membrane protein comprising 775 amino acids. The protein had a domain containing beta 3-glycosyltransferase motif but lacked a typical beta 4-glycosyltransferase motif, which is the same as chondroitin sulfate glucuronyltransferase, whereas chondroitin synthase had both domains. The putative catalytic domain was expressed in COS-7 cells as a soluble enzyme. Surprisingly, both glucuronyltransferase and N-acetylgalactosaminyltransferase activities were observed when chondroitin, chondroitin sulfate, and their oligosaccharides were used as the acceptor substrates. The reaction products were identified to have the linkage of GlcUA beta 1-3GalNAc and GalNAc beta 1-4GlcUA at the non-reducing terminus of chondroitin for glucuronyltransferase activity and N-acetylgalactosaminyltransferase activity, respectively. Quantitative real time PCR analysis revealed that the transcripts were ubiquitously expressed in various human tissues but highly expressed in the pancreas, ovary, placenta, small intestine, and stomach. These results indicate that this enzyme could synthesize chondroitin sulfate chains as a chondroitin sulfate synthase that has both glucuronyltransferase and N-acetylgalactosaminyltransferase activities. Sequence analysis based on three-dimensional structure revealed the presence of not typical but significant beta 4-glycosyltransferase architecture.