RESUMO
COVID-19 is a disease caused by SARS-CoV-2, which has led to more than 4 million deaths worldwide. As a result, there is a worldwide effort to develop specific drugs for targeting COVID-19. Papain-like protease (PLpro) is an attractive drug target because it has multiple essential functions involved in processing viral proteins, including viral genome replication and removal of post-translational ubiquitination modifications. Here, we established two assays for screening PLpro inhibitors according to protease and anti-ISGylation activities, respectively. Application of the two screening techniques to the library of clinically approved drugs led to the discovery of tanshinone IIA sulfonate sodium and chloroxine with their IC50 values of lower than 10 µM. These two compounds were found to directly interact with PLpro and their molecular mechanisms of binding were illustrated by docking and molecular dynamics simulations. The results highlight the usefulness of the two developed screening techniques for locating PLpro inhibitors.
Assuntos
Antivirais/farmacologia , Tratamento Farmacológico da COVID-19 , Proteases Semelhantes à Papaína de Coronavírus/antagonistas & inibidores , Inibidores de Protease de Coronavírus/farmacologia , Reposicionamento de Medicamentos , SARS-CoV-2/enzimologia , Antivirais/química , Sítios de Ligação , Cloroquinolinóis/química , Cloroquinolinóis/farmacologia , Proteases Semelhantes à Papaína de Coronavírus/genética , Proteases Semelhantes à Papaína de Coronavírus/isolamento & purificação , Inibidores de Protease de Coronavírus/química , Ensaios de Triagem em Larga Escala/métodos , Humanos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Fenantrenos/química , Fenantrenos/farmacologia , SARS-CoV-2/efeitos dos fármacosRESUMO
During viral RNA synthesis, Ebola virus (EBOV) nucleoprotein (NP) alternates between an RNA-template-bound form and a template-free form to provide the viral polymerase access to the RNA template. In addition, newly synthesized NP must be prevented from indiscriminately binding to noncognate RNAs. Here, we investigate the molecular bases for these critical processes. We identify an intrinsically disordered peptide derived from EBOV VP35 (NPBP, residues 20-48) that binds NP with high affinity and specificity, inhibits NP oligomerization, and releases RNA from NP-RNA complexes in vitro. The structure of the NPBP/ΔNPNTD complex, solved to 3.7 Å resolution, reveals how NPBP peptide occludes a large surface area that is important for NP-NP and NP-RNA interactions and for viral RNA synthesis. Together, our results identify a highly conserved viral interface that is important for EBOV replication and can be targeted for therapeutic development.
