Merbromin is a mixed-type inhibitor of 3-chyomotrypsin like protease of SARS-CoV-2.

3-chyomotrypsin like protease (3CLpro) has been considered as a promising target for developing anti-SARS-CoV-2 drugs. Herein, about 6000 compounds were analyzed by high-throughput screening using enzyme activity model, and Merbromin, an antibacterial agent, was identified as a potent inhibitor of 3CLpro. Merbromin strongly inhibited the proteolytic activity of 3CLpro but not the other three proteases Proteinase K, Trypsin and Papain. Michaelis-Menten kinetic analysis showed that Merbromin was a mixed-type inhibitor of 3CLpro, due to its ability of increasing the KM and decreasing the Kcat of 3CLpro. The binding assays and molecular docking suggested that 3CLpro possessed two binding sites for Merbromin. Consistently, Merbromin showed a weak binding to the other three proteases. Together, these findings demonstrated that Merbromin is a selective inhibitor of 3CLpro and provided a scaffold to design effective inhibitors of SARS-CoV-2.

Establishment of a pseudovirus neutralization assay based on SARS-CoV-2 S protein incorporated into lentiviral particles.

The coronavirus disease 2019 (COVID-19) is still causing a wide range of infections and deaths due to the high variability of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Therefore, it is necessary to establish a reliable and convenient pseudovirus-based neutralization assay to develop drug targeted variants of SARS-CoV-2. Based on the HIV-1 backbone, we generated a high titer luciferase (Luc)-expressing pseudovirus packaging system. Three dominant S mutant substitution pseudovirus were also established and identified compared to wide type in hACE2-overexpressing HEK-293T cells (293T-ACE2 cells). Compared to serine protease inhibitor camostat mesylate, the cysteine protease inhibitor E-64d could significantly block all SARS-CoV-2 mutant S pseudovirus infection in 293T-ACE2 cells. Furthermore, the neutralization ability of two antibodies targeted receptor-binding domain (RBD) of SARS-CoV-2 spike protein (S) was evaluated, which showed different inhibition dose-effect curves among four types of S pseudovirus. Overall, we developed a pseudovirus-based neutralization assay for SARS-CoV-2, which would be readily adapted to SARS-CoV-2 variants for evaluating antibodies.

SARS-CoV-2 envelope protein causes acute respiratory distress syndrome (ARDS)-like pathological damages and constitutes an antiviral target.

Cytokine storm and multi-organ failure are the main causes of SARS-CoV-2-related death. However, the origin of excessive damages caused by SARS-CoV-2 remains largely unknown. Here we show that the SARS-CoV-2 envelope (2-E) protein alone is able to cause acute respiratory distress syndrome (ARDS)-like damages in vitro and in vivo. 2-E proteins were found to form a type of pH-sensitive cation channels in bilayer lipid membranes. As observed in SARS-CoV-2-infected cells, heterologous expression of 2-E channels induced rapid cell death in various susceptible cell types and robust secretion of cytokines and chemokines in macrophages. Intravenous administration of purified 2-E protein into mice caused ARDS-like pathological damages in lung and spleen. A dominant negative mutation lowering 2-E channel activity attenuated cell death and SARS-CoV-2 production. Newly identified channel inhibitors exhibited potent anti-SARS-CoV-2 activity and excellent cell protective activity in vitro and these activities were positively correlated with inhibition of 2-E channel. Importantly, prophylactic and therapeutic administration of the channel inhibitor effectively reduced both the viral load and secretion of inflammation cytokines in lungs of SARS-CoV-2-infected transgenic mice expressing human angiotensin-converting enzyme 2 (hACE-2). Our study supports that 2-E is a promising drug target against SARS-CoV-2.

A computational framework of host-based drug repositioning for broad-spectrum antivirals against RNA viruses.

RNA viruses are responsible for many zoonotic diseases that post great challenges for public health. Effective therapeutics against these viral infections remain limited. Here, we deployed a computational framework for host-based drug repositioning to predict potential antiviral drugs from 2,352 approved drugs and 1,062 natural compounds embedded in herbs of traditional Chinese medicine. By systematically interrogating public genetic screening data, we comprehensively cataloged host dependency genes (HDGs) that are indispensable for successful viral infection corresponding to 10 families and 29 species of RNA viruses. We then utilized these HDGs as potential drug targets and interrogated extensive drug-target interactions through database retrieval, literature mining, and de novo prediction using artificial intelligence-based algorithms. Repurposed drugs or natural compounds were proposed against many viral pathogens such as coronaviruses including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), flaviviruses, and influenza viruses. This study helps to prioritize promising drug candidates for in-depth evaluation against these virus-related diseases.