Development of highly potent non-covalent inhibitors of SARS-CoV-2 3CLpro (preprint)

The SARS-CoV-2 virus is the causal agent of the ongoing pandemic of coronavirus disease 2019 (COVID-19). There is an urgent need for potent, specific antiviral compounds against SARS-CoV-2. The 3C-like protease (3CLpro) is an essential enzyme for the replication of SARS-CoV-2 and other coronaviruses, and thus is a target for coronavirus drug discovery. Nearly all inhibitors of coronavirus 3CLpro reported so far are covalent inhibitors. Here, we report the development of specific, non-covalent inhibitors of 3CLpro. The most potent one, WU-04, effectively blocks SARS-CoV-2 replications in human cells with EC 50 values in the 10-nM range. WU-04 also inhibits the 3CLpro of SARS-CoV and MERS-CoV with high potency, indicating that it is a pan-inhibitor of coronavirus 3CLpro. WU-04 showed anti-SARS-CoV-2 activity similar to that of PF-07321332 (Nirmatrelvir) in K18-hACE2 mice when the same dose was administered orally. Thus, WU-04 is a promising drug candidate for coronavirus treatment. One-Sentence Summary A oral non-covalent inhibitor of 3C-like protease effectively inhibits SARS-CoV-2 replication.

Novel sites for Cathepsin L cleavage in SARS-CoV-2 spike guide treatment strategies (preprint)

The spike (S) protein of SARS coronavirus 2 (SARS-CoV-2) is an ideal target for the development of specific vaccines or drugs. However, treatments targeting viruses with mutant S proteins that have recently emerged in many countries are limited. Cleavage of the S protein by host proteases is essential for viral infection. Here, we discovered two novel sites (CS-1 and CS-2) in the S protein for cleavage by the protease Cathepsin L (CTSL). Both sites are highly conserved among all SARS-CoV-2 variants of concern. Cryo-electron microscopy structural studies revealed that CTSL cleavage increases the dynamics of the receptor binding domain of S and induces novel conformations. In our pseudovirus (PsV) infection experiment, alteration of the cleavage site significantly reduced the infection efficiency, and CTSL inhibitors markedly inhibited infection with PsVs of both the wild-type and emerged SARS-CoV-2 variants. Furthermore, six highly efficient CTSL inhibitors were found to effectively inhibit live virus infection in human cells in vitro , and two of these were further confirmed to prevent live virus infection in human ACE2 transgenic mice in vivo . Our work suggested that the CTSL cleavage sites in SARS-CoV-2 S are emerging new but effective targets for the development of mutation-resistant vaccines and drugs.

Metabotropic glutamate receptor subtype 2 is a receptor of SARS-CoV-2 (preprint)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative pathogen of novel coronavirus disease 2019 (COVID-19)1. SARS-CoV-2 uses angiotensin converting enzyme 2 (ACE2) as a cellular receptor and enters cells via clathrin-mediated endocytosis (CME)2-4. However, the key molecules involved in internalizing and facilitating CME for virus entry remain unknown. Here, we found metabotropic glutamate receptor subtype 2 (mGluR2) is a key entry receptor for SARS-CoV-2 infection. mGluR2 directly interacts with the SARS-CoV-2 spike protein. Knockdown of mGluR2 decreases endocytosis of SARS-CoV-2 but not cell binding. mGluR2 cooperates with ACE2 to facilitate SARS-CoV-2 entry through CME. Knockout of the mGluR2 gene in mice abolished SARS-CoV-2 infection in the nasal turbinates and significantly reduced viral infection in the lungs. Importantly, mGluR2 also is important for severe acute respiratory syndrome coronavirus spike protein and Middle East respiratory syndrome coronavirus spike protein mediated endocytosis. Our study provides important insights into SARS-CoV-2 infection and reveals an important target for the development of novel approaches to limit coronavirus infection.

A single dose of an adenovirus-vectored vaccine provides complete protection of the upper and lower respiratory tracts against SARS-CoV-2 challenge (preprint)

The unprecedented coronavirus disease 2019 (COVID-19) epidemic has created a worldwide public health emergency, and there is an urgent need to develop an effective vaccine to control this severe infectious disease. Here, we found that a single vaccination with a replication-defective human type 5 adenovirus encoding the SARS-CoV-2 spike protein (Ad5-nCoV) protected mice completely against SARS-CoV-2 infection in the upper and lower respiratory tracts. Additionally, a single vaccination with Ad5-nCoV protected ferrets from SARS-CoV-2 infection in the upper respiratory tract. This study suggested that a combination of intramuscular and mucosal vaccination maybe provide a desirable protective efficacy and different Ad5-nCoV delivery modes are worth further investigation in human clinical trials.