Cysteine peptidase inhibition: The way to impair SARS-CoV-2 infection and replication

Since its outbreak, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) caused one of the most severe pandemics in recent history and had enormous effects on the lives of millions of people worldwide. Peptidases, both viral and host, have been described as critical enzymes in mechanisms underlying SARS-CoV-2 infection and replication. Among host peptidases, the involvement in activation of viral glycoproteins and processing of the SARS-CoV spike protein has been described for endosomal/lysosomal cysteine peptidases cathepsins B and L, which therefore represent promising targets for development of effective drugs for treatment of COVID-19. To date, a large number of cathepsin B and L inhibitors have been identified and evaluated for treatment of various pathological processes. In this study, we have evaluated well-established known potent selective and reversible cathepsin B inhibitors for their potential to act against SARS-CoV-2. Cathepsin B inhibitors showed significant activity in preventing viral entry and replication of SARS-CoV-2. Next, we observed that antiviral activity of compounds was dependent on the cell type and correlated well with the intracellular amount of the targeted cathepsin. Taken together, we have demonstrated the important role of host cysteine peptidase cathepsin B during SARS-CoV-2 infection and identified its inhibitors as potential new therapeutic agents for treatment of SARS-CoV-2 infection.

EXPRESSION OF EPITHELIAL PROTEASES PREDICTS SARS-COV-2 ENTRY IN HUMAN ENTEROIDS

Introduction: Coronavirus Disease 2019 (COVID-19) is an ongoing public health crisis that has sickened or precipitated death in millions. The etiologic agent of COVID-19, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), infects the intestinal epithelium and can persist long after the respiratory infection has cleared. We previously observed that intestinal SARS-CoV-2 infection levels varied by individual donors and did not correlate positively with ACE2, the cognate SARS-CoV-2 receptor. Therefore we aimed to delineate host factors that influence viral infection in the intestine. Methods: Published dataset GSE75214 was downloaded and expression levels of select genes were querried. Primary human ileal spheroids (enteroids), derived from healthy donors and patients with Crohn's disease (CD), were grown on 2D transwells until confluent. Cells were differentiated for 3d before infection with a modified vesicular stomatitis virus expressing the SARS-CoV-2 spike protein (VSV-SARS-CoV-2) and GFP for 1h at a multiplicity of infection of ~0.5. Cells were harvested pre-infection and 24h after infection and expression of select genes was performed by qRT-PCR. Expression data were fit to a linear regression model to predict viral RNA levels. Results: Small intestine biopsy samples from CD patients demonstrated a reduction in ACE and an increase in CTSB and CTSL expression during active inflammation compared to healthy controls. Viral RNA expression did not correlate with ACE2 expression in CD enteroids. A subset of CD enteroids exhibited enhanced protease expression (TMPRSS2, TMPRSS4, CTSL), each of which correlated with higher viral RNA levels (P=0.04, P=0.002, P=0.006, respectively). Expression of these proteases was higher in the pre-infection for the sample subset. Principle component analysis of uninfected expression data demonstrated these samples clustered separately from the others, with the difference driven by TMPRSS2, TMPRSS4, and CTSL. Modeling viral RNA levels based on gene expression revealed expression levels of these proteases are a predictive expression signature. Conclusions: Host protease expression can predict SARS-CoV-2 infection and represent potential therapeutic targets for COVID-19. This is consistent with the recent report showing that cathepsin inhibition reduces SARS-CoV-2 spike-mediated syncytia formation. High expression of these proteases in the intestine may also be a novel biomarker for the risk of intestinal complications associated with COVID-19.(Figure Presented)RNA data from dataset GSE75214 demonstrating reduced ACE2 and increased CTSB and CTSL in patients with Crohn's disease during active inflammation compared to healthy controls. (Figure Presented) Enteroids from healthy control donors and patients with Crohn's disease were grown in 2D transwells and expression of indicated genes was assessed in pre-infection (A) and after infection with VSV-SARS-CoV-2 (B)

Progress on SARS-CoV-2 cell entry inhibitors

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the COVID-19 pandemic. The life cycle of SARS-CoV-2 is not clear, which is one of the reasons that only Remdesivir has been approved by FDA for treating COVID-19. Although some new vaccines have been a- vailable, the quick mutations of SARS-CoV-2 affect the effectiveness of vaccines, calling for further assessment of the persistence and safety of vaccines. Therefore, drug treatment and prevention are still effective ways to deal with the epidemic of SARS-CoV-2. The article briefly summarizes the molecular mechanism of SARS-CoV-2 entry based on the existing literature. This virus enters the cell through two main ways, that is, spike protein mediating membrane fusion with plasma membrane or endosome membrane. According to the targets, the article summarizes the reported inhibitors of SARS-CoV-2 entry into cells, aiming to provide a reference for following research and clinical application of anti-SARS-CoV-2 drugs.

After one year of the COVID-19 pandemic and hundreds of suggested drugs, will cathepsin L inhibitors be the solution?

Cysteine cathepsins are defined as lysosomal enzymes that are members of the papain family. Cysteine cathepsins (Cts) prevalently exist in whole organisms, varying from prokaryotes to mammals, and possess greatly conserved cysteine residues in their active sites. Cts are engaged in the digestion of cellular proteins, activation of zymogens, and remodeling of the extracellular matrix (ECM). Host cells are entered by SARS-CoV-2 via endocytosis. Cathepsin L and phosphatidylinositol 3-phosphate 5-kinase are crucial in endocytosis by cleaving the spike protein, which permits viral membrane fusion with the endosomal membrane and succeeds in the release of the viral genome to the host cell. Therefore, inhibition of cathepsin L may be advantageous in terms of decreasing infection caused by SARS-CoV-2. Coordinate inhibition of multiple Cts and lysosomal function by different drugs and biological agents might be of value for some purposes, such as a parasite or viral infections and antineoplastic applications. Zn2+ deficiency or dysregulation leads to exaggerated cysteine cathepsin activity, increasing the autoimmune/inflammatory response. For this purpose, Zn2+ metal can be safely combined with a drug that increases the anti-proteolytic effect of endogenous Zn2+, lowering the excessive activity of some CysCts. Biguanide derivative complexes with Zn2+ have been found to be promising inhibitors of CysCts protease reactions. Molecular docking studies of cathepsin L inhibited by the metformin-Zn+2 complex have been performed, showing two strong key interactions (Cys-25&His-163) and an extra H-bond with Asp-163 compared to cocrystallized Zn+2 (PDB ID 4axl).