Ubiquitin variants potently inhibit SARS-CoV-2 PLpro and viral replication via a novel site distal to the protease active site

Abstract

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has made it clear that combating coronavirus outbreaks benefits from a combination of vaccines and therapeutics. A promising drug target common to all coronaviruses—including SARS-CoV, MERS-CoV, and SARS-CoV-2—is the papain-like protease (PLpro). PLpro cleaves part of the viral replicase polyproteins into non-structural protein subunits, which are essential to the viral replication cycle. Additionally, PLpro can cleave both ubiquitin and the ubiquitin-like protein ISG15 from host cell substrates as a mechanism to evade innate immune responses during infection. These roles make PLpro an attractive antiviral drug target. Here we demonstrate that ubiquitin variants (UbVs) can be selected from a phage-displayed library and used to specifically and potently block SARS-CoV-2 PLpro activity. A crystal structure of SARS-CoV-2 PLpro in complex with a representative UbV reveals a dimeric UbV bound to PLpro at a site distal to the catalytic site. Yet, the UbV inhibits the essential cleavage activities of the protease in vitro and in cells, and it reduces viral replication in cell culture by almost five orders of magnitude.

Synthetic Methodologies and Therapeutic Potential of Indole-3-Carbinol (I3C) and Its Derivatives

“…The importance of E3 ligases in the ubiquitination of some viral proteins has recently been confirmed by combined multi-omics studies that have allowed the demonstration of the ability of SARS-CoV-2 not only in the remodeling of innate immunity, but also in promoting viral infection, by hijacking specific processes of ubiquitination [ 138 ]. The role of ubiquitination processes in the survival of the virus have also been very recently confirmed by a study that demonstrated how ubiquitin variants are able to inhibit the production of new viral particles after infection, thus preventing the virus from spreading from one cell to another and wreaking havoc in the body [ 139 ]. Furthermore, treatment of human lung organoids (hLORGs) with I3C at different conditions significantly reduces viral entry and the expression of genes involved in innate immunity and inflammation response [ 11 ].…”

What Strengthens Protein-Protein Interactions: Analysis and Applications of Residue Correlation Networks

“…The strategy is transformative for identifying key mutation sites and specific residues to guide rational design with high efficiency for many disease-linked DUBs and Ub-bound proteins 31,32 , including USP4 43 , USP7 44 , USP11 45 , and PLpro of SARS-CoV-2 31 . In contrast to producing variant-specific antibodies/vaccines for diverse spike proteins, blocking functions of viral nonstructural proteins is an alternative therapeutic solution to fight against the COVID-19.…”

“…The re-engineered Ub also has potential advantages such as good binding specificity to PLpro and easy synthesis as compared with chemical compounds. Phage-display screened Ub variants (UbVs) against cognate enzymes including MERS PLpro demonstrated their feasibility in regulating the activities of E3 ligases and DUBs [30][31][32] . The phage-display screening technique focused on three surface patches of Ub to iteratively mutate and select tight binders.…”

“…The phage-display screening technique focused on three surface patches of Ub to iteratively mutate and select tight binders. The raised DUB UbVs are great inhibitors exhibiting IC50 at a range of 1-30 nM [31][32][33] . Alternatively, computational results were used to rationally design a screening library for identifying tight binding regulator UbVs for USP7 34 and USP21 35 .…”

What Strengthens Protein-Protein Interactions: Analysis and Applications of Residue Correlation Networks

“…The protein drug also has potential advantages such as good binding specificity to PLpro and easy synthesis as compared with chemical compounds. Phage-display screened Ub variants (UbVs) against cognate enzymes including MERS PLpro demonstrated their feasibility in regulating the activities of E3 ligases and DUBs 21,22 . Computational results were used to rationally design a screening library for identifying tight binding regulator UbVs for USP7 23 and USP21 24 .…”