Discovery of Novel Allosteric Sites of SARS-CoV-2 Papain-Like Protease (PLpro)

Abstract Papain-like protease (PLpro) is a viral protease found in some coronaviruses, including SARS-CoV-2, the virus that causes COVID-19. Inhibition of PLpro activity could potentially limit viral replication, making it an attractive target for antiviral drug development. This work describes the discovery of novel allosteric residues of SARS-CoV-2 PLpro that can be targeted with antiviral drugs. First, a computational analysis was performed to identify potential druggable pockets on the surface of SARS-CoV-2 PLpro. The computational analysis predicted three druggable pockets around PLpro’s surface and are located at the interfaces of its four domains. Pocket 1 is located at the interface between the Ub1 and thumb domains, pocket 2 is at the interface between the thumb, finger, and palm domains, and pocket 3 is at the interface between the finger and palm domains. Targeted alanine mutagenesis of selected residues with important structural interactions revealed 14 residues that are essential for maintaining a catalytically active and thermodynamically stable PLpro. Namely, these residues are T10, D12, T54, Y72, and Y83 in pocket 1; Q122, S239, Q237, H275, and S278 in pocket 2; and S212, Y213, Y251, K254, and Y305, in pocket 3. In addition to this, residues that were found important to catalysis were recovered by mutating them to amino acids of similar physicochemical properties to further elucidate the mechanistic properties of the local interactions they make. Experimental work confirms essential contacts are present in the allosteric sites of PLpro that could be targeted with non-competitive inhibitors as novel therapeutics against COVID-19.