ABSTRACT
SARS-CoV-2 Nsp15 is a uridine-specific endoribonuclease with C-terminal catalytic domain belonging to the EndoU family that is highly conserved in coronaviruses. As endoribonuclease activity seems to be responsible for the interference with the innate immune response, Nsp15 emerges as an attractive target for therapeutic intervention. Here we report the first structures with bound nucleotides and show how the enzyme specifically recognizes uridine moiety. In addition to a uridine site we present evidence for a second base binding site that can accommodate any base. The structure with a transition state analog, uridine vanadate, confirms interactions key to catalytic mechanisms. In the presence of manganese ions, the enzyme cleaves unpaired RNAs. This acquired knowledge was instrumental in identifying Tipiracil, an FDA approved drug that is used in the treatment of colorectal cancer, as a potential anti-COVID-19 drug. Using crystallography, biochemical, and whole-cell assays, we demonstrate that Tipiracil inhibits SARS-CoV-2 Nsp15 by interacting with the uridine binding pocket in the enzyme's active site. Our findings provide new insights for the development of uracil scaffold-based drugs.
Subject(s)
Antiviral Agents/pharmacology , COVID-19 Drug Treatment , COVID-19/virology , Endoribonucleases/antagonists & inhibitors , Enzyme Inhibitors/pharmacology , Pyrrolidines/pharmacology , SARS-CoV-2/drug effects , SARS-CoV-2/enzymology , Thymine/pharmacology , Viral Nonstructural Proteins/antagonists & inhibitors , A549 Cells , Antiviral Agents/chemistry , Antiviral Agents/pharmacokinetics , Catalytic Domain , Crystallography, X-Ray , Endoribonucleases/chemistry , Endoribonucleases/metabolism , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacokinetics , Humans , Ligands , Models, Molecular , Protein Conformation , Pyrrolidines/chemistry , Pyrrolidines/pharmacokinetics , Thymine/chemistry , Thymine/pharmacokinetics , Uridine/metabolism , Viral Nonstructural Proteins/chemistry , Viral Nonstructural Proteins/metabolismABSTRACT
The pandemic caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) continues to expand. Papain-like protease (PLpro) is one of two SARS-CoV-2 proteases potentially targetable with antivirals. PLpro is an attractive target because it plays an essential role in cleavage and maturation of viral polyproteins, assembly of the replicase-transcriptase complex, and disruption of host responses. We report a substantive body of structural, biochemical, and virus replication studies that identify several inhibitors of the SARS-CoV-2 enzyme. We determined the high resolution structure of wild-type PLpro, the active site C111S mutant, and their complexes with inhibitors. This collection of structures details inhibitors recognition and interactions providing fundamental molecular and mechanistic insight into PLpro. All compounds inhibit the peptidase activity of PLpro in vitro, some block SARS-CoV-2 replication in cell culture assays. These findings will accelerate structure-based drug design efforts targeting PLpro to identify high-affinity inhibitors of clinical value.