Tipiracil binds to uridine site and inhibits Nsp15 endoribonuclease NendoU from SARS-CoV-2

Abstract

SARS-CoV-2 Nsp15 is a uridylate-specific endoribonuclease with C-terminal catalytic domain belonging to the EndoU family. It degrades the polyuridine extensions in (−) sense strand of viral RNA and some non-translated RNA on (+) sense strand. This activity seems to be responsible for the interference with the innate immune response and evasion of host pattern recognition. Nsp15 is highly conserved in coronaviruses suggesting that its activity is important for virus replication. Here we report first structures with bound nucleotides and show that SARS-CoV-2 Nsp15 specifically recognizes U in a pattern previously predicted for EndoU. In the presence of manganese ions, the enzyme cleaves unpaired RNAs. Inhibitors of Nsp15 have been reported but not actively pursued into therapeutics. The current COVID-19 pandemic brought to attention the repurposing of existing drugs and the rapid identification of new antiviral compounds. Tipiracil is an FDA approved drug that is used with trifluridine in the treatment of colorectal cancer. Here, we combine crystallography, biochemical and whole cell assays, and show that this compound inhibits SARS-CoV-2 Nsp15 and interacts with the uridine binding pocket of the enzyme’s active site, providing basis for the uracil scaffold-based drug development.

Cryo-EM Structures of the SARS-CoV-2 Endoribonuclease Nsp15

“…5B and Table S2). This observation is in-line with 5¢-UMP-induced ordering of the Nsp15 endoU domain and a previous report that the addition of mononucleotide and dinucleotide analogs significantly increase Nsp15 stability by differential scanning fluorometry (20). Thus, 5¢-UMP binding locks the endoU domain into a fixed position that correlates its motion with the rest of the Nsp15 higherorder assembly.…”

“…The series of structures presented in this manuscript can be used to aid in the development of effective inhibitors which are urgently needed as reported cases of Covid-19 continue to rise. Nucleotide analogues have shown great promise as viral inhibitors against SARS-CoV-2, thus uridine derivatives that could bind the Nsp15 active site should continue to be explored as new therapeutic targets (20). Moreover, recent in silico-based approaches have identified potential Nsp15 inhibitors that await structural and biochemical validation (26,27).…”

“…Yet, mutation to an alanine reduces cleavage activity by more than 5-fold and abrogates uridine specificity such that a single rU or rC containing deoxyribonucleotide are cleaved at comparable rates (5). While this manuscript was in preparation a series of additional Nsp15 crystal structures with different ligands were deposited in the Protein Data Bank (20). One of these structures includes a 5¢-UMP in the active site.…”

Identification of evolutionarily stable sites across the SARS-CoV-2 proteome

“…Remdesivir has been shown to speed up the recovery of COVID-19 patients in clinical trials (50), while the α-ketoamide inhibitor 13b can suppress SARS-CoV-2 replication in cell lines (51). Vir251 and tipiracil were also shown to effectively inhibit the enzymatic activities of their targets (7,8). The remaining drug, sinefungin, is a pan-MTnase (NSP16) inhibitor that inhibits the growth of yeast cells ectopically expressing NSP16 from SARS-CoV (45).…”

“…and is more distantly related to the human infectious alphacoronaviruses HCoV 229E and HCoV NL63 (6). Finding ways to control and prevent further infection are top priorities which include the targeted discovery of drugs that impair viral mechanisms (7)(8)(9) and antigenic epitopes through which vaccines raise immunity (10)(11)(12). This study addresses both by utilizing evolutionary information from SARS-CoV-2 sequence and structural data to search for actionable functional sites for each protein in the SARS-CoV-2 genome.…”