Allosteric regulation and crystallographic fragment screening of SARS-CoV-2 NSP15 endoribonuclease

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Godoy, Andre Schutzer ; Nakamura, Aline Minalli ; Douangamath, Alice ; Song, Yun ; Noske, Gabriela Dias ; Gawriljuk, Victor Oliveira ; Fernandes, Rafaela Sachetto ; Pereira, Humberto D. Muniz ; Oliveira, Ketllyn Irene Zagato ; Fearon, Daren ; Dias, Alexandre ; Krojer, Tobias ; Fairhead, Michael ; Powell, Alisa ; Dunnet, Louise ; Brandao-Neto, Jose ; Skyner, Rachael ; Chalk, Rod ; Bajusz, David ; Bege, Miklos ; Borbas, Aniko ; Keseru, Gyoergy Miklos ; von Delft, Frank ; Oliva, Glaucius

• Document type: Journal article
• Source: Nucleic Acids Research; v. 51, n. 10, p. 16-pg., 2023-04-28.

Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019 (COVID-19). The NSP15 endoribonuclease enzyme, known as NendoU, is highly conserved and plays a critical role in the ability of the virus to evade the immune system. NendoU is a promising target for the development of new antiviral drugs. However, the complexity of the enzyme's structure and kinetics, along with the broad range of recognition sequences and lack of structural complexes, hampers the development of inhibitors. Here, we performed enzymatic characterization of NendoU in its monomeric and hexameric form, showing that hexamers are allosteric enzymes with a positive cooperative index, and with no influence of manganese on enzymatic activity. Through combining cryo-electron microscopy at different pHs, X-ray crystallography and biochemical and structural analysis, we showed that NendoU can shift between open and closed forms, which probably correspond to active and inactive states, respectively. We also explored the possibility of NendoU assembling into larger supramolecular structures and proposed a mechanism for allosteric regulation. In addition, we conducted a large fragment screening campaign against NendoU and identified several new allosteric sites that could be targeted for the development of new inhibitors. Overall, our findings provide insights into the complex structure and function of NendoU and offer new opportunities for the development of inhibitors. (AU)

• FAPESP's process: 13/07600-3 - CIBFar - Center for Innovation in Biodiversity and Drug Discovery
• Grantee: Glaucius Oliva
• Support Opportunities: Research Grants - Research, Innovation and Dissemination Centers - RIDC
• FAPESP's process: 15/16811-3 - Multi-user equipament aproved in grant 2014:15546-1: crystallization robot Gryphon
• Grantee: Richard Charles Garratt
• Support Opportunities: Multi-user Equipment Program
• FAPESP's process: 16/19712-9 - Structural characterization of Zika virus proteins and search for antiviral agents
• Grantee: Andre Schutzer de Godoy
• Support Opportunities: Scholarships in Brazil - Post-Doctoral