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

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.

Inhibition of SARS–CoV–2 NSP–15 by Uridine–5′–Monophosphate Analogues Using QSAR Modelling, Molecular Dynamics Simulations, and Free Energy Landscape

“…Furthermore, these compounds exhibited interactions with key residues Thr341, Tyr343, and Ser294 within the active site, establishing crucial hydrogen bonds with these pivotal residues, as well as with the catalytic triad residues His235, His250, and Lys290. These findings provide compelling evidence of the compounds' strong and specific interactions with the Nsp15 protein, emphasizing their potential as potent inhibitors.These insights aligned with prior studies ( Godoy et al, 2023 , Hong et al, 2021 , Sinha et al, 2020 ). It also coincides with the binding site residues (residues His235, His250, Lys290, Ser294, and Tyr343) of uridine 5′–monophosphate (5′–UMP) in the active site of Nsp15 ( Pillon et al, 2021 ).…”

“…Numerous prior investigations have delved into the intricate interactions between the identified compounds and specific amino acid residues, namely Gln245, Gly248, Lys290, Ser294, Thr341, and Tyr343. These insights were gleaned from the collective findings of three distinct studies ( Godoy et al, 2023 , Hong et al, 2021 , Sinha et al, 2020 ). In a distinct analysis, researchers explored the binding of uridine 5′–monophosphate (5′–UMP) to the active site of Nsp15, elucidating the formation of hydrogen bonds with residues His235, His250, Lys290, Ser294, and Tyr343 ( Pillon et al, 2021 ).…”

“…It is well known that Nsp15 (nonstructural protein–15) is crucial to the development of the pathology caused by coronaviruses. The C–terminal 'EndoU' domain of Nsp15 serves as a uridylate–specific endoribonuclease in the context of single– and double–stranded RNA molecules by cleaving the 3′ end of pyrimidines, specifically uridylates ( Deng and Baker, 2018 , Godoy et al, 2023 , Ivanov et al, 2004 , Kang et al, 2007 ). Evidence suggests that this RNase degrades dsRNA intermediates produced by viruses, thereby blocking their recognition by the host ( Deng et al, 2017 , Kindler et al, 2017 ).…”

“…Several studies showed identification of successful inhibitors against SARS–CoV–2 infections that targeted Nsp15 protein ( Chandra et al, 2021 , Hong et al, 2021 , Kim et al, 2021 , Saramago et al, 2022 , Sinha et al, 2020 ). The catalytic triad of Nsp15′s active site consists of two histidines and a lysine, and it bears a striking resemblance to the active site of the well–studied endoribonuclease RNase A ( Godoy et al, 2023 , Kim et al, 2020b , Pillon et al, 2021 ). Since Nsp15 appears to be the endoribonuclease responsible for suppressing the innate immune response, it presents itself as a promising therapeutic target.…”