A COVID moonshot: assessment of ligand binding to the SARS-CoV-2 main protease by saturation transfer difference NMR spectroscopy

Anastassia L Kantsadi,Georgios A Spyroulias,Emma Cattermole,Minos-Timotheos Matsoukas,Ioannis Vakonakis

doi:10.1007/s10858-021-00365-x

Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiological cause of the coronavirus disease 2019, for which no effective antiviral therapeutics are available. The SARS-CoV-2 main protease (Mpro) is essential for viral replication and constitutes a promising therapeutic target. Many efforts aimed at deriving effective Mpro inhibitors are currently underway, including an international open-science discovery project, codenamed COVID Moonshot. As part of COVID Moonshot, we used saturation transfer difference nuclear magnetic resonance (STD-NMR) spectroscopy to assess the binding of putative Mpro ligands to the viral protease, including molecules identified by crystallographic fragment screening and novel compounds designed as Mpro inhibitors. In this manner, we aimed to complement enzymatic activity assays of Mpro performed by other groups with information on ligand affinity. We have made the Mpro STD-NMR data publicly available. Here, we provide detailed information on the NMR protocols used and challenges faced, thereby placing these data into context. Our goal is to assist the interpretation of Mpro STD-NMR data, thereby accelerating ongoing drug design efforts.

Highlights

Infections by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) resulted in approximately 1.8 million deaths in 2020 (WHO 2021) and led to the coronavirus 2019 (COVID-19) pandemic (Kucharski et al 2020; Wu et al 2020; Zhu et al 2019)
The 3C-like protease of SARS-CoV-2, known as the viral main protease (Mpro), has been the target of intense study owing to its centrality in viral replication. Mpro studies have benefited from previous structural analyses of the SARC-CoV 3C-like protease and the earlier development of putative inhibitors (Ghosh et al 2007; Verschueren et al 2008; Yang et al 2003,2005)
Nuclear magnetic resonance (NMR), 1H spectra of the protease readily showed the presence of multiple up-field shifted (< 0.5 ppm) peaks corresponding to protein methyl groups (Fig. 1a)

Summary

Introduction

Infections by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) resulted in approximately 1.8 million deaths in 2020 (WHO 2021) and led to the coronavirus 2019 (COVID-19) pandemic (Kucharski et al 2020; Wu et al 2020; Zhu et al 2019). Key viral enzymes and factors, including the reverse-transcriptase machinery, The 3C-like protease of SARS-CoV-2, known as the viral main protease (Mpro), has been the target of intense study owing to its centrality in viral replication. Mpro studies have benefited from previous structural analyses of the SARC-CoV 3C-like protease and the earlier development of putative inhibitors (Ghosh et al 2007; Verschueren et al 2008; Yang et al 2003,2005). The active sites of these proteases are highly conserved, and peptidomimetic inhibitors active against Mpro are potent against the SARS-CoV 3C-like protease (Zhang et al 2020; Rut et al 2020). In order to accelerate M pro inhibitor development, an international, crowd-funded, open-science project was formed under the banner of COVID Moonshot (Achdout et al 2020), combining high-throughput crystallographic screening (Douangamath et al 2020), computational

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