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

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.

Computationally driven discovery of SARS-CoV-2 Mproinhibitors: from design to experimental validation

“…61) -Fig. 1A) shows one of the highest binding affinities 62 and therefore provides a reasonable starting point for fragmentbased design of novel M pro inhibitors.…”

NMR Spectroscopy of the Main Protease of SARS‐CoV‐2 and Fragment‐Based Screening Identify Three Protein Hotspots and an Antiviral Fragment

“…Most of the unassigned proton amides lie in the first two β‐barrel domains or at the dimerization interface (Figure S2). Whereas previous attempts to record multidimensional NMR data on SARS‐CoV [38] and SARS‐CoV‐2 [39] 3CLp have failed, these new NMR data open the field to a large range of future studies of the dimeric 3CLp in solution and at temperature close to physiological, an important parameter when considering dynamics. To assess the potential of our experimental system, we analyzed the 3CLp spectral perturbations upon binding of either boceprevir or GC376 (Figures S3,S4).…”

“…Whereas structural biology plays a central role in drug discovery and drug development, up to date, NMR spectroscopy has not successfully been pushed forward to study the 3CLp from coronaviruses. [ 37 , 38 , 39 ] In this work, we used solution‐state NMR spectroscopy to study the dimeric 3CLp protease of the SARS‐CoV‐2, which is one of the main targets to develop efficient antivirals to fight against the COVID‐19 pandemic. Considering the high sequence conservation between the 3CLps,[ 20 , 40 ] our data will also be valuable for others β‐coronaviruses, such as MERS‐CoV and SARS‐CoV (67 % and 98 % sequence similarity, respectively), and possibly for future emerging β‐coronaviruses.…”

An Experimental Toolbox for Structure‐Based Hit Discovery for P. aeruginosa FabF, a Promising Target for Antibiotics

“…It was recently shown that the STD ratio is roughly inversely proportional to K D . [41] While the ratio is influenced by the extent of NOE magnetisation transfer from protein to ligand, dissociation rate and effective ligand concentration, these influences should be minimized across variable fragment scaffolds by only considering the strongest STD signal in the aromatic region and using presumably weakly binding, fast exchanging ligands. Six compounds ( 1 , 2 , and 5 – 8 , Table 1 ) also showed binding in WaterLOGSY experiments and were progressed to competition experiments (Figure 7 b, Figure S6).…”

“…In combination with a competing ligand or the use of different enzyme variants they can also deliver information about the binding sites of the compounds. In addition, STD ratio [41] and % displacement can be used to rank compounds by affinity. In the case of the compounds were both values could be determined ( 1 , 2 , 5 and 7 ) they showed the same trends indicating that these measures are in agreement (Table 1 ).…”