Comprehensive Fragment Screening of the SARS‐CoV‐2 Proteome Explores Novel Chemical Space for Drug Development

Abstract

SARS‐CoV‐2 (SCoV2) and its variants of concern pose serious challenges to the public health. The variants increased challenges to vaccines, thus necessitating for development of new intervention strategies including anti‐virals. Within the international Covid19‐NMR consortium, we have identified binders targeting the RNA genome of SCoV2. We established protocols for the production and NMR characterization of more than 80% of all SCoV2 proteins. Here, we performed an NMR screening using a fragment library for binding to 25 SCoV2 proteins and identified hits also against previously unexplored SCoV2 proteins. Computational mapping was used to predict binding sites and identify functional moieties (chemotypes) of the ligands occupying these pockets. Striking consensus was observed between NMR‐detected binding sites of the main protease and the computational procedure. Our investigation provides novel structural and chemical space for structure‐based drug design against the SCoV2 proteome.

Crystal structure of the CoV-Y domain of SARS-CoV-2 nonstructural protein 3

“…To gain insights into functions of the nsp3 CoV-Y domain, we partnered with the international COVID19-NMR consortium ( https://covid19-nmr.de/ ) in a large-scale NMR-based ligand screening campaign that aimed to identify poised fragments targeting SARS-CoV-2 proteins 38 . The DSI (Diamond-SGC-iNEXT)-poised library consisting of 768 fragments represents a small collection of diverse compounds (< 300 Da) that bind promiscuously but cover a large chemical space and facilitate streamlined downstream hit optimization 39 .…”

“…The DSI (Diamond-SGC-iNEXT)-poised library consisting of 768 fragments represents a small collection of diverse compounds (< 300 Da) that bind promiscuously but cover a large chemical space and facilitate streamlined downstream hit optimization 39 . Four different 1 H-based NMR experiments of ligands, including chemical shift perturbation, waterLOGSY, saturation transfer difference and differential T 2 -relaxation, in the presence and absence of the unlabeled CoV-Y protein were recorded and analyzed 38 . In total, 81 displayed changes in at least one of the four NMR experiments.…”

“…Among them, 17 fragments were qualified as binders for CoV-Y based on the spectral changes observed in at least two out of four NMR experiments (Supplementary Table S3 ). Following the computational mapping strategy developed by the consortium 38 , we next employed FTMap 40 to identify accessible surface cavities in the CoV-Y structure. Using 16 small organic compounds provided by the FTMap server as scanning probes, we identified ten ligand-binding hot spots (clusters 0–9) that are clustered largely around the deep cleft in the middle of the V-shaped structure crossing the interfaces between Y2/Y3 and Y2/Y4 (Fig.…”

“…Ligand-based fragment screening by NMR spectroscopy using unlabeled CoV-Y was conducted previously at BMRZ at the Goethe University in Frankfurt, Germany as described 38 . The DSi-Poised library with 768 diverse fragments was screened in 64 mixes each containing 12 compounds as described previously 39 .…”

“…Binding of the compounds to the protein was evaluated by four different 1 H-based NMR measurements in the presence and absence of the protein: chemical shift perturbation, waterLOGSY effects, saturation transfer differences and signal intensity change in T 2 -relaxation experiments. In total 17 compounds were identified as CoV-Y binders based on the spectrum changes observed in at least two out of four NMR experiments 38 (Supplementary Table S3 ). The surface cavities of CoV-Y were analyzed using the default settings at the FTMap web server 40 ( https://ftmap.bu.edu/login.php ).…”