Ma, Shumeng; (2024) Fragment-based X-ray screening targeting SARS-CoV-2 Nsp1 and inhibitor discovery for SARS-CoV-2 Nsp5 and Nsp15. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Coronaviruses (CoVs) have caused global pandemics with high morbidity and mortality. Non-structural protein 1 (nsp1), a unique and conserved leader protein in β-CoVs, including SARS, MERS, and SARS-CoV-2, is a crucial virulence factor for suppressing host gene expression and modulating the host immune response. Therefore, it is regarded as an exploitable target for antiviral agent discovery. However, to date, no inhibitors of nsp1 for SARS-CoV-2 or other CoVs have been identified, and there is limited knowledge regarding its druggable sites. Herein, the structure of N-terminal (NT-) SARS-CoV-2 nsp1 at an atomic resolution was reported. Using fragment-based screening via X-ray crystallography, several fragment hits bound into two distinct binding sites of NT-SARS-CoV-2 nsp1 were identified. Subsequently, the binding affinities were determined by orthogonal assays. Long-wavelength experiments and PanDDA were conducted to determine the binding orientation(s) of fragments. The sequence and functional similarities observed among nsp1s from SARS, MERS, and SARS-CoV-2 suggest the potential for identified fragment hits to exhibit a cross-inhibitory effect against their nsp1s. However, the crystal structures of nsp1s of SARS and MERS and the C-terminal (CT-) SARS-CoV-2 nsp1 remain undetermined. Therefore, crystallisation trials were conducted for these proteins. Additionally, the room temperature structure of NT-SARS-CoV-2 nsp1 was compared with its cryogenic structure. The potential interactions between nsp1 and nsp10, as well as between nsp1 and its potential inhibitor Montelukast, were explored. In view of the indispensable role of SARS-CoV-2 nsp15 in viral RNA processing facilitating immune evasion from the host immune system and nsp5 in processing of polyproteins translated from the viral RNA, inhibitor discovery was conducted for both proteins, and the protein-protein interaction of nsp15 with other nsps was also explored. In conclusion, the structural characterisation, fragment/inhibitor discovery for the three nsps, and intermolecular interaction studies with other nsps enhance the understanding of their biological functions and contribute to the drug discovery process for these (potential) drug targets.

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