Lou, Jiaqi; (2024) Fragment-based drug discovery targeting SARS-CoV-2 nsp10 and ATP catalytic cycle study on human microtubule depolymerase kinesin MCAK, with biophysical characterisations of ligand interactions with Dengue virus RdRp and Keap1 proteins. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Fragment-based drug discovery (FBDD) is one of the most successful ways of identifying and optimising fragment hits for protein and nucleotide targets. It has been significantly developed over the past 20-30 years and has become a common alternative to more traditional drug discovery methods such as high-throughput screening. In the concept of FBDD, the chemical space of a biomolecule is more efficiently probed by screening through fragments rather than through small molecules. Therefore, by screening through a collection of fragments with up to 12 heavy atoms, there is a greater chance to identify fragment hits to the biomolecule target. In this PhD study, we mainly focused on two protein targets – SARS-CoV-2 Non-Structural Protein 10 (nsp10), and human kinesin depolymerase Kif2C protein, also known as Mitotic Centromere-Associated Kinesin (MCAK). Nsp10 is an important modulator in SARS-CoV-2. It has been shown to be a critical activator of two viral proteins – nsp14 and nsp16, which contribute to viral RNA proofreading and cap formation. Therefore, drugs that target nsp10 can potentially interfere with RNA replication, which represents an intervention strategy to inhibit coronavirus replication. The second project focused on MCAK, a protein integral to cellular processes and mitotic cycles. It is a key modulator of microtubule length and is involved in spindle formation, correcting microtubule-kinetochore attachments and it plays a crucial role in chromosome movements. Studies have shown that MCAK is overexpressed in various cancers, such as breast, colon and prostate. Targeting MCAK alone or combined with tubulin poisons can significantly reduce cancer cell proliferation, suggesting that MCAK is a potential target for drug discovery in cancer chemotherapy. In our study, we applied fragment screening methods to discover fragment hits to SARS-CoV-2 nsp10, and by applying the FBDD strategies, we employed ‘SAR-by-catalogue' approach to identify more potent analogues of our initial fragment hits targeting the proteins of interest. Moreover, for the MCAK project, we utilised protein x-ray crystallography to study its ATP catalytic cycle in various nucleotide-bound and nucleotide-free states, aiming to understand the conformational changes throughout the ATP catalytic cycle and how they contribute to the interactions with microtubules.

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