Shi, Xinyue;
(2025)
Use of fragment-based drug
discovery to identify potential
inhibitors targeting SARS-CoV-2 non-structural protein 14 and DENV-3
methyltransferase.
Doctoral thesis (Ph.D), UCL (University College London).
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Abstract
The work described in the thesis focuses on identifying fragments and analogues as starting points to develop inhibitors targeting Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) non-structural protein 14 (nsp14) and Dengue Virus serotype 3 (DENV-3) non-structural protein 5 (NS5). Both nsp14 and NS5 have a methyltransferase (MTase) domain that is involved in the capping of the 5’ end of viral mRNA. Attempts to crystallise the SARS-CoV-2 nsp14 MTase through designing more stable constructs and trying various crystallisation optimisation techniques are first discussed. S-(5’-Adenosyl)-L-methionine (SAM), S-(5’-Adenosyl)-L-homocysteine (SAH) and cap analogues are crucial substrates during methylation. Their interaction with nsp14 MTase was studied using thermal shift assays (TSA), microscale thermophoresis (MST) and small-angle X-ray scattering. The second part of the thesis covers the screening of 100 fragments from the Maybridge library using MST. The fragment hits were later tested by X-ray crystallography once nsp14 crystals had been obtained. Nsp14 was crystallised using published conditions and optimised for larger crystals and better reproducibility by seeding. 78 commercially available structural analogues of the six fragment hits were then purchased and soaked with nsp14 crystals, resulting in nine analogue hits. The analysis of the 19 binding sites across nsp14 and the binding affinity measurements of fragments and analogues to nsp14 provided information for further optimisation of the analogue hits. Finally, two short DENV-3 MTase constructs were crystallised and used to verify 20 fragment hits identified by a previous PhD student Shymaa Damfo. Crystals obtained from a longer construct were twinned. Less than 5% of the tested crystals obtained from the two shorter constructs did not show twinning. Other crystallisation conditions from seeding and screening were also tested but no improvement in crystal quality was observed. Additional MTase constructs and crystallisation conditions are required to further study the fragment hits.
| Type: | Thesis (Doctoral) |
|---|---|
| Qualification: | Ph.D |
| Title: | Use of fragment-based drug discovery to identify potential inhibitors targeting SARS-CoV-2 non-structural protein 14 and DENV-3 methyltransferase |
| Language: | English |
| Additional information: | Copyright © The Author 2025. Original content in this thesis is licensed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International (CC BY-NC 4.0) Licence (https://creativecommons.org/licenses/by-nc/4.0/). Any third-party copyright material present remains the property of its respective owner(s) and is licensed under its existing terms. Access may initially be restricted at the author’s request. |
| UCL classification: | UCL UCL > Provost and Vice Provost Offices > UCL BEAMS UCL > Provost and Vice Provost Offices > UCL BEAMS > Faculty of Engineering Science > Dept of Biochemical Engineering |
| URI: | https://discovery.ucl.ac.uk/id/eprint/10203483 |
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