Thesis
Reactive fragment screening : sulfur(VI) fluorides
- Creator
- Rights statement
- Awarding institution
- University of Strathclyde
- Date of award
- 2023
- Thesis identifier
- T17370
- Person Identifier (Local)
- 201979998
- Qualification Level
- Qualification Name
- Department, School or Faculty
- Abstract
- New methods for the identification of ligands with the potential to be developed into chemical probes and novel medicines are essential for the expansion of the liganded, and hence druggable, proteome. Reactive fragment libraries present an innovative approach for the discovery of protein ligands. These libraries are comprised of compounds bearing reactive groups for the targeting of proteinaceous amino acids, and the resultant covalent modification of proteins enables the use of mass spectrometry to identify hits. Previous research efforts in this area have focussed on photoreactive functionalities and cysteine targeting electrophiles. Photoreactive functionalities are capable of covalently crosslinking to any amino acid residue, but the approach is limited by low modification yields which make hits challenging to detect. Cysteine-targeting electrophiles give high modification yields, making hits easier to detect. However, the rarity of cysteine in the proteome (~2%) limits the broader application of this approach. The contents of this thesis describe a reactive fragment screening approach for ligand identification that harnesses the unique properties of sulfur(VI) fluorides. These have been shown to react with a broad range of amino acids, including: serine, threonine, lysine, tyrosine, and histidine. The use of SFs addresses the limitations of the established photoreactive and cysteine-targeting approaches. Consequently, sulfur(VI) fluorides may be used as electrophiles for the covalent modification of proteins, without reliance on the presence of a cysteine residue within a protein pocket. Furthermore, due to the inherent electrophilic mechanism, this approach gives rise to high covalent modification yields, making hits easier to detect by mass spectrometry. Further to this, to address the synthetic bottleneck associated with traditional medicinal chemistry, the research documented here marries the use of SF reactive fragments with a high-throughput chemistry direct-to-biology workflow. This workflow involved the screening of crude reaction mixtures against purified proteins. In this work, a succinimide-activated amide coupling strategy was taken to link a 352-membered library of amine-functionalised fragments to a carboxylic acid-containing sulfur(VI) fluoride reactive functionality. The reactions were carried out in 384-well plates, and the resultant crude libraries were directly screened against a panel of purified proteins, mitigating the time required for compound purification. Follow-up studies were conducted on four hits identified against CAII, and four hits identified against BCL6. Follow-up studies included kinetic analyses to measure modification efficiencies, and identification of the site(s) of binding, which revealed tyrosine and histidine modifications. The CAII hits were also screened in cells by chemoproteomics to assess target engagement, which highlighted the importance of electrophile intrinsic reactivity for cell based screening with SF reactive functionalities. Additionally, the rapid nature of the screening approach was exploited in iterative screens to efficiently drive toward compounds of greater modification efficiency. Further biophysical characterisations were also conducted for BCL6 hits, including X-ray crystallography. Overall, the research described here exemplifies the rapid and robust nature of reactive fragment screening with sulfur(VI) fluorides for hit identification and ligand discovery. It is anticipated that this work can contribute to the expansion of the druggable proteome, and the accelerated discovery of ‘beyond cysteine’ covalent inhibitors.
- Advisor / supervisor
- Tomkinson, Nicholas C. O.
- Bush, Jacob T.
- Resource Type
- Note
- Previously held under moratorium in the Chemistry department (GSK) from 12/04/2023 until 09/06/2025. The confidentiality statement on each page of this thesis DOES NOT apply.
- DOI
- Funder
- Managing organisation
- GlaxoSmithKline
- Embargo Note
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