Design and Synthesis of Novel Action Inhibitors for Bruton's Tyrosine Kinase (BTK)

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Bruton's Tyrosine Kinase (BTK) has a pivotal role in the BCR signalling pathway which is upregulated in B cell malignancies. Activation is dependent on two phosphorylation events, at Y551 and Y223 in the kinase and SH3 domains respectively. Of the remaining three domains, the Pleckstrin Homology (PH) domain is essential for the membrane recruitment which allows for phosphorylation. Conventional therapies target the ATP binding site in the kinase domain, specifically residue C481, to prevent BTK activation. However, this region is relatively conserved across the kinome and prolonged use leads to resistance and relapse as patients develop a C481S mutation. To overcome these issues, this project aims to exploit fragment-based drug discovery (FBDD) against the PH domain. Literature precedent justifies targeting this domain, which proved effective for a similar protein (AKT), as well as utilising FBDD against proteins with a significant role in cancer (CK2). Fragment elaboration led to a parent compound and the project is now well-established with three generations of compounds already synthesised by linking two hit fragments. Further generations of analogues will be synthesised and tested, using a synthetic route in need of optimisation. The scaffold will be investigated by replacing the ketone and phenyl groups and measuring affinity. Biochemical validation will be achieved in collaboration with the Hyvönen Group through mass spectrometry, differential scanning fluorimetry (DSF), X-ray crystallography and fluorescence polarisation (FP). Since the compounds are enantiomeric, key analogues will be sent to Astra Zeneca for separation. Testing the enantiomers individually in conjunction with molecular modelling and covalent docking will help deduce whether their binding affinities are competitive. Biological approaches to confirm inhibition will be investigated given the available facilities, but could include GFP-tagging, immunoprecipitation and blotting or flow cytometry. The research will contribute significantly to the field of covalent inhibition whilst providing a novel solution to the problem of resistance in targeting BTK.