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

Bruton‘s酪氨酸激酶(BTK)在BCR信号通路中起关键作用，BCR信号通路在B细胞恶性肿瘤中上调。激活依赖于两个磷酸化事件，分别位于KK和SH3结构域的Y551和Y223。在剩下的三个结构域中，Pleckstrin同源(PH)结构域对于允许磷酸化的膜募集是必不可少的。传统的治疗方法以激活域中的ATP结合位点为靶点，特别是残基C481，以防止BTK激活。然而，该区域在整个基因组中相对保守，当患者发生C481S突变时，长期使用会导致耐药性和复发。为了克服这些问题，该项目旨在开发针对PH域的基于片段的药物发现(FBDD)。文献的先例证明了瞄准这一领域的合理性，这被证明对类似的蛋白质(AKT)有效，以及利用FBDD来对抗在癌症中具有重要作用的蛋白质(CK2)。片段精加工导致了母体化合物，该项目现在已经很好地建立了，通过连接两个热门片段已经合成了三代化合物。未来几代类似物将使用需要优化的合成路线进行合成和测试。将通过取代酮和苯基并测量亲和力来研究支架。将与Hyvönen集团合作，通过质谱学、差示扫描荧光法(DSF)、X射线结晶学和荧光偏振(FP)实现生化验证。由于这些化合物是对映体，关键类似物将被送往阿斯特拉捷利康进行分离。结合分子建模和共价对接分别测试对映体将有助于推断它们的结合亲和力是否具有竞争性。在现有设施的情况下，将研究确认抑制的生物学方法，但可能包括GFP标记、免疫沉淀和印迹或流式细胞术。这项研究将对共价抑制领域做出重大贡献，同时为靶向BTK的耐药性问题提供新的解决方案。

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.