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

布鲁顿酪氨酸激酶（BTK）在BCR信号传导途径中具有关键作用，其在B细胞恶性肿瘤中上调。激活依赖于两个磷酸化事件，分别在激酶和SH3结构域中的Y551和Y223处。在其余三个结构域中，Pleckstrin同源（PH）结构域对于允许磷酸化的膜募集是必需的。常规疗法靶向激酶结构域中的ATP结合位点，特别是残基C481，以防止BTK活化。然而，该区域在激酶组中相对保守，并且随着患者发生C481S突变，长期使用会导致耐药性和复发。为了克服这些问题，该项目旨在利用基于片段的药物发现（FBDD）对PH域。文献先例证明靶向该结构域是合理的，这证明对类似蛋白质（AKT）有效，以及利用FBDD对抗在癌症中具有重要作用的蛋白质（CK2）。片段精细化导致母体化合物，该项目现在已经建立了三代化合物，通过连接两个命中片段已经合成。将使用需要优化的合成路线合成和测试更多代的类似物。将通过替换酮和苯基并测量亲和力来研究支架。将与Hyvönen Group合作，通过质谱法、差示扫描荧光法（DSF）、X射线晶体学和荧光偏振法（FP）进行生化验证。由于化合物是对映体，关键类似物将被送往阿斯利康进行分离。结合分子建模和共价对接单独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。将在现有设施的情况下研究确认抑制作用的生物学方法，但可能包括GFP标记、免疫沉淀和印迹或流式细胞术。该研究将为共价抑制领域做出重大贡献，同时为靶向BTK的抗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.