The Development of a Piperlongumine PROTAC for Targeted TRPV2 Degradation

已发现钙通道、瞬时受体电位香草酸2（TRPV 2）的过度表达的程度与多形性胶质母细胞瘤（GBM）中肿瘤等级的严重程度正相关。GBM是一种非常具有侵袭性的脑癌，其治疗选择已经持续了30多年。Bernardes Group先前的工作已经通过使用机器学习和冷冻电子显微镜确定了天然产物piperlongumine（PL）是TRPV 2的选择性变构拮抗剂。在GBM的原位小鼠模型中，用PL治疗诱导的肿瘤，将其配制成水凝胶以改善药物递送，导致肿瘤几乎完全缓解。该项目的目的是通过开发PL蛋白水解靶向嵌合体（PROTAC）来降解而不是抑制TRPV 2。用PL PROTAC降解TRPV 2将是一种有价值的研究工具，以破译TRPV 2在生理和病理过程中的（未充分研究的）作用，因为TRPV 2仍然是TRP通道的TRPV亚家族中最不被表征和理解的成员，尽管其具有许多病理生理作用/功能。PL PROTAC还可以为具有巨大未满足需求的侵袭性疾病提供比单独的TRPV 2拮抗剂更持久的治疗益处。最后，它可以潜在地用于其他疾病，其中异常TRPV 2表达与观察到的疾病表型密切相关。需要PL类似物来探索其可逆TRPV 2结合的结构-活性关系（SAR），增加其选择性并使其功能化以用于PROTAC开发。为此，PL和选择的类似物进行了合成，并开发了其他类似物的合成方法。与Klenerman实验室合作，测试了细胞内钙成像方法。最后，与Scherman Group合作，证明了PL类似物与新型水凝胶的相容性，以改善药物递送。

The extent of overexpression of the calcium channel, transient receptor potential vanilloid 2 (TRPV2) has been found to positively correlate with the degree of severity of the tumour grade in glioblastoma multiforme (GBM). GBM is a very aggressive brain cancer where therapeutic options have remained intransient for more than 30 years. Previous work in the Bernardes Group has identified, through the use of machine learning and cryo-electron microscopy, that the natural product, piperlongumine (PL), is a selective allosteric antagonist of TRPV2. In an orthotopic mouse model of GBM, treatment of induced tumours with PL, formulated into a hydrogel for improved drug delivery, led to near complete tumour remission.The project aim is to degrade, rather than inhibit, TRPV2 through the development of a PL proteolysis-targeting chimera (PROTAC). Degradation of TRPV2 with a PL PROTAC would be a valuable research tool in order to decipher the (understudied) role of TRPV2 in both physiological and pathological processes since TRPV2 remains the least characterised and understood member of the TRPV subfamily of TRP channels, despite its numerous pathophysiological roles/functions. A PL PROTAC could also provide more longer-lasting therapeutic benefits than solely a TRPV2 antagonist for an aggressive disease with great unmet need. Lastly, it could potentially be used for other diseases where aberrant TRPV2 expression is intimately implicated in the observed disease phenotype.Analogues of PL are required to explore its structure-activity relationship (SAR) for reversible TRPV2 binding, increase its selectivity and functionalise it for PROTAC development. To this end, PL and a selection of analogues were synthesised, and synthetic approaches developed for other analogues. An intracellular calcium imaging method was tested, in collaboration with the Klenerman Lab. Lastly, the compatibility of a PL analogue with a novel hydrogel for improved drug delivery was demonstrated, in collaboration with the Scherman Group.