利用水相威悌和點擊化學反應多樣化組裝共價抑制劑及蛋白降解靶向嵌合體

Abstract

標靶型共價抑制劑(Targeted covalent inhibitors，TCIs)及蛋白降解靶向嵌合體(proteolysis targeting chimera，PROTAC)為近年來備受關注的癌症治療策略之一。TCI的優勢在於和標靶蛋白的不可逆共價抑制，可以增強藥效。但其中最大問題為脫靶效應(off-target effect)，一旦形成錯誤不可逆鍵結便會導致嚴重的細胞毒性。另一方面，PROTAC則可以加速標靶蛋白降解，可應用於降解難成藥靶的蛋白及具抗藥性的突變蛋白。然而，較大的PROTAC分子會降低細胞穿膜度，導致生物應用受限。 在本研究中，我們利用二種水相相容的接續反應生成TCI藥物及PROTAC。首先，藉由含拉電子基磷鹽(stable phosphorus ylides)和醛衍生物，可在生理條件下進行威悌反應(Wittig reaction)形成丙烯醯胺(acrylamide)，開啟共價抑制活性。若使用帶有疊氮基團的醛衍生物，則可更進一步和可點擊E3徵招分子進行SPAAC反應，組裝成PROTAC。利用布魯頓酪氨酸激酶(Bruton's tyrosine kinase, BTK) 抑制劑Ibrutinib、Spebrutinib的磷鹽做為測試模型，在試管中我們成功展示上述多樣性的組裝策略。同時在細胞實驗中，抑制劑磷鹽及醛衍生物可成功自組裝並共價標記上目標蛋白BTK。若一併加入可點擊E3徵招分子，可觀察到目標蛋白BTK的降解。利用此策略，多樣化的TCI及PROTAC分子可以在生理條件下簡易地組裝，並進一步進行生物活性測試。

Targeted covalent inhibitors (TCIs) and proteolysis targeting chimera (PROTACs) are currently attractive cancer therapeutic strategies. TCIs irreversibly bind to targeted proteins to enhance their bioactivity. However, nonspecific covalent bond formation might cause off-target cytotoxicity. On the other hand, PROTACs are able to accelerate targeted protein degradation. Moreover, PROTACs potentially target undruggable proteins and drug-resistant mutant proteins. Nevertheless, the larger PROTACs might have reduced cell permeability, hence limiting their biomedical applications. In our work, we utilize two biocompatible reactions to assemble TCIs and PROTACs sequentially. First, under aqueous physiological conditions, stabilized phosphorus ylides and various functionalized aldehydes could undergo Wittig olefination to afford acrylamide inhibitors, which serve as the emerged electrophilic warheads for covalent inhibition. Furthermore, using azido-containing aldehydes, these acrylamide inhibitors could further ligate with clickable E3 ligase ligands through the strain-promoted alkyne-azide cycloaddition (SPAAC) to assemble PROTACs. Choosing Bruton's tyrosine kinase (BTK) inhibitors, Ibrutinib and Spebrutinib, as model compounds, we demonstrated our versatile assemble strategies of TCIs and PROTACs in vitro. In cellular conditions, the acrylamide-containing TCIs can be successfully assembled and in situ label BTK proteins. After ligating with clickable E3 ligase ligands, we can also observe degradation of BTK proteins. In short, through our versatile assemble strategy, various TCIs and PROTACs could be easily formed under mild conditions for subsequent bioactivity screening.