Targeting Rab27A with covalent inhibitors and Exocytosis TArgeting Chimeras (ExoTACs) for lung cancer treatment

小分子鸟苷三磷酸酶(GTP酶)是一组与鸟苷三磷酸(GTP)结合并水解鸟苷二磷酸(GDP)的水解酶。GTP结合的小GTP酶可以通过蛋白质-蛋白质相互作用(PPI)招募特定的效应分子来触发细胞内的信号级联反应。Rab27A是一种小的GTPase蛋白，在外体的分泌中起关键作用，外体是负责细胞间通讯和细胞迁移的膜结合小泡。GTP结合的Rab27A通过与其效应物如SLP2a结合来介导外切体的释放。一些研究表明，Rab27A表达的缺失会由于前侵袭性外切体的减少而损害癌细胞的侵袭和转移。因此，靶向Rab27A是克服肺癌转移和耐药的一种有前景的策略。然而，Rab27A是一个非常具有挑战性的靶点，因为它的构象灵活性以及在广泛保守的GTP结合位点之外缺乏规范的结合口袋。宿主实验室(Tate)使用共价配体方法针对Rab27亚型上效应器结合部位附近的两个非保守半胱氨酸，并鉴定了第一个真正的Rab27A配体系列，优化的HITS显示出对Cys123的高选择性，对Rab27A-SLp2效应器PPI的强烈干扰，以及中等的蛋白质组水平选择性(未发表结果)。在本项目中，我将(1)优化这些起点，设计第一个蛋白质组范围的选择性Rab27A抑制剂来干扰其与效应物的结合，随后(2)表征其对肺癌细胞外体分泌、侵袭和生长的影响。在项目的第二部分，我将(3)开发一类新的双功能分子，称为针对嵌合体的胞吐作用(ExoTAC)，将选择性的Rab27A配体与配体连接到目标蛋白上，首次实现化合物诱导的目标蛋白的胞吐作用。

Small monomeric guanosine triphosphatases (GTPases) are a group of hydrolase enzymes that bind to and hydrolyse a guanosine triphosphate (GTP) to form guanosine diphosphate (GDP). GTP-bound small GTPases can recruit specific effectors through protein-protein interactions (PPIs) to trigger signalling cascades in cells. Rab27A is a small GTPase protein playing a key role in secretion of exosomes, membrane-bound vesicles responsible for intercellular communication and cell migration. GTP-bound Rab27A mediates the release of exosomes by binding to its effectors such as Slp2a. Several studies have shown that loss of Rab27A expression impairs cancer cell invasion and metastasis due to reduction of pro-invasive exosomes. Therefore, targeting Rab27A is a promising strategy to overcome metastasis and drug resistance in lung cancer. However, Rab27A is a very challenging target due to its conformational flexibility and the lack of canonical binding pockets beyond the widely conserved GTP binding site. The host lab (Tate) used a covalent ligand approach to target two non-conserved cysteines in proximity to the effector binding site on Rab27 isoforms and identified the first bona fide Rab27A ligand series, with optimized hits exhibiting high selectivity for Cys123, strong interference with the Rab27A-Slp2 effector PPI, and moderate proteome-level selectivity (unpublished results). In this project, I will (1) optimize these starting points to design the first proteome-wide selective Rab27A inhibitor to interfere with its binding to effectors, subsequently (2) characterizing its impact on exosome secretion, invasion and growth of lung cancer cells. In the second part of the project, I will (3) develop a new class of bifunctional molecules termed Exocytosis TArgeting Chimeras (ExoTACs), linking a selective Rab27A ligand with a ligand to a target protein, enabling for the first time compound-induced exocytosis of a target protein.