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

小单体鸟苷三磷酸酶（GTP酶）是一组结合并水解鸟苷三磷酸（GTP）以形成鸟苷二磷酸（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）募集特异性效应物以触发细胞中的信号级联。Rab 27 A是一种小的GT3蛋白，在分泌外泌体、负责细胞间通讯和细胞迁移的膜结合囊泡中起关键作用。GTP结合的Rab 27 A通过与其效应物如Slp 2a结合来介导外泌体的释放。几项研究表明，Rab 27 A表达的缺失由于促侵袭外泌体的减少而损害癌细胞侵袭和转移。因此，靶向Rab 27 A是克服肺癌转移和耐药的一个有希望的策略。然而，Rab 27 A是一个非常具有挑战性的目标，由于其构象的灵活性和缺乏典型的结合口袋以外的广泛保守的GTP结合位点。宿主实验室（Tate）使用共价配体方法靶向Rab 27同种型上效应物结合位点附近的两个非保守半胱氨酸，并鉴定了第一个真正的Rab 27 A配体系列，优化的命中表现出对Cys 123的高选择性，对Rab 27 A-Slp 2效应物PPI的强干扰，以及中等的蛋白质组水平选择性（未发表的结果）。在这个项目中，我将（1）优化这些起点，设计第一个蛋白质组选择性Rab 27 A抑制剂，以干扰其与效应子的结合，随后（2）表征其对肺癌细胞外泌体分泌，侵袭和生长的影响。在该项目的第二部分，我将（3）开发一类新的双功能分子，称为胞吐Targeting嵌合体（ExoTACs），将选择性Rab 27 A配体与靶蛋白的配体连接起来，首次实现化合物诱导的靶蛋白胞吐。

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selectivity for Cys123, strong interference with the Rab27A-Slp2 effector PPI, and moderate proteome-level selectivity (unpublished results).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.