Abstract 7014: Characterization and validation of PROTACs that recruit KLHL12

Abstract Proteolysis targeting chimeras (PROTACs) have emerged as a promising strategy for cancer therapy in the field of drug discovery. PROTACs are heterobifunctional small molecules that bind to both a target protein and an E3 ubiquitin ligase, thus facilitating degradation of the target protein of interest. Benefits of PROTACs as an alternative therapeutic strategy include the ability to target inaccessible proteins, the regulation of target protein signaling pathways, and overcoming drug resistance in certain types of cancer. There are more than 600 known E3 ligases in the human genome, but current PROTAC technology has been limited to the use of CRBN, VHL, XIAP, and MDM2. Therefore, the discovery of novel ligands for different E3 ligases could further improve current PROTAC technology. An application that our research group has focused on is exploring the possibility of tissue selective E3 ligases that are overexpressed in cancer cells compared to normal tissue to design tissue selective PROTACs for a target of interest. We have observed that protein levels of the E3 ligase KLHL12 are low in normal tissue and high in cancer cells. Using structure-activity relationship (SAR) by nuclear magnetic resonance (NMR), we identified hits that bound to the KLHL12 E3 ligase, which were then optimized to bind in the range of 10-100 nM Kd by SPR. We then generated KLHL12 based PROTACS that target BRD4 and Bcl-xL using thalidomide and A-115463 derivatives, respectively. PROTAC activity was optimized by measuring protein target degradation in HT29 cells by an in-cell western experiment where our most potent degrader had an average DC50 of 5.05 nM and achieved 89.65% degradation. Mechanism studies were carried out to verify that the PROTACs behaved in both a proteasome and target dependent manner. Future studies will support further characterization of KLHL12 by investigating its expression patterns across various cancer tissues, its functional essentiality in cells, and its involvement in protein-protein interactions. Citation Format: Zoe A. Petros, John Lee Sensintaffar, Anish Kizhakkekkara Vadukoot, Kwangho Kim, Manikandan Thangaraj, Alex G. Waterson, Stephen W. Fesik. Characterization and validation of PROTACs that recruit KLHL12 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 7014.