Abstract 1209 Structural And Biochemical Studies Of Proteolysis-Targeting Chimera (PROTAC) Against Oncogenic Anti-Apoptotic Proteins, BCL-xl And Bcl-2

PROteolysis-TArgeting Chimeras (PROTAC) are heterobifunctional small molecules, which binds both an E3 ligase and protein of interest (POI) and brings them in close proximity followed by degradation of POI via the ubiquitin proteasome system. PROTACs provide many advantages compared to the conventional small molecule inhibitors (SMI) like ability to target "undruggable" proteins, required in sub-stoichiometric amounts for POI degradation, is "event-drive" i.e. it doesn't require to inhibit the POI, etc. Moreover, the ability to degrade a POI in a particular cell/tissue using a specific E3 ligases makes it far superior as it overcomes the issues of on-target and dose-limiting toxicity found in case of SMI. Hence PROTACs serves as a very powerful and promising approach for drug development against diseases like cancer. Formation of a successful E3 ligase-PROTAC-POI ternary complex serves as an important but not the only criteria for an efficient POI degradation. One of the key elements that could influence the aforementioned points is the linker molecule that connects the warhead/ligand for E3 ligase and POI respectively. Hence in this respect, a ternary complex structure could be very informative of how modifications in the linker could influence the efficacy of the PROTAC. Both BCL-xL and BCL-2 belong to anti-apoptotic family of proteins, which are notorious for tumor initiation, progression and drug resistance by preventing apoptosis of tumor cells. Although numerous SMIs have been thoroughly investigated as a suitable drug targets but only one has achieved regulatory approval, but suffers with toxicity/resistance issues. In this study, we set out to determine the molecular basis by which 753b (PROTAC molecule) mediates targeted degradation of BCL-xL/BCL-2 and determined crystal structures of both VHL/753b/BCL-xL and VHL/753b/BCL-2 ternary complexes. Interestingly, the two ternary complexes exhibit markedly different architectures that are accompanied by distinct networks of interactions at the VHL/753b-linker/target interfaces. We verified the newly created interfacial contacts within the ternary complexes through mutagenesis followed by in-vitro ubiquitination assays that were optimized in this study. Following this, we identified WH244 based on rationale design and informed structural insights derived from our ternary complex structures involving 753b. Importantly, WH244 degrades BCL-xL/2 with enhanced potency compared to 753b, as verified by our human cancer cell-based studies. Lastly, we determined a crystal structure of VHL/WH244/BCL-2 ternary complex, which provides insights into the molecular basis for its activity. With DT2216 already under clinical trial as the only PROTAC degrader for BCL-xL, our work here lays the foundation for advancing WH244 as a viable candidate for a BCL-xL/BCL-2 dual degrader for use as a cancer therapeutic. Research reported in this study was supported by NIH grants R01 GM115568 and R01 620 GM128731 and CPRIT Rising Star Award RR200030 (Shaun K Olsen). R01 CA242003, R01 CA241191 and R01 AG063801 (Guangrong Zheng and Daohong Zhou)