Targeted degradation of undruggable proteins using a novel heterobifunctional proteomimetic platform.

47 Background: Of the ~20,000 proteins in the human genome, only a subset can be modulated via traditional pharmacological approaches. “Undruggable” targets currently include proteins with pivotal roles in cancer pathogenesis. Our work aims to introduce a new paradigm for engaging these clinically relevant proteins using a modular platform we term the HYbrid DegRAding Copolymers (HYDRACs). HYDRACs multiplex targeting warheads with degradation inducers and have long circulation times plus high cell uptake. Preliminary studies on MYC and RAS, targets of great pharmaceutical interest, highlight the potential of this platform to revolutionize drug design. Methods: HYDRACs containing MYC or RAS targeting peptides randomly copolymerized with degrons were synthesized using ROMP. Fluorescent and biotin-tags were incorporated for use in uptake and pull-down assays. Viability following treatment with MYC-HYDRACs was performed in multiple cell lines with both cellular (MYC-independent lines) and polymer composition controls. Target engagement was assessed via immunoprecipitation and circular dichroism with protein degradation monitored by WB. Unbiased whole proteome analysis and RNAseq were done to confirm observed effects were selectively on-target. In vivo efficacy and biodistribution was assessed in tumor-bearing mice. Results: HYDRACs show high levels of cell uptake and antiproliferative effects at sub-micromolar concentrations in a formulation- and target-dependent manner. Cells treated with MYC-HYDRACs displayed reduced MYC protein levels, which were rescued by proteasome or neddylation inhibition. Swapping out the degron for various validated E3 ligase recruiters maintained MYC degradation, highlighting the “plug-and-play” nature of this approach. On-target activity was confirmed via RNAseq. Biophysical analysis showed strong HYDRAC-protein interactions, orthogonally supported by the presence of target protein following pull-down of biotin-terminated HYDRACs. Target protein degradation only occurred following treatment with an intact HYDRAC compound. Mice bearing MYC-CaP tumors showed delayed tumor growth following treatment with MYC-HYDRACs, with compound accumulation in the tumor for up to 72 h following a single injection. Generalizability was demonstrated against RAS, with RAS-HYDRACs capable of degrading KRAS across multiple different alleles, acting as a pan-KRAS degrader. Conclusions: We present a novel platform technology that addresses the challenges inherent to peptide delivery approaches. HYDRACs have the potential to dramatically alter the drug discovery landscape, allowing for the development of cancer-relevant target modulators for which there are no current viable therapies. We envision the HYDRAC platform as a generalizable approach to designing degraders of proteins of interest, greatly expanding the therapeutic armamentarium for TPD.