HYbrid DegRAding Copolymer (HYDRAC): Heterobifunctional polymers for targeted degradation of undruggable proteins.

Abstract

22 Background: Of the ~20,000 proteins encoded by the human genome, only a small subset can be modulated via traditional pharmacological approaches. Included in the list of “undruggable” proteins are ones with pivotal roles in cancer. We aim to expand the arsenal of compounds able to affect the activity of these therapeutically relevant targets by introducing a modular proteomimetic platform technology designed to carry side chains consisting of targeting warheads and recruiters of cellular protein degradation machinery. We term these compounds HYbrid DegRAding Copolymers (HYDRACs) in reference to the multiplexing of different protein binders and degradation inducing sequences. Peptides arranged as HYDRACs exhibit favorable emergent properties including resistance to proteolysis, high levels of cell uptake, and payload-specific activity. Initial studies were conducted focusing on a undruggable transcription factor MYC, which is widely dysregulated in human cancers. Methods: A library of HYDRACs and control polymers were synthesized and were cell-penetrating with antiproliferative effects at sub-micromolar IC50s in a formulation- and MYC-dependent manner across multiple cell lines. Results: HYDRAC treatment resulted in significant decreases in MYC protein levels, which was rescued by proteasome inhibitor MG132 and neddylation inhibitor MLN4924, without changes in mRNA levels. RNAseq showed selective overexpression of MYC pathway genes only in HYDRAC treated cells, confirming on-target effects. Circular dichroism showed strong HYDRAC-MYC interactions, which was supported by the presence of MYC following pull-down of biotin-terminated HYDRACs. Removal of the RRRG degron, scrambling the H1 sequence, and disassociation of the two domains on separate polymers all abrogated MYC-degradation. Mice bearing transplanted Myc-CaP tumors showed delayed tumor growth and reduced tumor cell proliferation following HYDRAC treatment. Mice bearing luminescent Luc-MV-411 tumors treated with Cy5.5-labeled HDRACs showed selective tumor accumulation with detectable fluorescent signal in the tumor up to 72 hours following a single IP administration. The versatility of the platform was demonstrated by substituting the degron for recruiters of three different E3 ligases (VHL, KEAP1, and CRBN), which all maintained ability to degrade MYC. HYDRACs were also able to selectively degrade other protein targets including alpha-syn and RAS. Conclusions: We present a platform technology that addresses challenges inherent to small molecule drug design. HYDRACs have the potential to dramatically alter the drug discovery landscape, allowing for development of modulators of "undruggable" targets. We envision the HYDRAC platform as a generalizable approach to designing degraders of proteins of interest, greatly expanding the therapeutic armamentarium for target protein degradation.