PROTEOLYSIS TARGETING CHIMERAS (PROTACS) AS ATTRACTIVE THERAPEUTICS IN IBD THROUGH THE REGULATION OF HYPOXIA-INDUCIBLE FACTORS

Abstract Inflammatory bowel disease (IBD) represents a group of chronic mucosal inflammatory disorders, including Crohn’s disease (CD) and ulcerative colitis (UC). Many IBD patients have limited therapeutic options. Accumulating literature has implicated a prominent role for O2 metabolism and hypoxia in innate immunity of healthy intestinal tissue (“physiologic hypoxia”) and shifts associated with active inflammation (“inflammatory hypoxia”). Reduction of oxygen levels triggers the temporal stabilization of the transcription factors hypoxia-inducible factors HIF1a and HIF2a, which are essential in intestinal homeostasis. HIF-1/2α are regulated by a family of prolyl hydroxylases (PHDs). Inhibition of PHDs results in transient stabilization of HIF-1/2α, and thus, PHD inhibitors (PHDi) have become attractive targets for the therapeutic treatment of IBD and are reported as protective in mouse models of colitis. We have pursued the development of PHDi using proteolysis targeting chimeras (PROTACs), heterobifunctional compounds that contain a protein of interest ligand, a linker, and a E3 ubiquitin ligase ligand. PROTACs hold advantages over small-molecule inhibitors, including enhanced specificity, higher potency, and sustained pathway inhibition. In ongoing studies, we have synthesized and tested a panel of PHD targeting PROTACs. In cultured intestinal epithelial cells, we have observed PROTAC activity at the nanomolar concentrations in vitro, representing a 2-3 log increase in potency compared to PHDi small molecules. For these purposes, we monitor PHD protein degradation and HIF1/2α stabilization through immunoblotting. We have confirmed HIF transcriptional activity in intestinal epithelial cells exposed to these PROTACs through the induction of various HIF target genes. In parallel, we have established a stably transduced GFP-PHD cell line as a high-throughput assay to monitor PHD degradation in real time. Thus, PHD-targeted PROTACs hold promise as viable alternatives to small molecules for the stabilization of HIF in intestinal epithelial cells.