TR-107, An Agonist of Caseinolytic Peptidase Proteolytic Subunit, Disrupts Mitochondrial Metabolism and Inhibits the Growth of Human Colorectal Cancer Cells
Oxidative phosphorylation (OXPHOS) is an essential metabolic process for cancer proliferation and therapy resistance. The ClpXP complex maintains mitochondrial proteostasis by degrading misfolded proteins. Madera Therapeutics has developed a class of highly potent and selective small-molecule activators (TR compounds) of the ClpXP component caseinolytic peptidase proteolytic subunit (ClpP). This approach to cancer therapy eliminates substrate recognition and activates non-specific protease function within mitochondria, which has shown encouraging preclinical efficacy in multiple cancer types. The class-leading compound, TR-107, has demonstrated significantly improved potency in ClpP affinity and activation and enhanced pharmacokinetic properties over the multi-targeting clinical agent ONC201. In this study, we investigate the in vitro efficacy of TR-107 against human colorectal cancer (CRC) cells. TR-107 inhibited CRC cell proliferation in a dose- and time-dependent manner and induced cell cycle arrest at low nanomolar concentrations. Mechanistically, TR-107 downregulated the expression of proteins involved in the mitochondrial unfolded protein response (UPRmt) and mtDNA transcription and translation. TR-107 attenuated oxygen consumption rate and glycolytic compensation, confirming inactivation of OXPHOS and a reduction in total cellular respiration. Multi-omics analysis of treated cells indicated a downregulation of respiratory chain complex subunits and an upregulation of mitophagy and ferroptosis pathways. Further evaluation of ferroptosis revealed a depletion of antioxidant and iron toxicity defenses that could potentiate sensitivity to combinatory chemotherapeutics. Together, this study provides evidence and insight into the subcellular mechanisms employed by CRC cells in response to potent ClpP agonism. Our findings demonstrate a productive approach to disrupting mitochondrial metabolism, supporting the translational potential of TR-107. Overall design: To investigate the mechanism of action and efficacy of TR-107 in vitro, we treated the human CRC cell line, HCT116, with vehicle control (DMSO, 0.1%) and 50nM TR-107 for 24 hours. Total RNA was isolated and RNA-seq was conducted. We then performed differential gene expression analysis using RNA-seq data from three biological replicates of each treatment condition to characterize significant transcriptomic changes in response to TR-107 treatment. Additionally, we conducted KEGG pathway analysis to identify upregulated and downregulated gene pathways following TR-107 treatment.