TR-107, an Agonist of Caseinolytic Peptidase Proteolytic Subunit, Disrupts Mitochondrial Metabolism and Inhibits the Growth of Human Colorectal Cancer Cells

Abstract

Oxidative phosphorylation 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 nonspecific protease function within mitochondria, which has shown encouraging preclinical efficacy in multiple malignancies. The class-leading compound TR-107 has demonstrated significantly improved potency in ClpP affinity and activation and enhanced pharmacokinetic properties over the multitargeting clinical agent ONC201. In this study, we investigate the in vitro efficacy of TR-107 against human colorectal cancer cells. TR-107 inhibited colorectal cancer 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 and mitochondrial DNA transcription and translation. TR-107 attenuated oxygen consumption rate and glycolytic compensation, confirming inactivation of oxidative phosphorylation and a reduction in total cellular respiration. Multiomics 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 colorectal cancer cells in response to potent ClpP agonism. Our findings demonstrate a productive approach to disrupting mitochondrial metabolism, supporting the translational potential of TR-107.

Figure 1.. TR-107 exerts potent inhibition of CRC viability.

Colorectal cancer cells (A-B) DLD-1 and LoVo (CMS1), (C-D) LS1034 and NCI-H508 (CMS2), (E-F) HT29 and LS 174T (CMS3), and (G-H) HCT116 and RKO (CMS4) were treated with vehicle control (0.1% DMSO) or various log-fold concentrations of TR-107 as described in Materials and Methods. CellTiter-Glo cell viability assay was performed 72 hours post-treatment. IC₅₀ values (nmol/L) were calculated with GraphPad Prism and listed on each respective graph. Data are presented as mean ± SD (N=6).

Figure 2.. TR-107 inhibits CRC cell proliferation in a dose- and time-dependent manner.

Colorectal cancer cells (A-B) DLD-1 and LoVo (CMS1), (C-D) LS1034 and NCI-H508 (CMS2), (E-F) HT29 and LS 174T (CMS3), and (G-H) HCT116 and RKO (CMS4) were treated with vehicle control (0.1% DMSO) or concentrations of TR-107 (10 nmol/L, 50 nmol/L, and 1 μmol/L) for 24, 48, and 72 hours. Cell proliferation was quantified at each time point with the Z-Series Coulter Counter. Data are presented as mean ± SD values (N=3). Each biological replicate was generated by averaging three technical replicates of proliferation count. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Figure 3.. TR-107 reduces mitochondrial metabolic function in CRC cells.

(A-D) Mitochondrial oxygen consumption rate (OCR) and (E-H) glycolytic proton efflux rate (PER) were evaluated in colorectal cancer cells DLD-1, LS1034, HT29, and HCT116 treated with vehicle control (0.1% DMSO) or indicated concentrations of TR-107 for 12 hours. Quantification of OCR and PER was measured with the Seahorse XF Real-Time ATP Rate Assay. Baseline rates were captured for the first three time points, followed by injection of indicated compounds at concentrations described in Materials and Methods. Data are presented as mean ± SD values (N=6).

Figure 4.. TR-107 downregulates the expression of essential mitochondrial proteins.

Colorectal cancer cells (A) DLD-1, (B) LS1034, (C) HT29, and (D) HCT116 were treated with vehicle control (0.1% DMSO) or TR-107 (50 nmol/L) for 24 hours. Immunoblot was performed for various mitochondrial regulatory and metabolic proteins. (E-H) Band densitometry analysis was conducted to quantify relative protein expression and normalized to β-actin control. Densitometry was measured with ImageJ and analyzed with GraphPad Prism as described in Materials and Methods. Data are presented as mean ± SD values (N=3). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Figure 5.. Multi-omics analysis reveals the downregulation of mitochondrial metabolism and the upregulation of mitophagy and ferroptosis pathways in CRC cells treated with TR-107.

Galaxy Reference-based RNA-Seq analysis was conducted on HCT116 cells treated with vehicle control (0.1% DMSO) or TR-107 (50 nmol/L) for 24 hours. (A) Close proximity comparison confirmed a strong correlation between up and downregulated transcripts with respect to biological replicate and treatment condition (B) Upregulated pathway analysis identified related transcripts, which were grouped by pathway and filtered by significance and strength of association (Odds ratio) with treatment condition. (C-D) Individual transcripts associated with two major upregulated pathways in treated cells, mitophagy and ferroptosis. Transcripts were filtered based on significance and fold change relative to control. (E-F) Individual proteins associated with downregulated mitochondrial processes in treated cells. Proteins were filtered based on significance and fold change relative to control.

Figure 6.. Co-treatment with TR-107 and Erastin leads to the synergistic inhibition of CRC cell viability.

(A-B) Immunoblotting and densitometry analysis of HCT116 cells treated with TR-107 (50 nmol/L) for 72 hours exhibited a significant loss of ferroptosis-related protein expression. (C-F) Dose response matrices were generated for the CRC cell lines DLD-1, LS1034, HT29, and HCT116 from each CMS classification. Cells were treated with independent TR-107 (10 nmol/L, 50 nmol/L), Erastin (1 μmol/L, 10 μmol/L), or combinations of the two drugs as described in Materials and Methods. Cell viability was measured with the CellTiter-Glo assay and values were converted to % inhibition for analysis. Analysis was conducted on RStudio using the synergyfinder package. (G-J) Drug synergy scores for Erastin and TR-107 co-treatment were calculated using the Highest Single Agent (HSA) score model as described in Materials and Methods. HSA scores less than −10 = antagonistic interaction, between −10 to 0 = additive interaction, and greater than 10 = synergistic interaction. Western blot data are presented as mean ± SD values (N=3). Dose response data are presented as mean ± SD values (N=6). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.