Characterization of TR-107, a Novel Chemical Activator of the Human Mitochondrial Protease ClpP

Abstract

We recently described the identification of a new class of small molecule activators of the mitochondrial protease ClpP. These compounds synthesized by Madera Therapeutics showed increased potency of cancer growth inhibition over the related compound ONC201. In this study, we describe chemical optimization and characterization of the next generation of highly potent and selective small molecule ClpP activators (TR compounds) and demonstrate their efficacy against breast cancer models in vitro and in vivo. One of these compounds (TR-107) was selected for further studies because of excellent potency, specificity and drug-like properties. Examination of TR-107 effects in Triple Negative Breast Cancer (TNBC) cell models showed growth inhibition in the low nanomolar range, equipotent to paclitaxel, in a ClpP-dependent manner. TR-107 reduced specific mitochondrial proteins including OXPHOS and TCA cycle components, in a time, dose and ClpP-dependent manner. Seahorse XF Analysis and glucose deprivation experiments confirmed inactivation of OXPHOS and demonstrated an increased dependence on glycolysis following TR-107 exposure. The pharmacokinetic properties of TR-107 were compared to other known ClpP activators including ONC201 and ONC212. TR-107 displayed excellent exposure and serum t1/2 after oral administration. Using an orthotopic xenograft murine model of MDA-MB-231 cells, the anti-tumor response to TR-107 was investigated. Oral administration of TR-107 resulted in reduction in tumor volume and extension of survival in the treated vs. vehicle control groups. In summary, these studies describe the identification of highly potent new ClpP agonists with improved efficacy against TNBC, through targeted inactivation of OXPHOS and disruption of mitochondrial metabolism.

Small Molecule CLPP Agonists Induce Senescence and Alter Trail-Mediated Apoptotic Response of Triple-Negative Breast Cancer Cells

“…2A). As previously described 9,33 , ClpP activation with ONC201 or TR-57 resulted in the loss of the mitochondrial protein TUFM but, as expected, abemaciclib had no effect on TUFM protein levels (Fig. 1D).…”

“…1A). Furthermore, treatment of SUM159 cells with another highly potent ClpP activator 33 , TR107, also caused a strong reduction in Rb phosphorylation (Supp Fig. 2A).…”

“…Lentivirus were produced in HEK293T cells. Transfection and clonal isolation of the CRISPR null mammalian cells was done as previously described 33 .…”