Development and evaluation of mitochondria-targeted caseinolytic protease (ClpP) agonist chemical probes in in vitro breast cancer models

Mitochondria carry out a plethora of functions that are vital for cellular physiology and metabolism. Mitochondrial caseinolytic protease (ClpP) is part of the larger CLPXP machinery that is critical for mitochondrial protein homeostasis and has emerged as a prospective molecular target in several types of cancer. ClpP agonists based around the imipridone scaffold have shown some promise in acute myeloid leukemia, but the polypharmacology of these compounds limits their utility. Effective methods to deliver drugs specifically to mitochondria have emerged, involving covalent linking of either a lipophilic cation, such as an alkyltriphenylphosphonium moiety, or a XJB peptide sequence to a pharmacophore of interest. Taking advantage of the evolutionary conservation of ClpP and combination with mitochondria targeting strategies, we designed, synthesized, and tested a series of mitochondria-targeted probe compounds based on the cyclic acyldepsipeptides (ADEP) scaffold, a natural product antibiotic and potent bacterial ClpP agonist. Herein, we report the synthesis and characterization of several ClpP agonist chemical probes that afford specific, and effective targeting of the mitochondrial protein in triple-negative breast cancer cell lines, with XJB conjugation significantly increasing cytotoxicity of the ADEP pharmacophore to triple-negative breast cancer BT549 cells, providing valuable chemical tools for further study.