Acyldepsipeptides (ADEPs) are potential antibiotics that dysregulate the activity of the highly conserved tetradecameric bacterial ClpP protease, leading to bacterial cell death. Here, we identified ADEP analogs that are potent dysregulators of the human mitochondrial ClpP (HsClpP). These ADEPs interact tightly with HsClpP, causing the protease to non-specifically degrade model substrates. Dysregulation of HsClpP activity by ADEP was found to induce cytotoxic effects via activation of the intrinsic, caspase-dependent apoptosis. ADEP-HsClpP co-crystal structure was solved for one of the analogs revealing a highly complementary binding interface formed by two HsClpP neighboring subunits but, unexpectedly, with HsClpP in the compact conformation. Given that HsClpP is highly expressed in multiple cancers and has important roles in cell metastasis, our findings suggest a therapeutic potential for ADEPs in cancer treatment.

UV-induced photoproducts are responsible for the pathological effects of sunlight. Mutations in nucleotide excision repair (NER) cause severe pathologies characterized by sunlight sensitivity, coupled to elevated predisposition to cancer and/or neurological dysfunctions. We have previously shown that in UV-irradiated non-cycling cells, only a particular subset of lesions activates the DNA damage response (DDR), and this requires NER and EXO1 activities. To define the molecular mechanism acting at these lesions, we demonstrate that Y family TLS polymerases are recruited at NER- and EXO1-positive lesion sites in non-S phase cells. The coordinated action of EXO1 and Y family TLS polymerases promotes checkpoint activation, leads to lesion repair, and is crucial to prevent cytotoxic double-strand break (DSB) formation.