Imipridone Anticancer Compounds Ectopically Activate the ClpP Protease and Represent a New Scaffold for Antibiotic Development

Abstract

Systematic genetic interaction profiles can reveal the mechanisms-of-action of bioactive compounds. The imipridone ONC201, which is currently in cancer clinical trials, has been ascribed a variety of different targets. To investigate the genetic dependencies of imipridone action, we screened a genome-wide clustered regularly interspaced short palindromic repeats (CRISPR) knockout library in the presence of either ONC201 or its more potent analog ONC212. Loss of the mitochondrial matrix protease CLPP or the mitochondrial intermediate peptidase MIPEP conferred strong resistance to both compounds. Biochemical and surrogate genetic assays showed that impridones directly activate CLPP and that MIPEP is necessary for proteolytic maturation of CLPP into a catalytically competent form. Quantitative proteomic analysis of cells treated with ONC212 revealed degradation of many mitochondrial as well as nonmitochondrial proteins. Prompted by the conservation of ClpP from bacteria to humans, we found that the imipridones also activate ClpP from Escherichia coli, Bacillus subtilis, and Staphylococcus aureus in biochemical and genetic assays. ONC212 and acyldepsipeptide-4 (ADEP4), a known activator of bacterial ClpP, caused similar proteome-wide degradation profiles in S. aureus. ONC212 suppressed the proliferation of a number of Gram-positive (S. aureus, B. subtilis, and Enterococcus faecium) and Gram-negative species (E. coli and Neisseria gonorrhoeae). Moreover, ONC212 enhanced the ability of rifampin to eradicate antibiotic-tolerant S. aureus persister cells. These results reveal the genetic dependencies of imipridone action in human cells and identify the imipridone scaffold as a new entry point for antibiotic development.

ONC201 and imipridones: Anti-cancer compounds with clinical efficacy

“…Ongoing exploration of the combinatorial expression of predictive biomarkers is underway, which may provide insight into subtypes or additional tumor types with enhanced imipridone sensitivity. It is possible that either the DRD2 or ClpP binding target may be more relevant depending on the tumor type [7] , [8] , [10] . It is also possible that ultimately the immediate drug binding targets may not predict response due to the numerous factors in cells that regulate cell death [31] .…”

“…Caseinolytic protease P (ClpP) is a serine protease located in the mitochondrial matrix that regulates several mitochondrial functions [7] , [8] , [9] , [10] . ClpP forms a large tetradecameric ATP-dependent protease complex involving two homo-heptamer rings, which not only interface with each other, but also each associate a ring of ClpX proteins that function as a regulatory cap [7] .…”

“…ClpP forms a large tetradecameric ATP-dependent protease complex involving two homo-heptamer rings, which not only interface with each other, but also each associate a ring of ClpX proteins that function as a regulatory cap [7] . This complex regulates oxidative phosphorylation (OXPHOS) by controlling the degradation of the respiratory chain complex subunits and can trigger the mitochondrial unfolded protein response [7] , [8] , [9] , [10] . The ClpP protease complex is conserved in bacteria and is also a target of interest for antibiotics [8] .…”

“…This complex regulates oxidative phosphorylation (OXPHOS) by controlling the degradation of the respiratory chain complex subunits and can trigger the mitochondrial unfolded protein response [7] , [8] , [9] , [10] . The ClpP protease complex is conserved in bacteria and is also a target of interest for antibiotics [8] . ClpP is structurally similar to the cytoplasmic/nuclear proteasome complex in human cells, a well-established therapeutic target used in the treatment of certain hematologic malignancies [7] , [11] .…”

“…In addition to DRD2/3 antagonism, direct activation of ClpP by ONC201 has recently been uncovered by chemical proteomics, chemical library screening, and acquired resistance approaches [7] , [8] , [10] . ONC201 interacts with human ClpP via non-covalent allosteric interactions that result in the protein complex opening specific areas of a substrate channel and altering the conformation of its active site.…”