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

The imipridones ONC201 and ONC212 selectively kill cancer cells and have been ascribed multiple mechanisms-of-action. Genome-wide CRISPR knockout screens revealed that loss of the mitochondrial proteases CLPP and MIPEP confer strong resistance to both compounds... 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.

[1]  H. Sahl,et al.  Dysregulation of bacterial proteolytic machinery by a new class of antibiotics , 2005, Nature Medicine.

[2]  Robert T. Sauer,et al.  Restriction of the Conformational Dynamics of the Cyclic Acyldepsipeptide Antibiotics Improves Their Antibacterial Activity , 2014, Journal of the American Chemical Society.

[3]  Adrian O. Olivares,et al.  Mechanical Protein Unfolding and Degradation. , 2018, Annual review of physiology.

[4]  K. Nagashima,et al.  ONC201 kills breast cancer cells in vitro by targeting mitochondria , 2018, Oncotarget.

[5]  S. Fracalanzza,et al.  Antimicrobial resistance in Neisseria gonorrhoeae: history, molecular mechanisms and epidemiological aspects of an emerging global threat , 2017, Brazilian journal of microbiology : [publication of the Brazilian Society for Microbiology].

[6]  M. Ryan,et al.  A mitochondrial specific stress response in mammalian cells , 2002, The EMBO journal.

[7]  E. White,et al.  Mitochondria and Cancer. , 2016, Molecular cell.

[8]  W. El-Deiry,et al.  Role of Dopamine Receptors in the Anticancer Activity of ONC20112 , 2017, Neoplasia.

[9]  S. Gottesman,et al.  Clp P represents a unique family of serine proteases. , 1990, The Journal of biological chemistry.

[10]  Olivier Elemento,et al.  A Bayesian machine learning approach for drug target identification using diverse data types , 2019, Nature Communications.

[11]  Roy R Chaudhuri,et al.  Comprehensive identification of essential Staphylococcus aureus genes using Transposon-Mediated Differential Hybridisation (TMDH) , 2009, BMC Genomics.

[12]  T. Lampe,et al.  Medicinal Chemistry Optimization of Acyldepsipeptides of the Enopeptin Class Antibiotics , 2006, ChemMedChem.

[13]  Ann E. Sizemore,et al.  Computational correction of copy-number effect improves specificity of CRISPR-Cas9 essentiality screens in cancer cells , 2017, Nature Genetics.

[14]  W. Houry,et al.  Chemical Modulation of the Human Mitochondrial ClpP: Potential Application in Cancer Therapeutics. , 2019, ACS chemical biology.

[15]  Mary Jane Ferraro,et al.  Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically : approved standard , 2000 .

[16]  E. Messaris,et al.  Dual Inactivation of Akt and ERK by TIC10 Signals Foxo3a Nuclear Translocation, TRAIL Gene Induction, and Potent Antitumor Effects , 2013, Science Translational Medicine.

[17]  W. El-Deiry,et al.  ONC201 kills solid tumor cells by triggering an integrated stress response dependent on ATF4 activation by specific eIF2α kinases , 2016, Science Signaling.

[18]  T. Baker,et al.  NOA1, a Novel ClpXP Substrate, Takes an Unexpected Nuclear Detour Prior to Mitochondrial Import , 2014, PloS one.

[19]  H. Chakrapani,et al.  Draft Genome Sequence of Methicillin-Sensitive Staphylococcus aureus ATCC 29213 , 2015, Genome Announcements.

[20]  A. Lin,et al.  Off-target toxicity is a common mechanism of action of cancer drugs undergoing clinical trials , 2019, Science Translational Medicine.

[21]  Neel S. Madhukar,et al.  Discovery and clinical introduction of first-in-class imipridone ONC201 , 2016, Oncotarget.

[22]  T. Baker,et al.  Proteomic discovery of cellular substrates of the ClpXP protease reveals five classes of ClpX-recognition signals. , 2003, Molecular cell.

[23]  B. Paw,et al.  Mutation in human CLPX elevates levels of δ-aminolevulinate synthase and protoporphyrin IX to promote erythropoietic protoporphyria , 2017, Proceedings of the National Academy of Sciences.

[24]  F. Denizot,et al.  ClpP of Bacillus subtilis is required for competence development, motility, degradative enzyme synthesis, growth at high temperature and sporulation , 1998, Molecular microbiology.

[25]  Jeff Piotrowski,et al.  Combining functional genomics and chemical biology to identify targets of bioactive compounds. , 2011, Current opinion in chemical biology.

[26]  A. Goldberg,et al.  Acyldepsipeptide antibiotics kill mycobacteria by preventing the physiological functions of the ClpP1P2 protease , 2016, Molecular microbiology.

[27]  Brian Raught,et al.  A High-Resolution Genome-Wide CRISPR/Cas9 Viability Screen Reveals Structural Features and Contextual Diversity of the Human Cell-Essential Proteome , 2017, Molecular and Cellular Biology.

[28]  P. Van der Veken,et al.  ClpP Protease, a Promising Antimicrobial Target , 2019, International journal of molecular sciences.

[29]  M. Mann,et al.  Comparative Proteomic Analysis of Eleven Common Cell Lines Reveals Ubiquitous but Varying Expression of Most Proteins* , 2012, Molecular & Cellular Proteomics.

[30]  The Gene Ontology Consortium,et al.  The Gene Ontology Resource: 20 years and still GOing strong , 2018, Nucleic Acids Res..

[31]  Neel S. Madhukar,et al.  Imipridone ONC212 activates orphan G protein-coupled receptor GPR132 and integrated stress response in acute myeloid leukemia , 2019, Leukemia.

[32]  Robert T Sauer,et al.  AAA+ proteases: ATP-fueled machines of protein destruction. , 2011, Annual review of biochemistry.

[33]  T. Walsh,et al.  Defining Fractional Inhibitory Concentration Index Cutoffs for Additive Interactions Based on Self-Drug Additive Combinations, Monte Carlo Simulation Analysis, and In Vitro-In Vivo Correlation Data for Antifungal Drug Combinations against Aspergillus fumigatus , 2009, Antimicrobial Agents and Chemotherapy.

[34]  R. Ghirlando,et al.  Acyldepsipeptide antibiotics induce the formation of a structured axial channel in ClpP: A model for the ClpX/ClpA-bound state of ClpP. , 2010, Chemistry & biology.

[35]  E. Brown,et al.  Antibacterial drug discovery in the resistance era , 2016, Nature.

[36]  K. Lewis.  Persister cells and the riddle of biofilm survival , 2005, Biochemistry (Moscow).

[37]  L. Rice.  Federal funding for the study of antimicrobial resistance in nosocomial pathogens: no ESKAPE. , 2008, The Journal of infectious diseases.

[38]  C. Benes,et al.  Preclinical evaluation of the imipridone family, analogs of clinical stage anti-cancer small molecule ONC201, reveals potent anti-cancer effects of ONC212 , 2017, Cell cycle.

[39]  P. Bross,et al.  Human ClpP protease: cDNA sequence, tissue‐specific expression and chromosomal assignment of the gene , 1995, FEBS letters.

[40]  H. Endo,et al.  Maintenance of mitochondrial genome distribution by mitochondrial AAA+ protein ClpX. , 2012, Experimental cell research.

[41]  G. Ippolito,et al.  Tuberculosis: progress and advances in development of new drugs, treatment regimens, and host-directed therapies. , 2018, The Lancet. Infectious diseases.

[42]  Martin Eisenacher,et al.  The PRIDE database and related tools and resources in 2019: improving support for quantification data , 2018, Nucleic Acids Res..

[43]  Cole M. Haynes,et al.  ClpP mediates activation of a mitochondrial unfolded protein response in C. elegans. , 2007, Developmental cell.

[44]  N. Strynadka,et al.  Aspergillomarasmine A overcomes metallo-b-lactamaseantibiotic resistance , 2014 .

[45]  C. López-Otín,et al.  New roles for mitochondrial proteases in health, ageing and disease , 2015, Nature Reviews Molecular Cell Biology.

[46]  A. Duerfeldt,et al.  Sclerotiamide: The First Non-Peptide-Based Natural Product Activator of Bacterial Caseinolytic Protease P. , 2016, Journal of natural products.

[47]  S. Glynn.  Multifunctional Mitochondrial AAA Proteases , 2017, Front. Mol. Biosci..

[48]  M. Tyers,et al.  Drug combinations: a strategy to extend the life of antibiotics in the 21st century , 2019, Nature Reviews Microbiology.

[49]  W. Houry,et al.  Development and Characterization of Potent Cyclic Acyldepsipeptide Analogues with Increased Antimicrobial Activity. , 2016, Journal of medicinal chemistry.

[50]  Laura E. Herring,et al.  Mitochondrial Protease ClpP is a Target for the Anticancer Compounds ONC201 and Related Analogues , 2019, ACS chemical biology.

[51]  Gary D Bader,et al.  Inhibition of the Mitochondrial Protease ClpP as a Therapeutic Strategy for Human Acute Myeloid Leukemia. , 2015, Cancer cell.

[52]  L. Rahme,et al.  A method for high throughput determination of viable bacteria cell counts in 96-well plates , 2012, BMC Microbiology.

[53]  R. Sauer,et al.  A Simple Fragment of Cyclic Acyldepsipeptides Is Necessary and Sufficient for ClpP Activation and Antibacterial Activity , 2014, Chembiochem : a European journal of chemical biology.

[54]  W. Houry,et al.  The Role of ClpP Protease in Bacterial Pathogenesis and Human Diseases. , 2018, ACS chemical biology.

[55]  Mike Tyers,et al.  Mubritinib Targets the Electron Transport Chain Complex I and Reveals the Landscape of OXPHOS Dependency in Acute Myeloid Leukemia. , 2019, Cancer cell.

[56]  Andrew B. Leber,et al.  Mitochondrial ClpP-Mediated Proteolysis Induces Selective Cancer Cell Lethality. , 2019, Cancer cell.

[57]  N. Strynadka,et al.  Aspergillomarasmine A overcomes metallo-β-lactamase antibiotic resistance , 2014, Nature.

[58]  J. D. Fishburn,et al.  PfClpC Is an Essential Clp Chaperone Required for Plastid Integrity and Clp Protease Stability in Plasmodium falciparum. , 2017, Cell reports.

[59]  Gerard D. Wright,et al.  Bacterial proteases, untapped antimicrobial drug targets , 2016, The Journal of Antibiotics.

[60]  Richard D. Smith,et al.  Activated ClpP kills persisters and eradicates a chronic biofilm infection , 2013, Nature.

[61]  Jessica M. Rusert,et al.  Dopamine Receptor D5 is a Modulator of Tumor Response to Dopamine Receptor D2 Antagonism , 2018, Clinical Cancer Research.

[62]  K. Lewis,et al.  Ureadepsipeptides as ClpP Activators. , 2019, ACS infectious diseases.

[63]  D. Speicher,et al.  The Mitochondrial Unfoldase-Peptidase Complex ClpXP Controls Bioenergetics Stress and Metastasis , 2016, PLoS biology.

[64]  James C. Hu,et al.  The Gene Ontology Resource: 20 years and still GOing strong , 2019 .

[65]  Michael L. Wang,et al.  ATF4 induction through an atypical integrated stress response to ONC201 triggers p53-independent apoptosis in hematological malignancies , 2016, Science Signaling.

[66]  S. Wodak,et al.  Activators of cylindrical proteases as antimicrobials: identification and development of small molecule activators of ClpP protease. , 2011, Chemistry & biology.

[67]  S. Phanse,et al.  Acyldepsipeptide Analogs Dysregulate Human Mitochondrial ClpP Protease Activity and Cause Apoptotic Cell Death. , 2018, Cell chemical biology.

[68]  M. Maurizi,et al.  Crystallography and mutagenesis point to an essential role for the N-terminus of human mitochondrial ClpP. , 2004, Journal of structural biology.

[69]  Derek J. Bailey,et al.  A Novel Differential Ion Mobility Device Expands the Depth of Proteome Coverage and the Sensitivity of Multiplex Proteomic Measurements* , 2018, Molecular & Cellular Proteomics.

[70]  Rahul Singh,et al.  Structure of the human aminopeptidase XPNPEP3 and comparison of its in vitro activity with Icp55 orthologs: Insights into diverse cellular processes , 2017, The Journal of Biological Chemistry.

[71]  P. Cavadini,et al.  Mitochondrial processing peptidases. , 2002, Biochimica et biophysica acta.

[72]  N. Boudebouch,et al.  Emergence and spread of resistant N. meningitidis implicated in invasive meningococcal diseases during the past decade (2008–2017) , 2018, The Journal of Antibiotics.

[73]  Hongtao Yu,et al.  Consequences of Depsipeptide Substitution on the ClpP Activation Activity of Antibacterial Acyldepsipeptides. , 2017, ACS medicinal chemistry letters.

[74]  S. Schneider,et al.  Selective Activation of Human Caseinolytic Protease P (ClpP). , 2018, Angewandte Chemie.