Imipridone anticancer compounds ectopically activate the ClpP protease and represent a new scaffold for antibiotic development
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ABSTRACT:
PROVIDER: PRJNA577699 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR10283186.fastq.gz | Fastqsanger.gz | |||
| SRR10283187.fastq.gz | Fastqsanger.gz | |||
| SRR10283188.fastq.gz | Fastqsanger.gz | |||
| SRR10283189.fastq.gz | Fastqsanger.gz | |||
| SRR10283190.fastq.gz | Fastqsanger.gz |
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Project description:Systematic genetic interaction profiles can reveal the mechanism-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 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 non-mitochondrial proteins. Prompted by the conservation of ClpP from bacteria to humans, we found that the imipridones also activate ClpP from Escherichia coli, B. subtilis and Staphylococcus aureus in biochemical and genetic assays. ONC212 and acyldepsipeptide (ADEP)-4, 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, Enterococcus faecium) and Gram-negative species (E. coli, 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.
2020-02-23 | GSE138894 | GEO
Project description: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.
| S-EPMC7153937 | biostudies-literature
Project description:The peptidase ClpP is conserved from bacteria to human. In the mitochondrial matrix, it multimerizes and forms a macromolecular proteasome-like cylinder. Because of its known relevance for the mitochondrial unfolded protein response during cell stress, we characterized two ClpP knock-out mouse founder lines and documented similar phenotypes. Ubiquitously, ClpP absence led to accumulation of its interactor protein ClpX without transcript upregulation. Interestingly, most wild-type tissues with substantial ClpP amounts had no detectable ClpX. This inverse correlation suggests that ClpX levels and degradation are regulated by ClpP. The expectation of similar protein levels, in view of a reported association of heptameric ClpP rings with hexameric ClpX rings, was confirmed only in testis of wild-type animals. Germline tissue was exceptional also in its vulnerability to ClpP deletion, with both founder lines showing complete infertility for males and females. Otherwise, ubiquitous mitochondrial dysfunction was apparent from severe growth retardation and reduced spontaneous motor activity of the animals, and from a pronounced decrease in pre-/postnatal survival. Spermatogenesis was found aborted at the spermatid stage, acrosomes and axonemes were not formed. Overall, tissue-specific roles of ClpP were evident by this massive effect for germ cells, mild bioenergetic deficits in muscle and liver tissues, and excellent compensation in brain. ClpX was previously reported to chaperone unfolded proteins and also DNA condensation in mitochondria, so it is likely that this pathway is particularly susceptible in germ cells. In conclusion, our study indicates that the role of ClpP in quality control is indispensable during development for cells with rapid changes of mitochondrial numbers, and is relevant during aging for growth and survival of the organism. Factorial design comparing ClpP knock-out mice with wild type littermates in five different tissues (brain, testis, liver, skeletal and heart muscle)
2013-07-17 | E-GEOD-40207 | biostudies-arrayexpress
Project description:The peptidase ClpP is conserved from bacteria to human. In the mitochondrial matrix, it multimerizes and forms a macromolecular proteasome-like cylinder. Because of its known relevance for the mitochondrial unfolded protein response during cell stress, we characterized two ClpP knock-out mouse founder lines and documented similar phenotypes. Ubiquitously, ClpP absence led to accumulation of its interactor protein ClpX without transcript upregulation. Interestingly, most wild-type tissues with substantial ClpP amounts had no detectable ClpX. This inverse correlation suggests that ClpX levels and degradation are regulated by ClpP. The expectation of similar protein levels, in view of a reported association of heptameric ClpP rings with hexameric ClpX rings, was confirmed only in testis of wild-type animals. Germline tissue was exceptional also in its vulnerability to ClpP deletion, with both founder lines showing complete infertility for males and females. Otherwise, ubiquitous mitochondrial dysfunction was apparent from severe growth retardation and reduced spontaneous motor activity of the animals, and from a pronounced decrease in pre-/postnatal survival. Spermatogenesis was found aborted at the spermatid stage, acrosomes and axonemes were not formed. Overall, tissue-specific roles of ClpP were evident by this massive effect for germ cells, mild bioenergetic deficits in muscle and liver tissues, and excellent compensation in brain. ClpX was previously reported to chaperone unfolded proteins and also DNA condensation in mitochondria, so it is likely that this pathway is particularly susceptible in germ cells. In conclusion, our study indicates that the role of ClpP in quality control is indispensable during development for cells with rapid changes of mitochondrial numbers, and is relevant during aging for growth and survival of the organism.
2013-07-17 | GSE40207 | GEO
Project description:By integration of transcriptome and metabolite analyses, we showed that pharmacological activation of the mitochondrial ClpP protease through utilization of enhanced imipridone compounds (ONC206 and ONC212) in combination with global (Panobinostat) and selective (romidepsin) HDAC – inhibitors caused synergistic reduction of viability in established and patient-derived xenograft (PDX) cultures of human GBM. This effect occurred independent of TP53 status and was partially mediated by activation of a cell death with apoptotic features accompanied by activation of initiator and effector caspases as well as cleavage of PARP. Consistently, the combination treatment altered the expression of anti-apoptotic and pro-apoptotic Bcl-2 family members, resulting in down-regulation of Bcl-xL and Mcl-1. Knockdown experiments targeting Noxa, BIM, Bcl-xL and Mcl-1 confirmed a functional implication of these proteins in the reduction of cellular viability mediated by the combination treatment. Importantly, knockdown of the ClpP protease significantly rescued the reduction of cellular viability by ClpP activators and the combination treatment, respectively, suggesting critical involvement of this protein. We used microarrays to detail the global programme of gene expression underlying cellularization and identified distinct classes of up-regulated and down-regulated genes in cells treated with ONC206 and Romidepsin.
2022-01-24 | GSE166111 | GEO
Project description:Ectopic activation of the conserved ClpP protease by chemical activators causes toxicity in bacteria and human cells due to unrestrained proteolysis. The imipridone ONC201 has been ascribed multiple mechanisms of action and is currently in cancer clinical trials. To systematically investigate the genetic dependencies of imipridone action, we screened a genome-wide CRISPR knockout library in the presence of ONC201 and its more potent analog ONC212. Loss of the mitochondrial matrix protease CLPP conferred strong resistance to both compounds, consistent with recent reports that ONC201 directly activates CLPP in cancer cells. Biochemical assays and surrogate genetic assays in yeast confirmed activation of CLPP in the absence of its regulatory subunits. Imipridone toxicity was bypassed by loss of only one other gene, the mitochondrial intermediate peptidase MIPEP, which we showed is necessary for proteolytic maturation of a CLPP precursor form. Quantitative proteomic analysis of cells treated with ONC212 revealed degradation of many mitochondrial proteins as well as cell cycle regulators. Prompted by the conservation of ClpP across kingdoms, we showed that the imipridones activate Escherichia coli ClpP in vitro and Staphylococcus aureus ClpP in a surrogate yeast assay. ONC212 and acyldepsipeptide (ADEP)-4, a known activator of bacterial ClpP, caused similar proteomic degradation profiles in S. aureus. ONC212 suppressed the proliferation of a number of Gram-positive (S. aureus, Bacillus subtilis, Enterococcus faecium) and Gram-negative species (E. coli, Neisseria gonorrhoeae). Moreover, a combination of ONC212 and rifampicin eradicated 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.
2020-03-26 | PXD016119 | Pride
Project description:Classic susbtrate-trapping screen for ClpXP substrates using MEF cell lines overexpressing FLAG-tagged CLPP (WT), proteolytically inactive CLPP (TRAP) or vector control (NEG)
2020-06-01 | PXD015399 | Pride
Project description:Targeting the mitochondrial caseinolytic protease P (ClpP) is a potential novel strategy for treating leukemia. However, little is known about the therapeutic effects of ClpP in solid tumors. In this study, we identify a strong activator of the mitochondrial ClpP for subsequent use in targeted pancreatic ductal adenocarcinoma (PDAC) therapy. Analysis of clinical data demonstrated a positive association between ClpP expression and prognosis in PDAC patients. We validated that aberrant ClpP activation can lead to growth arrest in PDAC cells and tumors. We then performed high-throughput screening and synthetic optimization to develop a potent ClpP activator, ZG111, which promoted ClpP to degrade respiratory chain complexes and occurred in a ClpP-dependent manner. This resulted in impaired oxidative phosphorylation in the mitochondria and activation of JNK/c-Jun pathway and endoplasmic reticulum stress response. ZG111 also produced significant antitumor effects on tumors in cell-derived and patient-derived xenografted mice models. Altogether, our identification of ZG111 demonstrated that the aberrant activation of ClpP hydrolysis can be used as a potential therapeutic strategy to treat PDAC cancer.
2021-10-21 | GSE186091 | GEO
Project description:We performed RNAseq analysis to determine the effect of ClpP-deletion on cumulus cells global gene expression profile in mature (3 month-old) and (6 month-old) mice. RNA sequencing revealed about 13,000 genes (FPKM > 0.1) expressed in cumulus cells. A total of 1025 genes were significantly differentially expressed (p<0.05) in 3-month ClpP-/- cumulus cells compared to WT (548 up-regulated and 477 down-regulated). At 6-month, a total of 555 genes were significantly differentially expressed in ClpP-/- cumulus cells compared to WT (464 up-regulated and 91 down-regulated). In addition, 1012 and 1034 genes were significantly differentially expressed in both 6 month ClpP-/- and WT cumulus cells compared to younger ClpP-/- cumulus cells, respectively. In this study, we assessed the effect of ClpP global deletion on cumulus cells gene expression. Our findings provide new insight into the role of CLPP in the cumulus cells, and may help understand the potential mechanism of infertility and reproductive aging associated with ClpP-deficiency.
2020-08-15 | GSE102756 | GEO
Project description:We performed RNAseq analysis to determine the effect of ClpP-deletion on cumulus cells global gene expression profile in mature (3 month-old) and (6 month-old) mice. RNA sequencing revealed about 13,000 genes (FPKM > 0.1) expressed in cumulus cells. A total of 1025 genes were significantly differentially expressed (p<0.05) in 3-month ClpP-/- cumulus cells compared to WT (548 up-regulated and 477 down-regulated). At 6-month, a total of 555 genes were significantly differentially expressed in ClpP-/- cumulus cells compared to WT (464 up-regulated and 91 down-regulated). In addition, 1012 and 1034 genes were significantly differentially expressed in both 6 month ClpP-/- and WT cumulus cells compared to younger ClpP-/- cumulus cells, respectively. In this study, we assessed the effect of ClpP global deletion on cumulus cells gene expression. Our findings provide new insight into the role of CLPP in the cumulus cells, and may help understand the potential mechanism of infertility and reproductive aging associated with ClpP-deficiency.
2017-12-01 | GSE102269 | GEO