Imipridone anticancer compounds ectopically activate the ClpP protease and represent a new scaffold for antibiotic development
ABSTRACT: 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. Overall design: Nalm-6 cells infected with the EKO sgRNA library (Bertomeu et al. 2017, PMID:29038160), after 7 days of Cas9 induction with doxycycline, were exposed to either 150nM ONC212, 10uM ONC201/TIC10 or media only for 8 days. sgRNA abundance was then assessed by high-throughput sequencing. Cells exposed to media only for two days served as controls.
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.
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.
Project description:The worldwide spread of antibiotic-resistant bacteria has lent urgency to the search for antibiotics with new modes of action that are devoid of preexisting cross-resistances. We previously described a unique class of acyldepsipeptides (ADEPs) that exerts prominent antibacterial activity against Gram-positive pathogens including streptococci, enterococci, as well as multidrug-resistant Staphylococcus aureus. Here, we report that ADEP prevents cell division in Gram-positive bacteria and induces strong filamentation of rod-shaped Bacillus subtilis and swelling of coccoid S. aureus and Streptococcus pneumoniae. It emerged that ADEP treatment inhibits septum formation at the stage of Z-ring assembly, and that central cell division proteins delocalize from midcell positions. Using in vivo and in vitro studies, we show that the inhibition of Z-ring formation is a consequence of the proteolytic degradation of the essential cell division protein FtsZ. ADEP switches the bacterial ClpP peptidase from a regulated to an uncontrolled protease, and it turned out that FtsZ is particularly prone to degradation by the ADEP-ClpP complex. By preventing cell division, ADEP inhibits a vital cellular process of bacteria that is not targeted by any therapeutically applied antibiotic so far. Their unique multifaceted mechanism of action and antibacterial potency makes them promising lead structures for future antibiotic development.
Project description:Acyldepsipeptide (ADEP) is an exploratory antibiotic with a novel mechanism of action. ClpP, the proteolytic core of the caseinolytic protease, is deregulated towards unrestrained proteolysis. Here, we report on the mechanism of ADEP resistance in Firmicutes. This bacterial phylum contains important pathogens that are relevant for potential ADEP therapy. For Staphylococcus aureus, Bacillus subtilis, enterococci and streptococci, spontaneous ADEP-resistant mutants were selected in vitro at a rate of 10-6 . All isolates carried mutations in clpP. All mutated S. aureus ClpP proteins characterised in this study were functionally impaired; this increased our understanding of the mode of operation of ClpP. For molecular insights, crystal structures of S. aureus ClpP bound to ADEP4 were determined. Well-resolved N-terminal domains in the apo structure allow the pore-gating mechanism to be followed. The compilation of mutations presented here indicates residues relevant for ClpP function and suggests that ADEP resistance will occur at a lower rate during the infection process.
Project description:Antibiotic acyldepsipeptides (ADEPs) deregulate ClpP, the proteolytic core of the bacterial Clp protease, thereby inhibiting its native functions and concomitantly activating it for uncontrolled proteolysis of nonnative substrates. Importantly, although ADEP-activated ClpP is assumed to target multiple polypeptide and protein substrates in the bacterial cell, not all proteins seem equally susceptible. In Bacillus subtilis, the cell division protein FtsZ emerged to be particularly sensitive to degradation by ADEP-activated ClpP at low inhibitory ADEP concentrations. In fact, FtsZ is the only bacterial protein that has been confirmed to be degraded in vitro as well as within bacterial cells so far. However, the molecular reason for this preferred degradation remained elusive. Here, we report the unexpected finding that ADEP-activated ClpP alone, in the absence of any Clp-ATPase, leads to an unfolding and subsequent degradation of the N-terminal domain of FtsZ, which can be prevented by the stabilization of the FtsZ fold via nucleotide binding. At elevated antibiotic concentrations, importantly, the C terminus of FtsZ is notably targeted for degradation in addition to the N terminus. Our results show that different target structures are more or less accessible to ClpP, depending on the ADEP level present. Moreover, our data assign a Clp-ATPase-independent protein unfolding capability to the ClpP core of the bacterial Clp protease and suggest that the protein fold of FtsZ may be more flexible than previously anticipated.IMPORTANCE Acyldepsipeptide (ADEP) antibiotics effectively kill multidrug-resistant Gram-positive pathogens, including vancomycin-resistant enterococcus, penicillin-resistant Streptococcus pneumoniae (PRSP), and methicillin-resistant Staphylococcus aureus (MRSA). The antibacterial activity of ADEP depends on a new mechanism of action, i.e., the deregulation of bacterial protease ClpP that leads to bacterial self-digestion. Our data allow new insights into the mode of ADEP action by providing a molecular explanation for the distinct bacterial phenotypes observed at low versus high ADEP concentrations. In addition, we show that ClpP alone, in the absence of any unfoldase or energy-consuming system, and only activated by the small molecule antibiotic ADEP, leads to the unfolding of the cell division protein FtsZ.
Project description:Mitochondria-targeted antioxidants are known to alleviate mitochondrial oxidative damage that is associated with a variety of diseases. Here, we showed that SkQ1, a decyltriphenyl phosphonium cation conjugated to a quinone moiety, exhibited strong antibacterial activity towards Gram-positive Bacillus subtilis, Mycobacterium sp. and Staphylococcus aureus and Gram-negative Photobacterium phosphoreum and Rhodobacter sphaeroides in submicromolar and micromolar concentrations. SkQ1 exhibited less antibiotic activity towards Escherichia coli due to the presence of the highly effective multidrug resistance pump AcrAB-TolC. E. coli mutants lacking AcrAB-TolC showed similar SkQ1 sensitivity, as B. subtilis. Lowering of the bacterial membrane potential by SkQ1 might be involved in the mechanism of its bactericidal action. No significant cytotoxic effect on mammalian cells was observed at bacteriotoxic concentrations of SkQ1. Therefore, SkQ1 may be effective in protection of the infected mammals by killing invading bacteria.
Project description:The continuing rise of multidrug resistant pathogens has made it clear that in the absence of new antibiotics we are moving toward a "postantibiotic" world, in which even routine infections will become increasingly untreatable. There is a clear need for the development of new antibiotics with truly novel mechanisms of action to combat multidrug resistant pathogens. Experimental evolution to resistance can be a useful tactic for the characterization of the biochemical mechanism of action for antibiotics of interest. Herein, we demonstrate that the use of a diverse panel of strains with well-annotated reference genomes improves the success of using experimental evolution to characterize the mechanism of action of a novel pyrrolizidinone antibiotic analog. Importantly, we used experimental evolution under conditions that favor strongly polymorphic populations to adapt a panel of three substantially different Gram-positive species (lab strain Bacillus subtilis and clinical strains methicillin-resistant Staphylococcus aureus MRSA131 and Enterococcus faecalis S613) to produce a sufficiently diverse set of evolutionary outcomes. Comparative whole genome sequencing (WGS) between the susceptible starting strain and the resistant strains was then used to identify the genetic changes within each species in response to the pyrrolizidinone. Taken together, the adaptive response across a range of organisms allowed us to develop a readily testable hypothesis for the mechanism of action of the CJ-16?264 analog. In conjunction with mitochondrial inhibition studies, we were able to elucidate that this novel pyrrolizidinone antibiotic is an electron transport chain (ETC) inhibitor. By studying evolution to resistance in a panel of different species of bacteria, we have developed an enhanced method for the characterization of new lead compounds for the discovery of new mechanisms of action.
Project description:It is often difficult to cure endocarditis, osteomyelitis, and device-associated infections caused by Gram-positive pathogens, despite therapy with clinically appropriate antibiotics. This may be due to antibiotic tolerance or resistance development. Acyldepsipeptides (ADEPs) are a class of bactericidal compounds active against a variety of clinically important Gram-positive bacteria, including staphylococci, streptococci, and enterococci. ADEPs activate caseinolytic protease P (ClpP), killing high-density, nondividing cultures of bacteria that are tolerant to approved classes of antibiotics. Acyldepsipeptide analog 4 (ADEP4) was active against a panel of drug-resistant Gram-positive pathogens in MIC assays, with no preexisting resistance detected. Killing of stationary-phase cultures was observed when ADEP4 was combined with multiple classes of approved antibiotics. Additionally, a hollow-fiber infection model was used to assess the effects of ADEP4 antibiotic combinations on bacterial killing and resistance development. These studies were performed on high-density cultures of methicillin-resistant S. aureus (MRSA), methicillin-susceptible S. aureus (MSSA), and vancomycin-resistant Enterococcus faecalis (VRE). None of the approved antibiotics linezolid, ampicillin, and oxacillin tested alone had bactericidal activity under these conditions. ADEP4 initially caused killing, but regrowth of the culture was apparent within 96?h due to resistance. Combinations of ADEP4 with linezolid or oxacillin caused substantially improved killing of MRSA or MSSA cultures, respectively, and no regrowth due to resistance was observed. The combination of ADEP4 and ampicillin eradicated cultures of VRE to the limit of detection within 52?h. These data suggest that combining ClpP activators with traditional antibiotics may be a good strategy to treat complicated Gram-positive infections.
Project description:The WalK (histidine kinase)/WalR (response regulator) two-component signal transduction system is a master regulatory system for cell wall metabolism and growth. This system is conserved in low G+C Gram-positive bacteria, including Bacillus subtilis, Staphylococcus aureus, Enterococcus faecalis, and Streptococcus mutans. In this study, we found the first antibiotic that functions as a WalK inhibitor (signermycin B) by screening 10,000 Streptomyces extracts. The chemical structure (C(23)H(35)NO(4); molecular weight, 389.5) comprises a tetramic acid moiety and a decalin ring. Signermycin B exhibited antimicrobial activity, with MIC values ranging from 3.13 ?g/ml (8 ?M) to 6.25 ?g/ml (16 ?M) against Gram-positive bacteria that possess the WalK/WalR two-component signal transduction system, including the drug-resistant bacteria methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus faecalis. The half-maximal inhibitory concentrations of signermycin B against WalK in these organisms ranged from 37 to 61 ?M. To determine the mechanism of action of signermycin B, surface plasmon resonance response analysis with the two WalK domains of Bacillus subtilis and competition assay with ATP were performed. The results showed that signermycin B binds to the dimerization domain but not the ATP-binding domain of WalK. In the presence of the cross-linker glutaraldehyde, signermycin B did not cause protein aggregation but interfered with the cross-linking of WalK dimers. These results suggest that signermycin B targets the conserved dimerization domain of WalK to inhibit autophosphorylation. In Bacillus subtilis and Staphylococcus aureus, signermycin B preferentially controlled the WalR regulon, thereby inhibiting cell division. These phenotypes are consistent with those of cells starved for the WalK/WalR system.
Project description:The increasing threat posed by multiresistant bacterial pathogens necessitates the discovery of novel antibacterials with unprecedented modes of action. ADEP1, a natural compound produced by Streptomyces hawaiiensis NRRL 15010, is the prototype for a new class of acyldepsipeptide (ADEP) antibiotics. ADEP antibiotics deregulate the proteolytic core ClpP of the bacterial caseinolytic protease, thereby exhibiting potent antibacterial activity against Gram-positive bacteria, including multiresistant pathogens. ADEP1 and derivatives, here collectively called ADEP, have been previously investigated for their antibiotic potency against different species, structure-activity relationship, and mechanism of action; however, knowledge on the biosynthesis of the natural compound and producer self-resistance have remained elusive. In this study, we identified and analyzed the ADEP biosynthetic gene cluster in S. hawaiiensis NRRL 15010, which comprises two NRPSs, genes necessary for the biosynthesis of (4S,2R)-4-methylproline, and a type II polyketide synthase (PKS) for the assembly of highly reduced polyenes. While no resistance factor could be identified within the gene cluster itself, we discovered an additional clpP homologous gene (named clpP ADEP) located further downstream of the biosynthetic genes, separated from the biosynthetic gene cluster by several transposable elements. Heterologous expression of ClpPADEP in three ADEP-sensitive Streptomyces species proved its role in conferring ADEP resistance, thereby revealing a novel type of antibiotic resistance determinant.IMPORTANCE Antibiotic acyldepsipeptides (ADEPs) represent a promising new class of potent antibiotics and, at the same time, are valuable tools to study the molecular functioning of their target, ClpP, the proteolytic core of the bacterial caseinolytic protease. Here, we present a straightforward purification procedure for ADEP1 that yields substantial amounts of the pure compound in a time- and cost-efficient manner, which is a prerequisite to conveniently study the antimicrobial effects of ADEP and the operating mode of bacterial ClpP machineries in diverse bacteria. Identification and characterization of the ADEP biosynthetic gene cluster in Streptomyces hawaiiensis NRRL 15010 enables future bioinformatics screenings for similar gene clusters and/or subclusters to find novel natural compounds with specific substructures. Most strikingly, we identified a cluster-associated clpP homolog (named clpP ADEP) as an ADEP resistance gene. ClpPADEP constitutes a novel bacterial resistance factor that alone is necessary and sufficient to confer high-level ADEP resistance to Streptomyces across species.