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Evaluation of 3- and 4-Phenoxybenzamides as Selective Inhibitors of the Mono-ADP-Ribosyltransferase PARP10.

ABSTRACT: Intracellular ADP-ribosyltransferases catalyze mono- and poly-ADP-ribosylation and affect a broad range of biological processes. The mono-ADP-ribosyltransferase PARP10 is involved in signaling and DNA repair. Previous studies identified OUL35 as a selective, cell permeable inhibitor of PARP10. We have further explored the chemical space of OUL35 by synthesizing and investigating structurally related analogs. Key synthetic steps were metal-catalyzed cross-couplings and functional group modifications. We identified 4-(4-cyanophenoxy)benzamide and 3-(4-carbamoylphenoxy)benzamide as PARP10 inhibitors with distinct selectivities. Both compounds were cell permeable and interfered with PARP10 toxicity. Moreover, both revealed some inhibition of PARP2 but not PARP1, unlike clinically used PARP inhibitors, which typically inhibit both enzymes. Using crystallography and molecular modeling the binding of the compounds to different ADP-ribosyltransferases was explored regarding selectivity. Together, these studies define additional compounds that interfere with PARP10 function and thus expand our repertoire of inhibitors to further optimize selectivity and potency.

SUBMITTER: Korn P

PROVIDER: S-EPMC8485830 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Development of Anti-Virulence Therapeutics against Mono-ADP-Ribosyltransferase Toxins.

Project description:Mono-ADP-ribosyltransferase toxins are often key virulence factors produced by pathogenic bacteria as tools to compromise the target host cell. These toxins are enzymes that use host cellular NAD+ as the substrate to modify a critical macromolecule target in the host cell machinery. This post-translational modification of the target macromolecule (usually protein or DNA) acts like a switch to turn the target activity on or off resulting in impairment of a critical process or pathway in the host. One approach to stymie bacterial pathogens is to curtail the toxic action of these factors by designing small molecules that bind tightly to the enzyme active site and prevent catalytic function. The inactivation of these toxins/enzymes is targeted for the site of action within the host cell and small molecule therapeutics can function as anti-virulence agents by disarming the pathogen. This represents an alternative strategy to antibiotic therapy with the potential as a paradigm shift that may circumvent multi-drug resistance in the offending microbe. In this review, work that has been accomplished during the past two decades on this approach to develop anti-virulence compounds against mono-ADP-ribosyltransferase toxins will be discussed.

| S-EPMC7824265 | biostudies-literature

Photox, a novel actin-targeting mono-ADP-ribosyltransferase from Photorhabdus luminescens.

Project description:Photorhabdus luminescens is a pathogenic bacterium that produces many toxic proteins. The mono-ADP-ribosyltransferases (mARTs) are an enzyme class produced by numerous pathogenic bacteria and participate in disease in plants and animals, including humans. Herein we report a novel mART from P. luminescens called Photox. This 46-kDa toxin shows high homology to other actin-targeting mARTs in hallmark catalytic regions and a similar core catalytic fold. Furthermore, Photox shows in vivo cytotoxic activity against yeast, with protection occurring when catalytic residues are substituted with alanine. In vitro, enzymatic activity (k(cat), 1680 +/- 75 min(-1)) is higher than that of the related iota toxin, and diminishes by nearly 14,000-fold following substitution of the catalytic Glu (E355A). This toxin specifically ADP-ribosylates monomeric alpha-skeletal actin and nonmuscle beta- and gamma-actin at Arg(177), inhibiting regular polymerization of actin filaments. These results indicate that Photox is indeed an ADP-ribosyltransferase, making it the newest member of the actin-targeting mART family.

| S-EPMC2859513 | biostudies-literature

Mono-ADP-ribosylation by PARP10 inhibits Chikungunya virus nsP2 proteolytic activity and viral replication.

Project description:Replication of viruses requires interaction with host cell factors and repression of innate immunity. Recent findings suggest that a subset of intracellular mono-ADP-ribosylating PARPs, which are induced by type I interferons, possess antiviral activity. Moreover, certain RNA viruses, including Chikungunya virus (CHIKV), encode mono-ADP-ribosylhydrolases. Together, this suggests a role for mono-ADP-ribosylation (MARylation) in host-virus conflicts, but the relevant substrates have not been identified. We addressed which PARP restricts CHIKV replication and identified PARP10 and PARP12. For PARP10, this restriction was dependent on catalytic activity. Replication requires processing of the non-structural polyprotein nsP1-4 by the protease located in nsP2 and the assembly of the four individual nsP1-nsP4 into a functional replication complex. PARP10 and PARP12 inhibited the production of nsP3, indicating a defect in polyprotein processing. The nsP3 protein encodes a macrodomain with de-MARylation activity, which is essential for replication. In support for MARylation affecting polyprotein processing, de-MARylation defective CHIKV replicons revealed reduced production of nsP2 and nsP3. We hypothesized that MARylation regulates the proteolytic function of nsP2. Indeed, we found that nsP2 is MARylated by PARP10 and, as a consequence, its proteolytic activity was inhibited. NsP3-dependent de-MARylation reactivated the protease. Hence, we propose that PARP10-mediated MARylation prevents polyprotein processing and consequently virus replication. Together, our findings provide a mechanistic explanation for the role of the viral MAR hydrolase in CHIKV replication.

| S-EPMC9959937 | biostudies-literature

Newly discovered and characterized antivirulence compounds inhibit bacterial mono-ADP-ribosyltransferase toxins.

Project description:The mono-ADP-ribosyltransferase toxins are bacterial virulence factors that contribute to many disease states in plants, animals, and humans. These toxins function as enzymes that target various host proteins and covalently attach an ADP-ribose moiety that alters target protein function. We tested compounds from a virtual screen of commercially available compounds combined with a directed poly(ADP-ribose) polymerase (PARP) inhibitor library and found several compounds that bind tightly and inhibit toxins from Pseudomonas aeruginosa and Vibrio cholerae. The most efficacious compounds completely protected human lung epithelial cells against the cytotoxicity of these bacterial virulence factors. Moreover, we determined high-resolution crystal structures of the best inhibitors in complex with cholix toxin to reveal important criteria for inhibitor binding and mechanism of action. These results provide new insight into development of antivirulence compounds for treating many bacterial diseases.

| S-EPMC3067067 | biostudies-literature

Loss of the Mono-ADP-ribosyltransferase, Tiparp, Increases Sensitivity to Dioxin-induced Steatohepatitis and Lethality.

Project description:The aryl hydrocarbon receptor (AHR) mediates the toxic effects of the environmental contaminant dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin; TCDD). Dioxin causes a range of toxic responses, including hepatic damage, steatohepatitis, and a lethal wasting syndrome; however, the mechanisms are still unknown. Here, we show that the loss of TCDD-inducible poly(ADP-ribose) polymerase (Tiparp), an ADP-ribosyltransferase and AHR repressor, increases sensitivity to dioxin-induced toxicity, steatohepatitis, and lethality. Tiparp(-/-) mice given a single injection of 100 ?g/kg dioxin did not survive beyond day 5; all Tiparp(+/+) mice survived the 30-day treatment. Dioxin-treated Tiparp(-/-) mice exhibited increased liver steatosis and hepatotoxicity. Tiparp ADP-ribosylated AHR but not its dimerization partner, the AHR nuclear translocator, and the repressive effects of TIPARP on AHR were reversed by the macrodomain containing mono-ADP-ribosylase MACROD1 but not MACROD2. These results reveal previously unidentified roles for Tiparp, MacroD1, and ADP-ribosylation in AHR-mediated steatohepatitis and lethality in response to dioxin.

| S-EPMC4505429 | biostudies-literature

Human alpha-defensins neutralize toxins of the mono-ADP-ribosyltransferase family.

Project description:Various bacterial pathogens secrete toxins, which are not only responsible for fatal pathogenesis of disease, but also facilitate evasion of host defences. One of the best-known bacterial toxin groups is the mono-ADP-ribosyltransferase family. In the present study, we demonstrate that human neutrophil alpha-defensins are potent inhibitors of the bacterial enzymes, particularly against DT (diphtheria toxin) and ETA (Pseudomonas exotoxin A). HNP1 (human neutrophil protein 1) inhibited DT- or ETA-mediated ADP-ribosylation of eEF2 (eukaryotic elongation factor 2) and protected HeLa cells against DT- or ETA-induced cell death. Kinetic analysis revealed that inhibition of DT and ETA by HNP1 was competitive with respect to eEF2 and uncompetitive against NAD+ substrates. Our results reveal that toxin neutralization represents a novel biological function of HNPs in host defence.

| S-EPMC1609915 | biostudies-other

The orthologue of the "acatalytic" mammalian ART4 in chicken is an arginine-specific mono-ADP-ribosyltransferase.

Project description:BackgroundHuman ART4, carrier of the GPI-(glycosyl-phosphatidylinositol) anchored Dombrock blood group antigens, is an apparently inactive member of the mammalian mono-ADP-ribosyltransferase (ART) family named after the enzymatic transfer of a single ADP-ribose moiety from NAD+ to arginine residues of extracellular target proteins. All known mammalian ART4 orthologues are predicted to lack ART activity because of one or more changes in essential active site residues that make up the R-S-EXE motif. So far, no other function has been detected.ResultsHere we report the identification and characterisation of ART4 in chicken, which to our knowledge is the first true non-mammalian orthologue of a mammalian ART family member. The chicken ART4 gene has the same physical structure as its mammalian counterparts (three coding exons separated by two introns in phase 0 and phase 1, respectively) and maps to a region of conserved linkage synteny on chromosome 1. Its mRNA encodes a 289 amino acid protein with predicted N-terminal signal peptide and C-terminal GPI-anchor sequences and 47% sequence identity to human ART4. However, in striking contrast to its mammalian orthologues, the chicken protein contains an intact R-S-EXE motif. Upon ectopic expression in C-33A cells, recombinant chicken ART4 localized at the cell surface as a GPI-anchored, highly glycosylated protein, which displayed arginine-specific ART activity (apparent Km of the recombinant protein for etheno-NAD+ 1.0 +/- 0.18 microM).ConclusionThe avian orthologue of the "acatalytic" mammalian ART4 is a mono-ADP-ribosyltransferase with enzymatic activity comparable to that of other, catalytically active and GPI-anchored members of the mammalian ART family.

| S-EPMC2576468 | biostudies-literature

Catalytic and non-catalytic roles for the mono-ADP-ribosyltransferase Arr in the mycobacterial DNA damage response.

Project description:Recent evidence indicates that the mycobacterial response to DNA double strand breaks (DSBs) differs substantially from previously characterized bacteria. These differences include the use of three DSB repair pathways (HR, NHEJ, SSA), and the CarD pathway, which integrates DNA damage with transcription. Here we identify a role for the mono-ADP-ribosyltransferase Arr in the mycobacterial DNA damage response. Arr is transcriptionally induced following DNA damage and cellular stress. Although Arr is not required for induction of a core set of DNA repair genes, Arr is necessary for suppression of a set of ribosomal protein genes and rRNA during DNA damage, placing Arr in a similar pathway as CarD. Surprisingly, the catalytic activity of Arr is not required for this function, as catalytically inactive Arr was still able to suppress ribosomal protein and rRNA expression during DNA damage. In contrast, Arr substrate binding and catalytic activities were required for regulation of a small subset of other DNA damage responsive genes, indicating that Arr has both catalytic and noncatalytic roles in the DNA damage response. Our findings establish an endogenous cellular function for a mono-ADP-ribosyltransferase apart from its role in mediating Rifampin resistance.

| S-EPMC3138739 | biostudies-literature

2,3,7,8-Tetrachlorodibenzo-p-dioxin poly(ADP-ribose) polymerase (TiPARP, ARTD14) is a mono-ADP-ribosyltransferase and repressor of aryl hydrocarbon receptor transactivation.

Project description:2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD)-inducible poly(ADP-ribose) polymerase (TiPARP/ARTD14) is a member of the PARP family and is regulated by the aryl hydrocarbon receptor (AHR); however, little is known about TiPARP function. In this study, we examined the catalytic function of TiPARP and determined its role in AHR transactivation. We observed that TiPARP exhibited auto-mono-ADP-ribosyltransferase activity and ribosylated core histones. RNAi-mediated knockdown of TiPARP in T-47D breast cancer and HuH-7 hepatoma cells increased TCDD-dependent cytochrome P450 1A1 (CYP1A1) and CYP1B1 messenger RNA (mRNA) expression levels and recruitment of AHR to both genes. Overexpression of TiPARP reduced AHR-dependent increases in CYP1A1-reporter gene activity, which was restored by overexpression of AHR, but not aryl hydrocarbon receptor nuclear translocator. Deletion and mutagenesis studies showed that TiPARP-mediated inhibition of AHR required the zinc-finger and catalytic domains. TiPARP and AHR co-localized in the nucleus, directly interacted and both were recruited to CYP1A1 in response to TCDD. Overexpression of Tiparp enhanced, whereas RNAi-mediated knockdown of TiPARP reduced TCDD-dependent AHR proteolytic degradation. TCDD-dependent induction of AHR target genes was enhanced in Tiparp(-/-) mouse embryonic fibroblasts compared with wildtype controls. Our findings show that TiPARP is a mono-ADP-ribosyltransferase and a transcriptional repressor of AHR, revealing a novel negative feedback loop in AHR signalling.

| S-EPMC3562000 | biostudies-literature

Identification of two regulatory binding sites which confer myotube specific expression of the mono-ADP-ribosyltransferase ART1 gene.

Project description:BACKGROUND: Mono-ADP-ribosyltransferase (ART) 1 belongs to a family of mammalian ectoenzymes that catalyze the transfer of ADP-ribose from NAD+ to a target protein. ART1 is predominantly expressed in skeletal and cardiac muscle. It ADP-ribosylates alpha7-integrin which together with beta1-integrin forms a dimer and binds to laminin, a protein of the extracellular matrix involved in cell adhesion. This posttranslational modification leads to an increased laminin binding affinity. RESULTS: Using C2C12 and C3H-10T 1/2 cells as models of myogenesis, we found that ART1 expression was restricted to myotube formation. We identified a fragment spanning the gene 1.3 kb upstream of the transcriptional start site as the functional promoter of the ART1 gene. This region contains an E box and an A/T-rich element, two conserved binding sites for transcription factors found in the promoters of most skeletal muscle specific genes. Mutating the DNA consensus sequence of either the E box or the A/T-rich element resulted in a nearly complete loss of ART1 promoter inducibility, indicating a cooperative role of the transcription factors binding to those sites. Gel mobility shift analyses carried out with nuclear extracts from C2C12 and C3H-10T 1/2 cells revealed binding of myogenin to the E box and MEF-2 to the A/T-rich element, the binding being restricted to C2C12 and C3H-10T 1/2 myotubes. CONCLUSION: Here we describe the molecular mechanism underlying the regulation of the ART1 gene expression in skeletal muscle cells. The differentiation-dependent upregulation of ART1 mRNA is induced by the binding of myogenin to an E box and of MEF-2 to an A/T-rich element in the proximal promoter region of the ART1 gene. Thus the transcriptional regulation involves molecular mechanisms similar to those used to activate muscle-specific genes.

| S-EPMC2575215 | biostudies-literature

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