Characterization of Competitive Inhibitors for the Transferase Activity of Pseudomonas aeruginosa Exotoxin A

Armstrong, Souzan; Li, Jia He; Zhang, Jie; Merrill, A. Rod

doi:10.1080/1475636021000010914

Cited by 21 publications

(32 citation statements)

References 23 publications

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“…We demonstrated previously that NAP is a potent in vitro competitive inhibitor against the NAD + substrate of ExoA c (Armstrong et al , 2002) and, herein, we also show that NAP is an effective in vitro inhibitor of both DT c and cholix c (Table 1). More specifically, NAP mimics the nicotinamide moiety of NAD + and binds into a nonpolar substrate pocket within the enzymes as shown in Fig.…”

Section: Resultssupporting

confidence: 81%

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Yeast as a tool for characterizing mono-ADP-ribosyltransferase toxins

Turgeon¹,

White²,

Jørgensen³

et al. 2009

FEMS Microbiology Letters

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The emergence of bacterial antibiotic resistance poses a significant challenge in the pursuit of novel therapeutics, making new strategies for drug discovery imperative. We have developed a yeast growth-defect phenotypic screen to help solve this current dilemma. This approach facilitates the identification and characterization of a new diphtheria toxin (DT) group, ADP-ribosyltransferase toxins from pathogenic bacteria. In addition, this assay utilizes Saccharomyces cerevisiae, a reliable model for bacterial toxin expression, to streamline the identification and characterization of new inhibitors against this group of bacterial toxins that may be useful for antimicrobial therapies. We show that a mutant of the elongation factor 2 target protein in yeast, G701R, confers resistance to all DT group toxins and recovers the growth-defect phenotype in yeast. We also demonstrate the ability of a potent small-molecule toxin inhibitor, 1,8-naphthalimide (NAP), to alleviate the growth defect caused by toxin expression in yeast. Moreover, we determined the crystal structure of the NAP inhibitor-toxin complex at near-atomic resolution to provide insight into the inhibitory mechanism. Finally, the NAP inhibitor shows therapeutic protective effects against toxin invasion of mammalian cells, including human lung cells.

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Section: Resultssupporting

confidence: 81%

“…NAP was previously shown to be a potent inhibitor of ExoA c in vitro mART activity (Armstrong et al , 2002). To determine whether NAP could protect yeast cells expressing DT group mART toxins, yeast broth was supplemented with 50 μM of the inhibitor (in 1% DMSO) before toxin induction with Cu 2+ (Fig.…”

Section: Resultsmentioning

confidence: 99%

Yeast as a tool for characterizing mono-ADP-ribosyltransferase toxins

Turgeon¹,

White²,

Jørgensen³

et al. 2009

FEMS Microbiology Letters

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“…1). It was previously demonstrated that the P-series compounds compete with the NAD ϩ substrate in ExoA/cholix by binding within the nicotinamide binding site within these enzymes (2,36). The virtual screen approach identified 72 hits (prospective inhibitors), which were filtered based upon chemical stability and redundancy, and 31 compounds were then tested experimentally (Table 1; see also Table S1 in the supplemental material).…”

Section: Resultsmentioning

confidence: 99%

“…We recently developed an in silico approach based on fold recognition methods to identify prospective new mART members from bacterial genomes (13). These newly discovered toxins can now be exploited as targets in the development of new antivirulence therapeutics for treating bacterial diseases and infections (9,29).Here, we focus on two DT-group mARTs targeting elongation factor 2-ExoA, a well-characterized factor produced by P. aeruginosa, and cholix, a new mART toxin recently identified with our in silico approach from V. cholerae (16)-which, along with diphtheria toxin, show nearly identical enzyme activities and inhibitor specificities (2,16,31,35,36). Using the 1.25-Å cocrystal structure of cholix toxin with PJ34 [N-(6-oxo-5,6-dihydrophenanthridin-2-yl)-(N,N-dimethylamino)acet-* Corresponding author.…”

mentioning

confidence: 99%

“…Here, we focus on two DT-group mARTs targeting elongation factor 2-ExoA, a well-characterized factor produced by P. aeruginosa, and cholix, a new mART toxin recently identified with our in silico approach from V. cholerae (16)-which, along with diphtheria toxin, show nearly identical enzyme activities and inhibitor specificities (2,16,31,35,36). Using the 1.25-Å cocrystal structure of cholix toxin with PJ34 [N-(6-oxo-5,6-dihydrophenanthridin-2-yl)-(N,N-dimethylamino)acet-amide hydrochloride] inhibitor (Protein Data Bank [PDB] accession number 2Q6M) as a template, a virtual screen of over 500,000 commercial compounds identified 72 prospective inhibitors.…”

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confidence: 99%

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Newly Discovered and Characterized Antivirulence Compounds Inhibit Bacterial Mono-ADP-Ribosyltransferase Toxins

Turgeon

Jørgensen

Visschedyk

et al. 2011

Antimicrob Agents Chemother

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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.Bacteria use virulence factors as tools to facilitate disease in plants, animals, and humans (14,26,30,34); one strategy to combat infection is to inhibit these factors by small-molecule therapy, thereby helping to neutralize the offending microbe (5,6,12,19,22). It is now generally appreciated that an antivirulence approach is a powerful alternative strategy for antibacterial treatment and vaccine development (27) and that it may require multiple tactics to resolve the current drug resistance dilemma (6,8). Antivirulence compounds offer significant advantages over conventional antibiotics since these inhibitors are directed toward specific mechanisms (targets) in the offending pathogen that promote infection rather than against an essential metabolic factor (12). Neutralizing the cytotoxic properties of virulence factors from microorganisms without threatening their survival offers reduced selection pressure, making the induction of drug resistance mutations less likely (6). Additionally, virulence-specific therapeutics avoid the undesirable effects on the host microbiota that are associated with current antibiotics.The mono-ADP-ribosyltransferase (mART) family is a group of toxic bacterial enzymes, some of which possess a long history against human civilization. The best-characterized and wellknown members of this lethal family are cholera toxin (CT) from Vibrio cholerae, diphtheria toxin (DT) produced by Corynebacterium diphtheriae, pertussis toxin (PT) from Bordella pertussis, heat-labile enterotoxin from Escherichia coli, C3-like exoenzyme produced by Clostridium botulinum and Clostridium limosum, and exotoxin A (ExoA) from Pseudomonas aeruginosa. These enzymes act on NAD ϩ and facilitate the scission of the glycosidic bond (C-N) between nicotinamide and its conjugated ribose followed by the transfer of the ADPribose group to a nucleophilic residue on a target macromolecule (35). This family can be divided into the CT and DT groups. The CT group consists of an ExoS-like subgroup (enzymatic A domain alone or paired with ano...

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Gamma radiation coupled ADP-ribosyl transferase activity of Pseudomonas aeruginosa PE24 moiety

Morgan

Saleh

Farrag

et al. 2023

Appl Microbiol Biotechnol

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The ADP-ribosyl transferase activity of P. aeruginosa PE24 moiety expressed by E. coli BL21 (DE3) was assessed on nitrobenzylidene aminoguanidine (NBAG) and in vitro cultured cancer cell lines. Gene encoding PE24 was isolated from P. aeruginosa isolates, cloned into pET22b( +) plasmid, and expressed in E. coli BL21 (DE3) under IPTG induction. Genetic recombination was confirmed by colony PCR, the appearance of insert post digestion of engineered construct, and protein electrophoresis using sodium dodecyl-sulfate polyacrylamide gel (SDS-PAGE). The chemical compound NBAG has been used to confirm PE24 extract ADP-ribosyl transferase action through UV spectroscopy, FTIR, c13-NMR, and HPLC before and after low-dose gamma irradiation (5, 10, 15, 24 Gy). The cytotoxicity of PE24 extract alone and in combination with paclitaxel and low-dose gamma radiation (both 5 Gy and one shot 24 Gy) was assessed on adherent cell lines HEPG2, MCF-7, A375, OEC, and Kasumi-1 cell suspension. Expressed PE24 moiety ADP-ribosylated NBAG as revealed by structural changes depicted by FTIR and NMR, and the surge of new peaks at different retention times from NBAG in HPLC chromatograms. Irradiating recombinant PE24 moiety was associated with a reduction in ADP-ribosylating activity. The PE24 extract IC50 values were < 10 μg/ml with an acceptable R2 value on cancer cell lines and acceptable cell viability at 10 μg/ml on normal OEC. Overall, the synergistic effects were observed upon combining PE24 extract with low-dose paclitaxel demonstrated by the reduction in IC50 whereas antagonistic effects and a rise in IC50 values were recorded after irradiation by low-dose gamma rays. Key points • Recombinant PE24 moiety was successfully expressed and biochemically analyzed. • Low-dose gamma radiation and metal ions decreased the recombinant PE24 cytotoxic activity. • Synergism was observed upon combining recombinant PE24 with low-dose paclitaxel.

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Characterization of Competitive Inhibitors for the Transferase Activity of Pseudomonas aeruginosa Exotoxin A

Cited by 21 publications

References 23 publications

Yeast as a tool for characterizing mono-ADP-ribosyltransferase toxins

Yeast as a tool for characterizing mono-ADP-ribosyltransferase toxins

Newly Discovered and Characterized Antivirulence Compounds Inhibit Bacterial Mono-ADP-Ribosyltransferase Toxins

Gamma radiation coupled ADP-ribosyl transferase activity of Pseudomonas aeruginosa PE24 moiety

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