Identification of Inhibitors of Pseudomonas aeruginosa Exotoxin-S ADP-Ribosyltransferase Activity

Pinto, A.F.; Ebrahimi, Mahsa; Saleeb, Michael; Försberg, Åke; Elofsson, Mikael; Schüler, H.

doi:10.1177/1087057116629923

Cited by 14 publications

(13 citation statements)

References 27 publications

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“…Our current study shows that small molecular weight compounds inhibiting the ADP-ribosyltransferase activity of pertussis toxin might also have therapeutic potential. Similar findings have been published on some other ADP-ribosylating bacterial toxins, ExoS of Pseudomonas aeruginosa and cholix-toxin of Vibrio cholerae 34, 35 . We conclude that NSC228155 and NSC29193 are useful templates for future hit development to specifically inhibit pertussis toxin ADP-ribosyltransferase activity in whooping cough.…”

Section: Resultssupporting

confidence: 87%

Discovery of compounds inhibiting the ADP-ribosyltransferase activity of pertussis toxin

Ashok

Miettinen

Oliveira

et al. 2019

Preprint

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Targeted pathogen-selective approach to antibiotic development holds promise to minimize collateral damage to the beneficial microbiome. The AB5-topology pertussis toxin (PtxS1-S5, 1:1:1:2:1) is a major virulence factor of Bordetella pertussis, the causative agent of the highly contagious respiratory disease whooping cough. Once internalized into the host cell, PtxS1 ADP-ribosylates α-subunits of the heterotrimeric Gαi-superfamily, thereby disrupting G-protein-coupled receptor signaling. Here, we report the discovery of the first small molecules inhibiting the ADP-ribosyltransferase activity of pertussis toxin. We developed protocols to purify mg-levels of truncated but highly active recombinant B. pertussis PtxS1 from Escherichia coli and an in vitro high throughput-compatible assay to quantify NAD+ consumption during PtxS1-catalyzed ADP-ribosylation of Gαi. Two inhibitory compounds (NSC228155 and NSC29193) with low micromolar IC50-values (3.0 µM and 6.8 µM) were identified in the in vitro NAD+ consumption assay that also were potent in an independent in vitro assay monitoring conjugation of ADP-ribose to Gαi. Docking and molecular dynamics simulations identified plausible binding poses of NSC228155 and in particular of NSC29193, most likely owing to the rigidity of the latter ligand, at the NAD+-binding pocket of PtxS1. NSC228155 inhibited the pertussis AB5 holotoxin-catalyzed ADP-ribosylation of Gαi in living human cells with a low micromolar IC50-value (2.4 µM). NSC228155 and NSC29193 might prove useful hit compounds in targeted B. pertussis-selective drug development.

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Section: Resultssupporting

confidence: 87%

Discovery of compounds inhibiting the ADP-ribosyltransferase activity of pertussis toxin

Ashok

Miettinen

Oliveira

et al. 2019

Preprint

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“…1f, g ), and the heterotrimer with inhibitor STO1101 (ref. 30 ) at 3.24 Å resolution (Supplementary Fig. 2 ).…”

Section: Resultsmentioning

confidence: 99%

“…Z96229612). 30 PPI inhibitor STO1704 ( N -(2-ethyl-6-methylphenyl)-2-[2-(1-hydroxyethyl)-1H-benzimidazol-1-yl]- N -(2-methoxy-1-methylethyl)acetamide) was purchased from ChemBridge (cat. no.…”

Section: Methodsmentioning

confidence: 99%

“…Expression plasmids for other ExoS fragents were based on vector pNIC-Bsa4 or pET28a, and have been described. 30 GFP-tagged ExoS expression vectors were constructed by sub-cloning ExoS 233–419 , ExoS 233–435 , ExoS 233–453 , and ExoS 419–453 in pET-GFP1a. 44 An ExoS:14-3-3β co-expression vector was constructed by sub-cloning a cDNA fragment encoding the E379A,E381A mutant of ExoS 233–453 and the cDNA encoding 14-3-3β 1–239 into pET-Duet1 (Novagen).…”

Section: Methodsmentioning

confidence: 99%

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14-3-3 proteins activate Pseudomonas exotoxins-S and -T by chaperoning a hydrophobic surface

et al. 2018

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Pseudomonas are a common cause of hospital-acquired infections that may be lethal. ADP-ribosyltransferase activities of Pseudomonas exotoxin-S and -T depend on 14-3-3 proteins inside the host cell. By binding in the 14-3-3 phosphopeptide binding groove, an amphipathic C-terminal helix of ExoS and ExoT has been thought to be crucial for their activation. However, crystal structures of the 14-3-3β:ExoS and -ExoT complexes presented here reveal an extensive hydrophobic interface that is sufficient for complex formation and toxin activation. We show that C-terminally truncated ExoS ADP-ribosyltransferase domain lacking the amphipathic binding motif is active when co-expressed with 14-3-3. Moreover, swapping the amphipathic C-terminus with a fragment from Vibrio Vis toxin creates a 14-3-3 independent toxin that ADP-ribosylates known ExoS targets. Finally, we show that 14-3-3 stabilizes ExoS against thermal aggregation. Together, this indicates that 14-3-3 proteins activate exotoxin ADP-ribosyltransferase domains by chaperoning their hydrophobic surfaces independently of the amphipathic C-terminal segment.

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“…The 14-3-3 encoding cDNA YWHAZ was also sub-cloned in pNic28-Bsa4 to obtain an N-terminal hexahistidine fusion construct. Expression plasmids for other ExoS fragents were based on vector pNIC-Bsa4 or pET28a, and have been described 30. Green fluorescent protein (GFP)-tagged ExoS expression vectors were constructed by sub-cloning ExoS 233-419 , ExoS 233-435 , ExoS 233-453 , and ExoS 419-453 in pET-GFP1a 45.…”

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confidence: 99%

14-3-3 proteins activatePseudomonasexotoxins-S and –T by chaperoning a hydrophobic surface

Karlberg

Hornyák

Pinto

et al. 2018

Preprint

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Short summary -Crystal structures of Pseudomonas exotoxins-S and -T identify a novelhydrophobic interface with 14-3-3 proteins, and we show that 14-3-3 activates these toxins independent of their phosphopeptide groove binding C-termini, by preventing their aggregation.All rights reserved. No reuse allowed without permission.(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint . http://dx.doi.org/10.1101/342659 doi: bioRxiv preprint first posted online Jun. 8, 2018; AbstractPseudomonas are a common cause of hospital acquired infections that may be lethal. ADPribosyltransferase activities of Pseudomonas exotoxin-S and -T depend on 14-3-3 proteins inside the host cell. By binding in the 14-3-3 phosphopeptide binding groove, a hydrophobic C-terminal helix of ExoS and ExoT has been thought to be crucial for their activation. However, crystal structures of the 14-3-3:ExoS and -ExoT complexes presented here reveal an extensive novel binding interface that is sufficient for complex formation and toxin activation. We show that C-terminally truncated ExoS ADP-ribosyltransferase domain lacking the hydrophobic binding motif is active when co-expressed with 14-3-3. Moreover, swapping the hydrophobic C-terminus with a fragment from Vibrio Vis toxin creates a 14-3-3 independent toxin that ADP-ribosylates known ExoS targets. Finally, weshow that 14-3-3 stabilizes ExoS against thermal aggregation. Together, this indicates that 14-3-3 proteins activate exotoxin ADP-ribosyltransferase domains by chaperoning their hydrophobic surfaces independently of the hydrophobic C-terminal segment.Pseudomonas aeruginosa is an opportunistic pathogen that is infamous for causing hospital acquired airway and wound infections. To initiate the infection process, the bacterium uses a type III secretion system to deliver a small set of exotoxins into the host cell.1, 2 Two of these, exotoxins S and T (ExoS; ExoT) are homologous enzymes consisting of an N-terminal GTPase activating protein (GAP) domain (73% identity; 81% similarity) and a C-terminal ADPribosyltransferase (ART) domain (78% identity; 87% similarity). 3,4 Their GAP domains target Rho-family GTPases which leads to a remodeling of the host actin cytoskeleton. The activities of the ART domains are directed toward a more diverse set of proteins. ExoS targets Rasand Rho-family GTPases, 5,6 ezrin/radixin/moesin (ERM) proteins, 7 and the intermediate All rights reserved. No reuse allowed without permission.(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint . http://dx.doi.org/10.1101/342659 doi: bioRxiv preprint first posted online Jun. 8, 2018; filament protein, vimentin.8 ADP-ribosylation disturbs these targets presumably by placing the bulky ADP-ribose moiety in a protein-protein interaction site, and has multiple consequences including and disruption of actin polym...

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Identification of Inhibitors of Pseudomonas aeruginosa Exotoxin-S ADP-Ribosyltransferase Activity

Cited by 14 publications

References 27 publications

Discovery of compounds inhibiting the ADP-ribosyltransferase activity of pertussis toxin

Discovery of compounds inhibiting the ADP-ribosyltransferase activity of pertussis toxin

14-3-3 proteins activate Pseudomonas exotoxins-S and -T by chaperoning a hydrophobic surface

14-3-3 proteins activatePseudomonasexotoxins-S and –T by chaperoning a hydrophobic surface

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