ABSTRACT
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.
ABBREVIATIONS
- ART
- ADP-ribosyltransferase
- ADP
- adenosine diphosphate
- B. pertussis
- Bordetella pertussis
- BSA
- bovine serum albumin
- DMSO
- dimethyl sulfoxide
- DSF
- differential scanning fluorimetry
- E. coli
- Escherichia coli
- Gαi
- inhibitory α-subunit of heterotrimeric G-protein
- GPCR
- G-protein coupled receptor
- HRP
- horse radish peroxidase
- IC50
- half maximal inhibitory concentration
- LB
- Luria-Bertani
- MALS
- multi-angle light scattering
- MAR
- mono-ADP-ribose
- MDS
- molecular dynamic simulation
- NAD+
- nicotinamide adenine dinucleotide
- PAR
- poly-ADP-ribose
- PARP1
- poly(ADP-ribose) polymerase 1 (also known as ARTD1)
- PCR
- polymerase chain reaction
- PDB
- protein data bank
- PtxS1
- pertussis toxin S1 subunit
- rGαi
- recombinant inhibitory α-subunit of heterotrimeric G-protein
- RMSD
- root-mean-square deviation
- rPtxS1
- recombinant pertussis toxin S1 subunit
- SDS-PAGE
- sodium dodecyl sulphate polyacrylamide gel electrophoresis
- SEC
- size exclusion chromatography
- TBST
- Tris-buffered saline containing 0.05 % Tween
- Tm
- melting temperature