Discovery of Compounds Inhibiting the ADP-Ribosyltransferase Activity of Pertussis Toxin.

The targeted pathogen-selective approach to drug development holds promise to minimize collateral damage to the beneficial microbiome. The AB-topology pertussis toxin (PtxS1-S5) is a major virulence factor of , 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 milligram-levels of active recombinant PtxS1 from and an high throughput-compatible assay to quantify NAD consumption during PtxS1-catalyzed ADP-ribosylation of Gαi. Two inhibitory compounds (NSC228155 and NSC29193) with low micromolar IC-values (3.0 μM and 6.8 μM) were identified in the NAD consumption assay that also were potent in an independent 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 AB holotoxin-catalyzed ADP-ribosylation of Gαi in living human cells with a low micromolar IC-value (2.4 μM). NSC228155 and NSC29193 might prove to be useful hit compounds in targeted -selective drug development.