The targeted pathogen-selective approach to drug development holds
promise to minimize collateral damage to the beneficial microbiome. The
AB₅-topology pertussis toxin (PtxS1-S5) 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 milligram-levels of 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 to be useful hit compounds in targeted B. pertussis-selective drug development.