The rise of antibiotic-resistant pathogenic bacteria and the increasing understanding of the beneficial role of the bacterial microbiota, disrupted by antibiotics, prompt the development of new anti-bacterial therapies. One such approach targets bacterial virulence factors such as exotoxins, which are often the disease-causing virulence factor, making them appealing drug development targets. Detailed molecular understanding of exotoxins is crucial for efficient drug development efforts.

This study focuses on pertussis toxin (PT), a major virulence factor of Bordetella pertussis, the causative agent of whooping cough. PT, an ADP-ribosyltransferase (ART) toxin, disrupts cellular signaling by transferring ADP-ribose from nicotinamide adenine dinucleotide (NAD+) to inhibitory α-subunits of G proteins of the host cell, leading to variety of systemic pathologies. The aim of the study was to identify small molecules inhibiting the ART activity of PT, and to obtain atomic resolution structural insights of the binding poses of the inhibitors as well as of the ART activity of PT.

An in vitro high-throughput multiwell assay to screen small molecules inhibiting the ART activity of PT was developed. Two compounds, effective at low micromolar levels in vitro, with one also potent in living cells, were identified. No binding poses for the compounds were obtained with X-ray crystallography. However, crystal structures of PT in complex with NAD+, its hydrolysis products ADP-ribose and nicotinamide, NAD+ analog PJ34 (ART inhibitor), and a novel NAD+ analog formed upon crystallization with 3-aminobenzamide (ART inhibitor) and NAD+, were obtained. These structures provide novel insights into pre- and post-NAD+ hydrolysis steps of the ART activity of PT and provide a rational basis to develop ART inhibitors as well as the biocatalytic use of PT to produce the novel NAD+ analog for biochemical and structural experiments with NAD+ binding proteins.

In conclusion, this study identified small molecules inhibiting the ART activity of PT and obtained atomic resolution structural insights of the ART activity, as well as binding poses of the small molecules in PT, known to inhibit other ARTs. These findings could aid in the rational drug design approaches and development of PT-specific small-molecule inhibitors.