Yeast as a tool for characterizing mono-ADP-ribosyltransferase toxins

Abstract

The emergence of bacterial antibiotic resistance poses a significant challenge in the pursuit of novel therapeutics, making new strategies for drug discovery imperative. We have developed a yeast growth-defect phenotypic screen to help solve this current dilemma. This approach facilitates the identification and characterization of a new diphtheria toxin (DT) group, ADP-ribosyltransferase toxins from pathogenic bacteria. In addition, this assay utilizes Saccharomyces cerevisiae, a reliable model for bacterial toxin expression, to streamline the identification and characterization of new inhibitors against this group of bacterial toxins that may be useful for antimicrobial therapies. We show that a mutant of the elongation factor 2 target protein in yeast, G701R, confers resistance to all DT group toxins and recovers the growth-defect phenotype in yeast. We also demonstrate the ability of a potent small-molecule toxin inhibitor, 1,8-naphthalimide (NAP), to alleviate the growth defect caused by toxin expression in yeast. Moreover, we determined the crystal structure of the NAP inhibitor-toxin complex at near-atomic resolution to provide insight into the inhibitory mechanism. Finally, the NAP inhibitor shows therapeutic protective effects against toxin invasion of mammalian cells, including human lung cells.

The 1.8 Å Cholix Toxin Crystal Structure in Complex with NAD+ and Evidence for a New Kinetic Model

“…However, several crystal structures of the wild type cholix c catalytic domain show small molecule inhibitors consistently binding to the same secondary site (supplemental Fig. 8; PDBs 2Q6M, 3KI0, 3KI1, 3KI2, 3KI5) (2,11,12). This secondary site is occluded in the full-length structure by domain Ib, perhaps blocking NAD ϩ from binding there in the full-length crystal structure (but not the catalytic fragments used during the fluorescence-based assays).…”

“…Recently, our group characterized the structure and activity of several cholix inhibitors that fully protect human lung cells (2). Several cholix structures have now been reported, including the 2.1 Å fulllength cholix toxin structure and several high-resolution structures of the catalytic C-terminal domain (cholix c ) (2,11,12 (22), and cholera toxin (PDB 2A5F) (23). Key to understanding the toxin reaction mechanism, and how toxin inhibitors work, is to view toxins in the context of NAD ϩ , especially given that the NAD ϩ conformation is different in the toxins when compared with most other NAD ϩ binding proteins (24).…”

“…Cholix is active against eukaryotes, including crustaceans and mammals (11); it also kills yeast cells when expressed intracellularly (2,12). Cholix enters eukaryotic cells by receptormediated endocytosis (11).…”

Newly Discovered and Characterized Antivirulence Compounds Inhibit Bacterial Mono-ADP-Ribosyltransferase Toxins

“…We recently developed an in silico approach based on fold recognition methods to identify prospective new mART members from bacterial genomes (13). These newly discovered toxins can now be exploited as targets in the development of new antivirulence therapeutics for treating bacterial diseases and infections (9,29).Here, we focus on two DT-group mARTs targeting elongation factor 2-ExoA, a well-characterized factor produced by P. aeruginosa, and cholix, a new mART toxin recently identified with our in silico approach from V. cholerae (16)-which, along with diphtheria toxin, show nearly identical enzyme activities and inhibitor specificities (2,16,31,35,36). Using the 1.25-Å cocrystal structure of cholix toxin with PJ34 [N-(6-oxo-5,6-dihydrophenanthridin-2-yl)-(N,N-dimethylamino)acet-* Corresponding author.…”

“…Here, we focus on two DT-group mARTs targeting elongation factor 2-ExoA, a well-characterized factor produced by P. aeruginosa, and cholix, a new mART toxin recently identified with our in silico approach from V. cholerae (16)-which, along with diphtheria toxin, show nearly identical enzyme activities and inhibitor specificities (2,16,31,35,36). Using the 1.25-Å cocrystal structure of cholix toxin with PJ34 [N-(6-oxo-5,6-dihydrophenanthridin-2-yl)-(N,N-dimethylamino)acet-amide hydrochloride] inhibitor (Protein Data Bank [PDB] accession number 2Q6M) as a template, a virtual screen of over 500,000 commercial compounds identified 72 prospective inhibitors.…”