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

Abstract

The mono-ADP-ribosyltransferase toxins are bacterial virulence factors that contribute to many disease states in plants, animals, and humans. These toxins function as enzymes that target various host proteins and covalently attach an ADP-ribose moiety that alters target protein function. We tested compounds from a virtual screen of commercially available compounds combined with a directed poly(ADP-ribose) polymerase (PARP) inhibitor library and found several compounds that bind tightly and inhibit toxins from Pseudomonas aeruginosa and Vibrio cholerae. The most efficacious compounds completely protected human lung epithelial cells against the cytotoxicity of these bacterial virulence factors. Moreover, we determined high-resolution crystal structures of the best inhibitors in complex with cholix toxin to reveal important criteria for inhibitor binding and mechanism of action. These results provide new insight into development of antivirulence compounds for treating many bacterial diseases.Bacteria use virulence factors as tools to facilitate disease in plants, animals, and humans (14,26,30,34); one strategy to combat infection is to inhibit these factors by small-molecule therapy, thereby helping to neutralize the offending microbe (5,6,12,19,22). It is now generally appreciated that an antivirulence approach is a powerful alternative strategy for antibacterial treatment and vaccine development (27) and that it may require multiple tactics to resolve the current drug resistance dilemma (6,8). Antivirulence compounds offer significant advantages over conventional antibiotics since these inhibitors are directed toward specific mechanisms (targets) in the offending pathogen that promote infection rather than against an essential metabolic factor (12). Neutralizing the cytotoxic properties of virulence factors from microorganisms without threatening their survival offers reduced selection pressure, making the induction of drug resistance mutations less likely (6). Additionally, virulence-specific therapeutics avoid the undesirable effects on the host microbiota that are associated with current antibiotics.The mono-ADP-ribosyltransferase (mART) family is a group of toxic bacterial enzymes, some of which possess a long history against human civilization. The best-characterized and wellknown members of this lethal family are cholera toxin (CT) from Vibrio cholerae, diphtheria toxin (DT) produced by Corynebacterium diphtheriae, pertussis toxin (PT) from Bordella pertussis, heat-labile enterotoxin from Escherichia coli, C3-like exoenzyme produced by Clostridium botulinum and Clostridium limosum, and exotoxin A (ExoA) from Pseudomonas aeruginosa. These enzymes act on NAD ϩ and facilitate the scission of the glycosidic bond (C-N) between nicotinamide and its conjugated ribose followed by the transfer of the ADPribose group to a nucleophilic residue on a target macromolecule (35). This family can be divided into the CT and DT groups. The CT group consists of an ExoS-like subgroup (enzymatic A domain alone or paired with ano...

Scabin, a Novel DNA-acting ADP-ribosyltransferase from Streptomyces scabies

“…The side-chain oxygen of Asn 110 is situated 2.9 Å away from the tertiary amine of the R-group of PJ34, which forms another important H-bond. As reported earlier in other mART toxin structures with PJ34 (25,35,36), the electron density of the hetero-ring system of the inhibitor is clearly defined; however, the electron density is weaker for the R-group (tertiary amine) (supplemental Fig. S1), suggesting some flexibility in the "tail" of the inhibitor.…”

“…Several potent inhibitors were identified, including a previously characterized inhibitor of mART toxins known as PJ34 (25,35,36). The inhibitor structures are stabilized within the active site of Scabin, largely through hydrophobic interactions combined with a few critical H-bonds.…”

“…Scabin was tested against several in-house mART toxin libraries of inhibitors, which include the P6 series (25). Several potent inhibitors were identified, including a previously characterized inhibitor of mART toxins known as PJ34 (25,35,36).…”

“…6B). P6-E is a member of the P6 series of inhibitors (25), and this is the first report of its crystal complex bound to a mART enzyme. The P6-E-bound structure shows a low root mean square deviation from Scabin-apo (0.114 Å for 1053 atoms).…”

“…These compounds, PJ34, P6-C, P6-D, P6-E, and P6-F (Fig. 4), are known inhibitors of other mART toxins (23,25). Lineweaver-Burk analysis was performed for kinetic inhibition data of Scabin with PJ34 inhibitor to validate the mechanism of inhibition for the P-series inhibitors.…”