ABSTRACT
Acetaldehyde dehydrogenase (AdhE) is a bifunctional acetaldehyde-coenzyme A (CoA) dehydrogenase and alcohol dehydrogenase involved in anaerobic metabolism in gram-negative bacteria. This enzyme was recently found to be a key regulator of the type three secretion (T3S) system in Escherichia coli. AdhE inhibitors can be used as tools to study bacterial virulence and a starting point for discovery of novel antibacterial agents. We developed a robust enzymatic assay, based on the acetaldehyde-CoA dehydrogenase activity of AdhE using both absorption and fluorescence detection models (Z' > 0.7). This assay was used to screen ~11,000 small molecules in 384-well format that resulted in three hits that were confirmed by resynthesis and validation. All three compounds are noncompetitive with respect to acetaldehyde and display a clear dose-response effect with hill slopes of 1-2. These new inhibitors will be used as chemical tools to study the interplay between metabolism and virulence and the role of AdhE in T3S regulation in gram-negative bacteria, and as starting points for the development of novel antibacterial agents.
Subject(s)
Alcohol Dehydrogenase/antagonists & inhibitors , Aldehyde Oxidoreductases/antagonists & inhibitors , Anti-Bacterial Agents/pharmacology , Drug Evaluation, Preclinical , Enterohemorrhagic Escherichia coli/drug effects , Enterohemorrhagic Escherichia coli/enzymology , Enzyme Inhibitors/pharmacology , Escherichia coli Proteins/antagonists & inhibitors , Alcohol Dehydrogenase/genetics , Alcohol Dehydrogenase/metabolism , Aldehyde Oxidoreductases/genetics , Aldehyde Oxidoreductases/metabolism , Animals , Anti-Bacterial Agents/chemistry , Cell Line , Dose-Response Relationship, Drug , Drug Discovery/methods , Drug Evaluation, Preclinical/methods , Enterohemorrhagic Escherichia coli/genetics , Enzyme Activation/drug effects , Enzyme Inhibitors/chemistry , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , Humans , Mice , WorkflowABSTRACT
Pseudomonas aeruginosa is an opportunistic pathogen that can be very hard to treat because of high resistance to different antibiotics and alternative treatment regimens are greatly needed. An alternative or a complement to traditional antibiotic is to inhibit virulence of the bacteria. The salicylidene acylhydrazide, INP0341, belongs to a class of compounds that has previously been shown to inhibit virulence in a number of Gram-negative bacteria. In this study, the virulence blocking effect of INP0341 on P. aeruginosa was studied in vitro and in vivo. Two important and closely related virulence system were examined, the type III secretion system (T3SS) that translocates virulence effectors into the cytosol of the host cell to evade immune defense and facilitate colonization and the flagella system, needed for motility and biofilm formation. INP0341 was shown to inhibit expression and secretion of the T3SS toxin exoenzyme S (ExoS) and to prevent bacterial motility on agar plates and biofilm formation. In addition, INP0341 showed an increased survival of P. aeruginosa-infected mice. In conclusion, INP0341 attenuates P. aeruginosa virulence.
