PROJECT SUMMARY/ABSTRACTThe percent of Gram-negative bacterial infections that are resistant to common antibiotics has increased at analarming rate over the last decade, and there is now an acute need for the discovery of novel antibiotics effectiveagainst multidrug-resistant Gram-negative pathogens. The standard method of antibacterial discovery ? whole-cell screening of compound collections ? has met with repeated failure for Gram-negatives, and these failureshave been traced to the fact that very few compounds in standard collections can penetrate the Gram-negativecell membranes and accumulate in these pathogens. Unfortunately, there has been scant information about thetypes of compounds that are competent for accumulation in Gram-negatives. Excitingly, we recently assessed aunique collection of >180 diverse compounds for their ability to accumulate in E. coli, trained a random forestclassification model to analyze the results, and from this data we identified physicochemical properties importantfor accumulation and developed predictive guidelines for compound accumulation in E. coli. We then showedthe utility of these guidelines by converting a Gram-positive-only antibiotic into a broad-spectrum agent. We nowpropose to develop tools that will allow us to fully define the physicochemical traits that enable compoundsaccumulation in three of the most concerning Gram-negative bacteria, carbapenem-resistantEnterobacteriaceae (CRE), drug-resistant Acinetobacter, and drug-resistant P. aeruginosa (to be referred tocollectively as EAP pathogens). Specifically, we seek to develop novel tools in the area of chemical probes(compound collections), bacterial strains, and computational models. Using these tools in conjunction with ourwell-validated compound accumulation assay, we intend to define the physicochemical traits needed forcompound accumulation in the EAP pathogens, including assessment of the influence of porins and effluxpumps, and the relative contribution of the outer and inner-membranes to blocking compound penetrance. Ourpredictive guidelines will be utilized to convert several high-value Gram-positive-only compounds into broad-spectrum antibiotics.