Project Summary/Abstract Infections caused by Gram-negative bacterial pathogens are on the rise in hospital and non-hospital settings. Indeed 4 of the 6 “ESKAPE” pathogens – recently highlighted as responsible for the majority of hospital infections and being exceedingly difficult to treat – are Gram-negatives. The development of new antibiotics is complicated by the fact that Gram-negative bacteria have a highly impenetrable membrane that confers significant intrinsic resistance to antibacterial agents. Without advances, we will soon face a crisis situation whereby our current antibiotics can no longer effectively treat these infections. Although it is clear that novel antibiotics for Gram-negative infections are desperately needed, there has been minimal progress in this regard, and it has been over 50 years since a new class of drugs have been introduced for Gram-negative pathogens. Why is this? A chief reason is that no rules or guidelines have been developed that enable the accurate prediction of compound accumulation in Gram-negatives, thus it has been difficult to convert Gram-positive-only drugs into broad-spectrum agents, and impossible to create large collections of compounds that are biased for Gram-negative accumulation. We have been working to define the physicochemical features of small molecules that allow them to accumulate in E. coli. In important preliminary results we have assessed >180 diverse compounds for their ability to accumulate in E. coli; using a sophisticated computation analysis of the data, we have begun to discern the physicochemical traits that govern compound accumulation in E. coli, and we have used these guidelines to convert a Gram-positive-only antibiotic into one that also has activity against many Gram-negative pathogens. We now propose to gain a further understanding of compound accumulation in E. coli (especially with respect to porin penetration and pump-mediated efflux), and to extend these guidelines to other Gram-negative pathogens. We will also use the guidelines to convert important FDA-approved antibiotics that are currently only effective against Gram-positive bacteria into derivatives that are also active against Gram-negative organisms. Finally, we will use our guidelines to design and construct a collection of thousands of compounds all of which are heavily biased for accumulation in Gram-negative bacteria. Significant outputs of this work include a fundamental understanding of the types of compounds that accumulate in Gram-negative bacteria, and actionable guidelines to be used to discover novel antibacterials.