Using DNA-encoded Chemical Libraries to Develop Inhibitors of the MCR-1 Colistin Resistance Enzyme The increasing prevalence of bacterial pathogens resistant to multiple antibiotics is a serious threat to global health. The spread of multidrug-resistant Gram-negative bacterial pathogens is a particular concern. In this regard, the increased prevalence of carbapenemase enzymes such as the KPC and NDM b-lactamases has reduced the efficacy of b-lactam antibiotics. The limited treatment options has led to increased use of polymyxin antibiotics such as colistin. The recent emergence and spread of a plasmid-encoded, transferable resistance gene, mcr-1, is a cause for concern. The mcr-1 gene encodes an enzyme, MCR-1, that catalyzes the transfer of phosphoethanolamine (PEA) from phosphatidylethanolamine to the 1’ or 4’ phosphate of lipid A. The neutralization of the negative charge on lipid A reduces binding of the positively charged colistin, leading to resistance. The MCR-1 enzyme contains an N-terminal membrane domain and a soluble C-terminal catalytic domain. We have expressed and purified the full-length MCR-1 enzyme from E. coli and have shown it is active in vitro. The availability of the purified full-length enzyme provides the basis for a biochemical screen for small molecule inhibitors. DNA-encoded chemical libraries contain small molecules covalently attached to encoding DNA. These libraries have proven to be an efficient means of identifying small molecules that bind to a target protein. We screened a library containing >2 billion compounds against immobilized MCR-1 and identified potential hits by next-generation DNA sequencing. Several putative hit compounds were synthesized in the absence of the DNA tag and two of these molecules synergize with colistin to kill E. coli expressing the MCR-1 enzyme. Importantly, the compounds do not alter E. coli growth rate when used alone and do not synergize with colistin against a strain containing a catalytically inactive MCR-1 protein. We propose to validate these chemically unique compounds by showing that they bind and inhibit the MCR-1 enzyme. In addition, we will determine their spectrum of activity against a set of colistin-resistant clinical isolates containing the MCR- 1 enzyme as well as isolates containing the MCR-2, -3, -5, and -9 variant enzymes. Further, we will utilize medicinal chemistry approaches to determine structure-activity relationships and identify more potent derivatives of the compounds. Finally, we will screen new DNA-encoded chemical libraries to identify additional novel inhibitors. The end result of these studies will be validated, potent MCR-1 inhibitors that can be further developed with regard to pharmacological properties and efficacy in animal models in future studies. Our goal is to identify potent inhibitors of MCR-1 that will restore the effectiveness of current and future polymyxin antibiotics.