Project Summary/Abstract Infections with antibiotic-resistant strains of bacteria are a looming public health threat to the nation. The problem is especially pressing in the era of COVID-19, where secondary infections were identified in half of hospitalized patients who did not survive. More research on the identity of the secondary infections in the current outbreak still needs to be conducted, but in the SARS coronavirus outbreak, gram-negative bacteria were the most common source of the hospital-acquired infections. Gram-negative bacteria like Pseudomonas aeruginosa have few effective treatments, making any resistant strains particularly difficult to treat. Cationic antimicrobial peptides (CAPs) like colistin are currently used as last line therapies to treat infections with multidrug-resistant strains, however, colistin-resistant strains are increasingly encountered. P. aeruginosa becomes resistant to CAPs by modifying the lipids of its outer membrane to reduce the net negative charge. Without the favorable electrostatic interaction between the positively-charged CAP and the negatively- charged bacterial outer membrane, the antibiotic cannot enter and kill the bacterium. Our goal is to develop and characterize inhibitors of the CAP-resistance pathway in the bacterium. If the outer membrane remains negatively charged, the CAP should continue to work. The inhibitors could be used as an adjuvant in a combination therapy along with a CAP to treat resistant infections. In the first funding period, we identified a suite of compounds that act as antibiotic adjuvants, potentiating susceptibility to colistin. In the current work, we propose to characterize the efficacy of the combination treatment (Aim 1). We will investigate activity toward biofilms, resistant strains, and under resistance- inducing conditions. The kinetics of killing and the development of resistance will also be studied. Our current hypothesis is that the antibiotic adjuvant acts by inhibition of ArnA, a key bifunctional biosynthetic enzyme of the lipid modification pathway. We will study the adjuvant’s effects on the ArnA-mediated resistance pathway through analysis of the lipids by mass spectrometry and direct interactions with ArnA in kinetics and binding experiments (Aim 2). The proposed work will validate the further development of the combination therapy towards use in the clinic. Confirmation of the molecular target will also guide the optimization of even more potent inhibitors in future work. Deciphering the molecular target could also allow us to extend the therapy to other gram-negative bacteria like carbapenem-resistant Enterobacteriaceae and drug-resistant Salmonella as a long-term goal. The first-in-class inhibitor has the potential to widen the therapeutic index of colistin, minimizing the drug’s serious side effects, as well as to enable the treatment of colistin-resistant strains, ensuring the continued efficacy of the life-saving drug.