PROJECT SUMMARY Antibiotic resistance is one of the most serious public health challenges of our time. In this proposal, we focus on two common antibiotic resistance mechanisms that are: (i) alteration of drug target enzymes and (ii) modification of antibiotic molecules. Despite recent advances in biological sciences, clinically accessible antibiotics can still target only a handful of enzymes, such as DNA gyrases and dihydrofolate reductase (DHFR). Hence, it is important to better understand molecular evolution of these enzymes and accordingly develop effective drugs that target these enzymes without exacerbating the resistance problem. Similarly, modification of -lactam antibiotics through hydrolysis by -lactamases is currently the most concerning antibiotic resistance mechanism because -lactams account for nearly seventy percent of antibiotics currently used in clinics. To address this important health problem, we propose an innovative research plan to study evolution of the DHFR enzyme, and develop mutant-specific competitive and allosteric DHFR inhibitors to select against resistance- conferring DHFR mutations. Finally, to address the -lactam resistance problem, we will engineer a novel class of molecules that we named “-lactamase traps” (or BLTs) to select against -lactamase producing bacteria. Our first aim is to map epistatic interactions that shape DHFR evolution under antibiotic selection. DHFR is a ubiquitous enzyme with a central role in metabolism. We have previously shown that E. coli DHFR accumulates 3 to 5 resistance conferring mutations following a quasi-deterministic order, as a result of strong epistatic interactions between DHFR mutations. We will quantitatively map all epistatic interactions in the DHFR fitness landscape by using state-of-the-art genetic tools and the Hierarchical Model, a mathematical toolbox we developed to efficiently explore the fitness space and identify epistatic interactions between mutations. Our second goal is to engineer competitive and allosteric mutant-specific DHFR inhibitors. We have previously developed an L28R-specific competitive trimethoprim derivative (4’-dTMP) that impeded evolution of resistance by selecting against the L28R mutation. Following a similar procedure, we will engineer new competitive DHFR inhibitors that will target resistance-conferring mutations on the D27, W30, I94, and F153 residues. Similarly, we will identify allosteric inhibitors that can target a druggable DHFR cryptic site we discovered. This site has limited drug accessibility for the wild-type DHFR but always remains open for the D27E, I94L, and F153S variants of DHFR. We already showed that proglumetacin, a commonly used NSAID drug, allosterically slows down DHFR activity. Our third goal is to engineer a novel class of molecules that select against -lactamase genes. We will develop a novel class of molecules that we call BLTs to eliminate -lactamase producing Gram-negative bacteria. BLT molecules are inac...