Project Summary With numerous Gram-negative bacterial species demonstrating antimicrobial drug resistance, the identification of new inhibitors and the optimization of existing inhibitors is necessary to enable an effective treatment of illnesses. Recent research efforts in our lab and others have demonstrated that aminoglycosides have minimal impact on protein synthesis and in fact they potently bind to heptosytransferase I (HepI) in Escherichia coli. This is an important finding, because it may allow for this class of antibiotics to be dramatically redesigned to be better drugs with fewer side effects, because the HepI and ribosome binding sites have very different sizes and they have dramatic differences in charges (HepI is positively charged, while the ribosome is negatively charged). This proposal will advance efforts to redesign aminoglycoside antibiotics to enhance HepI binding and to reduce binding to other cellular targets that can lead to side effects like oto- and nephrotoxicity. Our investigation will address three hypotheses: (1) that aminoglycosides bind to other cellular targets beyond the ribosome including heptosyltransferase enzymes, (2) understanding the HepI-aminoglycoside interactions will enable structural modification and optimization of bactericidal activity, and (3) that computational methods can enhance aminoglycoside redesign. To date, efforts to redesign aminoglycosides for more potent binding to the ribosome has failed to lead to more potent drugs, and this is likely because the mechanism of action involves other enzymes like HepI. This work promises to enhance drug discovery efforts while also providing training for students in my lab and in two upper-level biochemistry courses at Wesleyan in 21st century drug discovery methods.