PROJECT SUMMARY Aminoglycosides are one of the cheapest and well-known antibiotics in clinical use for over 70 years, but one of the major limitations in their use is their ototoxicity. We are developing fast and low-cost methods to develop aminoglycosides with anti-ribosomal activities and reduced toxicity. In this project, we will identify novel aminoglycoside antibacterials, that show reduced ototoxicity. Complexes between ribosomal components will be exploited as targets for small molecule drug libraries that- inactivate the ribosome, stopping bacterial protein synthesis and causing bacterial death while reducing toxicity. This work addresses an important health issue, antibiotic ototoxicity, and presents creative steps towards a novel solution to this problem. Cases of multidrug-resistant (MDR, resistance to 2-3 classes), extensive drug resistance (XDR, resistance to most classes except colistin or tigecycline) and even pan drug resistance (PDR, resistance to all classes) nosocomial bacterial infections have skyrocketed in recent years, and the emergence of pan drug- resistant isolates are making these infections increasingly difficult to treat. Hospital-acquired infections like these account for up to 4% of all hospital stays in the United States and are incredibly diverse in causative pathogen, antibiotic resistance profile, and severity. A significant cause of nosocomial infection is the Enterobacteriaceae family, which includes Gram-negative bacilli that can be commensal or pathogenic. Enterobacteriaceae have a widespread clinical and economic impact due to the diversity of infections they cause; this family causes many infections such as pneumonia, bloodstream infections (BSIs), urinary tract infections (UTIs), and intra-abdominal infections (IAIs). The World Health Organization (WHO) lists carbapenem-resistant Enterobacteriaceae (CRE) as having a critical need for novel antibiotics on their Priority Pathogens list. Because the mortality of these multi drug-resistant infections is between 30 and 50% and there is such difficulty in finding viable treatments, the need for novel therapeutics for these pathogens must be addressed. Unless innovative strategies are developed to produce robust and effective new classes of antibiotics, health care costs will continue to climb and we will completely lose our ability to combat even the most common infection. Influenza and coronavirus (SARS and COVID-19) create an even more urgent need for targeting resistant bacteria related to lung infections, such as carbapenem-resistant Enterobacteriaceae (CRE), a common example of CRE being Klebsiella Pneumoniae (KP). A recent article by J. Gerberding, former CDC director states, “The patients at greatest risk from superbugs like CRE, CR-A. buamanii and CR-P. aeruginosa and other bacterial pathogens that can cause lung diseases, are the ones who are already more vulnerable to illness from viral lung infections like influenza, severe acute respiratory syndr...