Structural, functional, and microbiological exploration toward synergistic dual aminoglycoside combinations

PROJECT SUMMARY/ABSTRACT Aminoglycosides are a critically important class of antibiotics as designated by the World Health Organization, in part due to their activity against multidrug-resistant (MDR) Gram-negative bacteria. However, aminoglycoside use is currently impeded by the potential for resistance and dose-limiting toxicities to emerge during treatment. Aminoglycosides cause miscoding of the mRNA, leading to translation errors that contribute to their bactericidal effect. Although a majority of the antibiotics in this class bind to helix 44 (h44) of the ribosomal 30S subunit to cause miscoding, streptomycin can bind to an adjacent non-overlapping site on the ribosome. In preliminary studies, we found that combinations between aminoglycosides that bind separate sites on the ribosome ('dual- aminoglycoside combinations') were synergistic and bactericidal against MDR Enterobacter cloacae, Escherichia coli, and Klebsiella pneumoniae isolates. Dual-aminoglycoside combinations synergistically enhanced bacterial killing, suppressed resistance emergence, and caused higher rates of miscoding than individual aminoglycosides. These synergistic combinations may also retain their bactericidal activity at lower concentrations than monotherapies, which could enable the use of smaller doses that limit toxicity. Although other synergistic antibiotic combinations simultaneously bind to the ribosome (e.g., quinupristin and dalfopristin), there are no studies to evaluate activity of multiple aminoglycosides and the mechanism of their synergy is completely unknown. This structural, functional, and microbiological project will be the first to examine dual- aminoglycoside combinations against MDR Gram-negative bacteria, solve the structure of multiple aminoglycosides simultaneously bound to the bacterial ribosome, and provide critical insights into their mechanism of synergy. Our central hypothesis is that specific combinations of two aminoglycosides are synergistically bactericidal due to their ability to bind simultaneously to the bacterial ribosome and increase miscoding. To test this hypothesis, we will pursue the following specific aims. In Aim 1, we will identify dual- aminoglycoside combinations that are synergistic and maximally suppress resistance. An array of structurally unique aminoglycosides will be tested against a genetically diverse panel of MDR isolates to detect the most active combinations. Cytotoxicity of synergistic combinations will also be evaluated. In Aim 2, we will define the mechanism of synergy for dual-aminoglycoside combinations. We will determine X-ray crystal structures of the ribosome in complex with different aminoglycoside pairs, quantify miscoding in the presence of synergistic combinations, and define the importance of simultaneous binding for synergy. Structural insights will aid the development of next-generation aminoglycosides with unique binding sites. This project will deliver novel dual- aminoglycoside combinations and an understanding of their mechanism(s) of synergy, which will establish the foundation for future drug development and clinical explorations.