The World Health Organization has identified antimicrobial resistance as one of the top three threats to human health. In contrast with the fast evolution and spreading of drug-resistant pathogens, the number of effective antimicrobials that are available to healthcare workers is dwindling. The long-term goal of the research is to find solutions to treat bacterial infections caused by drug resistant pathogens. The biogenesis process of the primary multidrug efflux pump in bacterial pathogen will be used as a novel target. In Gram-negative bacteria such as Escherichia coli, the triplex AcrA-AcrB-TolC pump is a major player in both intrinsic and acquired multidrug resistance. Mutant strains of E. coli deficient in the pump exhibit drastically decreased tolerance to many drugs. Therefore, inhibitors that interrupt drug efflux should be very useful in reversing drug- resistance in pathogens. The central hypothesis is that the protein assembly process can be blocked to prevent or disrupt the production of functional MDR pumps. Specifically, the objective of the proposed study is to identify inhibitors to block the oligomerization of AcrB, which exists and functions as a homotrimer. The following Specific Aims will be pursued: 1) To develop an assay to identify compounds that promote AcrB trimer dissociation. 2) To develop an assay to identify compounds that prevent AcrB subunit association. 3) To screen a small collection of compounds to validate and optimize assays. In year one, the work focus will be the development of assays (Aims 1 and 2). In year two, approximately 5000 compounds will be screened to validate the assay and identify hit compounds. For identified active compounds, we will confirm their activity using bacteria culture and measure binding parameters of their interaction with the target protein. Outcomes of the proposed study include the establishment of useful assays and methodologies to interfere with membrane protein oligomerization. Similar methodologies will be directly applicable to AcrB homologues in other Gram- negative pathogens. In addition, the collection of hit compounds that will be obtained will serve as seed structures to generate follow up compound for future screening effort. Upon completion of the project, we will seek additional funding and collaborate with the University of Cincinnati Drug Discovery Center to adapt our assay for high throughput screening using 384-well plates. It would also include a collaborative medicinal chemistry synthetic effort based on the results of this R21 and results from the expanded screening. These extended efforts would also involve development of a biology component to test compound efficacy including mammalian cellular toxicity. Another option is broader screening through a proposal to the NIH Molecular Libraries Program or partnering with an interested pharmaceutical company. We expect the proposed research will lead to new compound classes and validate novel target mechanisms for the development of effective antimicrobial treatments for drug-resistant E. coli and other Gram-negative bacterial pathogens.
The rapid increase in the incidence of drug resistance in pathogenic strains of Gram-negative bacterium is an especially worrisome and critical global public health challenge. The goal of the proposed studies is to identify inhibitors to block the biogenesis of the major multidrug resistant pump AcrAB-TolC. Two assays will be developed to identify compounds that interfere with the trimerization of AcrB, which will disable the efflux pump and drastically increase the potency of antimicrobial treatment.
