Discovery of efflux pump inhibitors using a high-throughout bacterial assay

Project Summary Antibiotic resistance is a global health emergency that threatens advancements in modern medicine. Bacteria employ several mechanisms to evade the action of antibiotics, including efflux, reduced permeability, inactivation of antibiotics, and mutations to target proteins. Therapeutics that block resistance mechanisms would serve as adjuvants to antibiotics in the fight against pathogenic bacteria. One such class of resistance blockers are efflux pump inhibitors (EPIs), which are a promising therapeutic since these membrane transport proteins provide one of the broadest mechanisms of antibiotic resistance. Our team discovered Fab inhibitors of efflux pumps that block drug expulsion by inserting their CDRH3 loops into the substrate binding pocket. This finding and other discoveries of protein EPIs have been facilitated through display technologies, such as phage display and mRNA display. These screens require stable and pure efflux pumps and constitute a rather time-consuming process since they involve sample optimization prior to the screen. Moreover, the identified hits do not always correspond to inhibitors since screens are designed to detect binding and not function. The goal of this proposal is to develop a high-throughput screening approach to discover protein-based EPIs to block antibiotic efflux without the need for sample optimization. In our assay, the efflux pump is co-expressed with a library of Fabs in E. coli. Antibody expression is directed to the periplasm, such that binding occurs from the outside, a requirement to avoid membrane permeability in Gram-positive bacteria. Next, bacteria are treated with a fluorescent dye, which can be transported by the efflux pump and bind to DNA. Hence, E. coli harboring inhibited efflux pumps show greater fluorescence while uninhibited ones display reduced fluorescence. This feature enables bacteria to be sorted based on their fluorescence by using flow cytometry to obtain the identity of the binder. In Aim 1, we will explore several parameters with the goal of establishing the assay into a robust tool to be used for discovering protein EPIs. In Aim 2, we will use the method to screen against efflux pumps from pathogenic bacteria where no such specific inhibitor has been developed. By accomplishing the goals of the project, we will establish a high- throughput method for identifying protein-based EPIs, thereby expediting the discovery process.