Three Fluorophore Assay for Antibiotic Inhibitors of Ribosome Assembly

DESCRIPTION (provided by applicant): Each year millions of people die from infectious diseases worldwide. The rapid rise in antibiotic resistance poses a threat to human health, and there is a critical need for the development of novel anti-infectives. RiboNovix is developing several new technologies applicable to antibiotics, with a focus on the ribosome. This Phase I proposal outlines a plan to optimize and implement a novel three-color fluorescence assay that will allow the identification of small molecules that bind to RNA. Most high throughput assays used in drug screening have been developed based on protein targets. This new assay will allow the identification of inhibitors of protein-RNA interactions, and will represent a significant advance. Dr. James Williamson (Scripps Research Institute) has completed preliminary work on a three-fluorophore fluorescence resonance energy transfer assay (3F-FRET) that is based on the binding of the ribosomal protein S15 to a three helix junction in the small subunit 16S ribosomal RNA, and induces a conformational change in the RNA binding site. It is possible to monitor this conformational change using fluorescence resonance energy transfer (FRET) between Donor and Acceptor Dyes attached to the RNA. Introduction of a third chromophore attached to the S15 protein allows independent monitoring of the protein binding and the conformational change. RiboNovix' major goals of the Phase I proposal are to improve the fluorescence properties of the assay, to scale up preparation of the necessary reagents, and to adapt the assay for high throughput screening. Under Phase II of this program we have two primary aims. First, we plan to use the 3F-FRET assay to screen libraries to find compounds that inhibit the binding of S15 to rRNA and thus inhibit ribosomal subunit formation. RiboNovix will develop these into clinical candidates. Second, we will extend the use of the 3F-FRET assay to other rRNA target sites with known ribosomal protein interactions, including those in the SOS subunit that interact with the five primary rRNA binding proteins. As these additional assays are developed, compounds will be screened, leading to the potential for a new class of antibiotics to be discovered that interfere with protein synthesis.