Abstract B53: Utilizing bimolecular fluorescent complementation to identify inhibitors of RAL and KRAS

We have identified RAL activity to be vital for NSCLC survival independent of KRAS or EGFR mutation status in a large panel of adenocarcinomas and have developed a novel high throughput screening assay based on Bimolecular Fluorescent Complementation (BiFC) in order to identify small molecules that impair RAL function. By coupling RALA and RALB to the C-terminus of the Venus protein (VC) and coexpressing either with direct binding effector RALBP1 fused to the N-terminus of the Venus protein (VN) results in a fluorescent signal when RAL-RALBP1 binding occurs in cells. This allows for the interrogation of small molecules to disrupt RAL-RALBP1 binding interactions (resulting in loss of fluorescence) in a real time, high throughput manner. Our preliminary screen with a natural compound library identified 14 small molecules that demonstrated high activity in disrupting both RALA- and RALB-RALBP1 binding and fluorescence. We are currently investigating these compounds9 mode of action and have recently expanded our assay to screen for molecules that impair mutant KRAS activity (VC-KRAS / RAF1-VN). BiFC provides a rapid, more physiologic method to identify molecules that can disrupt protein-protein interactions of targets considered ‘undruggable’ including membrane bound small GTPases such as RAL and KRAS. Citation Format: Marytheresa Ifediba, David Burnett, Nicholas Hovda, William Burnett, Matthew VanBrocklin. Utilizing bimolecular fluorescent complementation to identify inhibitors of RAL and KRAS. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr B53. doi: 10.1158/1557-3125.RASONC14-B53