Abstract B53: Utilizing bimolecular fluorescent complementation to identify inhibitors of RAL and KRAS
2014
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
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