Design and application of in vivo FRET biosensors to identify protein prenylation and nanoclustering inhibitors

Protein prenylation is required for membrane anchorage of small GTPases. Correct membrane targeting is essential for their biological activity. Signal output of the prenylated proto-oncogene Ras in addition critically depends on its organization into nanoscale proteolipid assemblies of the plasma membrane, so called nanoclusters. While protein prenylation is an established drug target, only a handful of nanoclustering inhibitors are known, partially due to the lack of appropriate assays to screen for such compounds. Here, we describe three cell-based high-throughput screening amenable Förster resonance energy transfer NANOclustering and Prenylation Sensors (NANOPS) that are specific for Ras, Rho, and Rab proteins. Rab-NANOPS provides the first evidence for nanoclustering of Rab proteins. Using NANOPS in a cell-based chemical screen, we now identify macrotetrolides, known ionophoric antibiotics, as submicromolar disruptors of Ras nanoclustering and MAPK signaling.