Discovery of a dual Ras and ARF6 inhibitor from a GPCR endocytosis screen

Abstract Internalization and intracellular trafficking of hormone receptors, like receptor tyrosine kinases (RTKs) and G protein-coupled receptors (GPCRs), play pivotal roles in cell responsiveness homeostasis. Dysregulation in receptor trafficking can lead to aberrant signaling and cell behavior, prevalent in cancer. Here, using an endosomal BRET-based assay in a high-throughput screen with the prototypical GPCR angiotensin II type 1 receptor (AT1R), we sought to identify inhibitors of receptor trafficking from a library of ~115,000 small molecules. We identified a novel dual Ras and ARF6 inhibitor that blocks agonist-mediated internalization of AT1R and other GPCRs, which we named Rasarfin. Rasarfin also potently inhibited agonist-induced ERK1/2 signaling by GPCRs, and MAPK and Akt signaling by EGFR, as well as prevented cancer cell proliferation. In silico modeling and in vitro studies revealed a unique binding modality of Rasarfin within the SOS-binding domain of Ras. Our findings unveil a new class of dual small G protein inhibitors for receptor trafficking and signaling, useful for the inhibition of oncogenic cellular responses.