RESUMO
Internalization and intracellular trafficking of G protein-coupled receptors (GPCRs) play pivotal roles in cell responsiveness. Dysregulation in receptor trafficking can lead to aberrant signaling and cell behavior. 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 receptor trafficking inhibitors from a library of ~115,000 small molecules. We identified a novel dual Ras and ARF6 inhibitor, which we named Rasarfin, that blocks agonist-mediated internalization of AT1R and other GPCRs. Rasarfin also potently inhibits agonist-induced ERK1/2 signaling by GPCRs, and MAPK and Akt signaling by EGFR, as well as prevents cancer cell proliferation. In silico modeling and in vitro studies reveal a unique binding modality of Rasarfin within the SOS-binding domain of Ras. Our findings unveil a class of dual small G protein inhibitors for receptor trafficking and signaling, useful for the inhibition of oncogenic cellular responses.
Assuntos
Fatores de Ribosilação do ADP/antagonistas & inibidores , Endocitose/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Receptores Acoplados a Proteínas G/metabolismo , Proteínas ras/antagonistas & inibidores , Fator 6 de Ribosilação do ADP , Fatores de Ribosilação do ADP/metabolismo , Sítios de Ligação , Técnicas de Transferência de Energia por Ressonância de Bioluminescência , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Descoberta de Drogas , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Células HEK293 , Humanos , Simulação de Dinâmica Molecular , Receptores Proteína Tirosina Quinases/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteínas ras/química , Proteínas ras/metabolismoRESUMO
In addition to G protein-coupled receptor (GPCR) desensitization and endocytosis, ß-arrestin recruitment to ligand-stimulated GPCRs promotes non-canonical signalling cascades. Distinguishing the respective contributions of ß-arrestin recruitment to the receptor and ß-arrestin-promoted endocytosis in propagating receptor signalling has been limited by the lack of selective analytical tools. Here, using a combination of virtual screening and cell-based assays, we have identified a small molecule that selectively inhibits the interaction between ß-arrestin and the ß2-adaptin subunit of the clathrin adaptor protein AP2 without interfering with the formation of receptor/ß-arrestin complexes. This selective ß-arrestin/ß2-adaptin inhibitor (Barbadin) blocks agonist-promoted endocytosis of the prototypical ß2-adrenergic (ß2AR), V2-vasopressin (V2R) and angiotensin-II type-1 (AT1R) receptors, but does not affect ß-arrestin-independent (transferrin) or AP2-independent (endothelin-A) receptor internalization. Interestingly, Barbadin fully blocks V2R-stimulated ERK1/2 activation and blunts cAMP accumulation promoted by both V2R and ß2AR, supporting the concept of ß-arrestin/AP2-dependent signalling for both G protein-dependent and -independent pathways.