Developing a PROTAC to Degrade the Constitutively Active Onco-GPCR in Uveal Melanoma

Abstract ID 129435 Poster Board 254 G protein-coupled receptors (GPCRs) comprise a super-family of transmembrane signal transducers. GPCRs are common drug targets, but only one-third of non-olfactory GPCRs have been "drugged" to date and many GPCRs are considered "undruggable". Aberrant GPCR signaling is implicated in the pathophysiology of many cancers, but there are only a few anti-cancer drugs available that target GPCRs. New strategies are required to develop novel therapeutic agents for cancer-associated GPCRs. Proteolysis-targeting chimeras (PROTACs) exploit the ubiquitin-proteasome protein degradation system to target previously undruggable targets. PROTACs are bifunctional molecules with a ligand for the target protein linked to a warhead ligand that binds an E3 ubiquitin ligase. PROTACs bring the target protein into proximity of the E3 ligase, which enables the transfer of ubiquitin from an E3 ubiquitin-conjugating enzyme to the target protein, which can then be degraded at the proteasome. Our hypothesis is that PROTAC-dependent ubiquitination of GPCRs can trigger their proteasomal and lysosomal degradation. To test the hypothesis we designed a fusion receptor expression construct in which HiBiT and HaloTags are added to the cytoplasmic tail of CysLTR2 – a GPCR implicated as a driver oncoprotein in uveal melanoma. The HaloTag provides a binding site for the HaloPROTAC3 ligand, which recruits the von Hippel-Lindau (VHL) tumor suppressor – an E3 ligase often used for PROTACs. The HiBiT tag allows quantification of HaloPROTAC3-dependent degradation through a nanoluciferase protein complementation assay. We will use the CysLTR2 fusion constructs for a proof-of-concept study to evaluate the potential feasibility of using PROTACs to target GPCRs.