The proposed research supplements the HEAL grant Discovery and validation of a novel orphan GPCR as a target for therapeutic intervention in neuropathic pain (R01NS113257). Neuropathic pain conditions are exceedingly difficult to treat1-4 and novel non-narcotic analgesics are desperately needed. Our recent work led to the discovery that activation of the orphan G protein-coupled receptor 160 (oGPCR; GPR160) in the spinal cord contributes to the development of neuropathic pain states5. Since there are no small molecule antagonists of GPR160, contribution of GPR160 signaling was unraveled using genetic and immunopharmacological approaches. Blocking GPR160 blocks and reverses neuropathic pain with no effect in acute pain settings, suggesting that GPR160 is important in the transition from acute to chronic pain. We also de-orphanized GPR160 and identified cocaine- and amphetamine-regulated transcript peptide (CARTp) as a ligand.5 Blocking endogenous CARTp signaling in the spinal cord attenuates neuropathic pain, whereas intrathecal injection of CARTp evokes painful hypersensitivity in rodents through GPR160-dependent extracellular signal-regulated kinase (ERK) and cyclic AMP response element-binding pathways (CREB). Our findings are the first to de-orphanize GPR160, identify it as a determinant of neuropathic pain and potential therapeutic target with non-opioid based small molecule GPR160 antagonists, and provide insights to its signaling pathways.5 GPR160 has never been isolated and biochemically characterized and studies to understand the direct interaction of CARTp with the purified protein have never been done.5 Here, we propose to isolate and biochemically characterize GPR160, currently identified as one of the Understudied Druggable Genome targets, and to establish methods for biochemical characterization of GPR160 interaction with CARTp activator. We will miniaturize and optimize biochemical assay and scale up protein production for future high throughput biochemical screening to identify potent inhibitors of GPR160 activation. Specifically, we will develop GPR160 expression system(s) and purification protocol(s) and will characterize/optimize stability and solubility of the receptor for biochemical studies using a nanodisc approach. We will characterize direct interaction of GPR160 and CARTp using purified proteins and complementary binding assays such as fluorescence polarization (FP) with fluorescein-labeled (FAM-) CARTp and surface plasmon resonance (SPR) using unlabeled CARTp. These studies are critical for defining the molecular mechanism of CARTp/GPR160 interactions and initiating large-scale screens for new inhibitors to develop novel therapeutics. Impact: Discovery and development of small molecule GPR160 antagonists as non-addictive analgesics is anticipated to have a huge impact on the treatment of chronic pain patients.
Chronic neuropathic pain affects over 15-20 million people in the US. We discovered that GPR160 is involved in the development and maintenance of chronic neuropathic pain and have de-orphanized this receptor by identifying CARTp as its ligand. Results will bring novel insight into the molecular and biochemical interactions of GPR160/CARTp that are essential for our understanding of this mechanism in neuropathic pain states and for developing innovative non-addictive therapeutic interventions addressing a huge unmet medical need.
