High throughput screening at novel striatum orphan GPCRs identified by human transcriptomic profiling
Authors: Felsing Daniel Eric , Raval Sweta , Jain Manish , Xi Simon , Allen John A 
Published: June 2021

The striatum performs essential brain functions including movement control, regulation of attention and is a critical component of reward systems. Current druggable targets have displayed limited efficacy in treating striatal diseases including addiction, schizophrenia and Parkinson's disease. G protein‐coupled receptors (GPCRs) are essential drug targets with ~35% of FDA approved medicines targeting these receptors. Of the 350 non‐sensory GPCRs, nearly 120 remain orphan GPCRs, whose endogenous ligands and G protein signaling are unknown. A previous challenge with orphan receptors has been the design of reliable drug screens, but a recent advance in the PRESTO‐Tango assay now allows for a measurement of receptor activation for over 300 GPCRs. Here we pursue the objective of identifying new druggable GPCRs, selectively expressed in the human striatum, using human tissue transcriptomics and the PRESTO‐Tango platform. We conducted a comprehensive genome‐wide survey of human tissue‐selective gene expression using 1640 high‐quality RNA‐seq samples from the Genotype Tissue Expression (GTEx) project. To identify receptor function and screen ligands, human clones of receptors were expressed in HEK293 cells and signaling was assessed using the Glo‐sensor cAMP and Tango beta‐arrestin assay. As a ligand discovery proof‐of‐concept, the Tango assay was used to screen the LOPAC library against five striatal orphan GPCRs. Human gene expression analyses identified expression of 18 striatal‐selective GPCRs. 11 of these receptors have known ligands including established therapeutic drug targets (e.g. dopamine D1, D2 receptors). The remaining 7 human striatal GPCRs, namely, GPR6, 52, 55, 88, 101, 139 and 149 were identified as class A orphan receptors. Biochemical and CRISPR/Cas9 knockout studies confirmed GPR6, GPR52 and GPR101 increased cAMP via Gs/olf G proteins, while GPR88 decreased cAMP levels via Gi/o G proteins. Screening the LOPAC library in parallel against five orphans (6, 52, 88, 101, 149) using the Tango assay revealed 60 compounds that selectively induced a 2‐fold activity above or below vehicle treated cells for one of the five receptors. Dose response assays confirmed two compounds with agonist activity for GPR88 (clozapine and carbachol) and one with agonist activity for GPR149 (PDTC). Interestingly, carbachol and clozapine are FDA approved drugs. Clozapine is a classic atypical antipsychotic medication and inverse agonist at serotonin and DA receptors; and carbachol is a cholinergic agonist currently used for the treatment of glaucoma. PDTC agonist activity for GPR149 represents the first described agonist compound for this orphan receptor. This research provides a template for identifying brain region specific targets using human tissue RNAseq transcriptomic datasets. Our findings also suggest the seven identified human orphan GPCRs are potential drug targets amenable to screening, whose pharmacological modulation may be therapeutic for treating striatum‐related neurological diseases.

Support or Funding Information

Rising Star Award, UT System (JAA); UTMB institutional funding (JAA); T32 DA07287 (DEF)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.