PRESTO-Tango as an open-source resource for interrogation of the druggable human GPCRome

Abstract

G protein-coupled receptors (GPCRs) are essential mediators of cellular signaling and are important targets of drug action. Of the approximately 350 nonolfactory human GPCRs, more than 100 are still considered to be 'orphans' because their endogenous ligands remain unknown. Here, we describe a unique open-source resource that allows interrogation of the druggable human GPCRome via a G protein-independent β-arrestin-recruitment assay. We validate this unique platform at more than 120 nonorphan human GPCR targets, demonstrate its utility for discovering new ligands for orphan human GPCRs and describe a method (parallel receptorome expression and screening via transcriptional output, with transcriptional activation following arrestin translocation (PRESTO-Tango)) for the simultaneous and parallel interrogation of the entire human nonolfactory GPCRome.

Figure 1. Design, principle, and validation of selected TANGO assays

(a) Modular design of TANGO constructs (top); blue arrowheads indicate Cla I sites, and green arrowheads indicate Age I sites. General scheme for the β-arrestin (TANGO) recruitment assay (bottom). Upon activation of the GPCR by an agonist (1), β-arrestin is recruited to the C-terminus of the receptor (2). This is followed by cleavage of the GPCR fusion protein at the TEV protease site (3). Cleavage results in the release of the tTA transcription factor (4), which, after transport to the nucleus, activates transcription of the luciferase reporter gene (5). (b) Surface expression of two selected TANGO constructs as shown by immunofluorescence using an anti-FLAG antibody. Concentration-response curves of a prototypical non-orphan GPCR, the neuromedin B receptor stimulated by neuromedin B (NMB) in the TANGO assay (c) and in a calcium-release assay (d); data are shown as the mean ± SEM of typical experiments done in quadruplicate. Curves were fitted using Graphpad Prism 5.0.

Figure 2. First-time demonstration of arrestin mobilization using the TANGO assay

Shown are concentration-response curves for the response of (a) the CMRM5 muscarinic acetylcholine receptor to carbachol (EC50 = 133.8 nM, n = 4), (b) the CX3CR1 chemokine receptor to its ligand CX3CL1 (EC50 = 0.34 nM, n = 4), (c) the DRD4 dopamine receptor to lisuride (EC50 = 2.0 nM, n = 3), (d) the GAL3 galanin receptor to galanin (EC50 = 0.13 nM, n = 4), (e) the NMUR1 neuromedin receptor to neuromedin S (EC50 = 10.2 nM, n = 4), and (f) the NMUR2 neuromedin receptor to neuromedin S (EC50 = 15.4 nM, n = 4).

Figure 3. Constitutive (or basal) activity for TANGO-ized constructs

Data are expressed in RLU (relative luminescence units) for the orphan and peptide GPCRs (a) and the non-orphan (b) GPCRs (n = 4 for each target.

Figure 4. Compound profiling using the PRESTO-TANGO method at the human GPCR-ome

(a) General format in which compounds are screened against a large number of GPCRs in quadruplicate in 384-well plates; for clarity only the first row of individual GPCRs is shown. (b–d) Concentration-response curves of LSD at various targets; data are shown as mean ± SEM, and curves were fitted with Graphpad Prism. (b) DRD4-dopamine, (c) DRD5-dopamine, (d) ADRA2B-adrenergic.

Figure 5. Novel ligand-target interactions detected by parallel GPCR-ome screening

Results of screening of the 446-compound NCC-1 library at 91 GPCR targets in the TANGO assay, and follow-up studies are shown. (a) Heatmap of the entire matrix (red = stimulation of luminescence over background). (b) Closeup view of a section of the heatmap, showing the activity of nateglinide at MRGPRX4. (c) Concentration-response curve of nateglinide at MRGPRX4 in the TANGO assay (n = 4). (d) Concentration-response curve of nateglinide at MRGPRX4 by PI hydrolysis (n = 3). (e) Concentration-response curve of saquinavir at the BB3 bombesin receptor in the TANGO assay (n = 4). (f) Concentration-response curve of saquinavir at the BB3 bombesin receptor by PI hydrolysis (n = 3).

Fig. 6. TANGO-izing the druggable GPCR-ome

Tree-based phylogram of the non-olfactory GPCR-ome showing the status of the TANGO assay for each GPCR. Cyan circles represent those non-orphan GPCRs for which assays were validated; yellow circles represent those assays for which expression of orphan GPCRs were validated; blue circles represent those non-orphan GPCRs for which optimized assays are not yet available, and black circles represent those GPCRs for which no TANGO-ized construct is available.