CID 1375606 is a surrogate agonist specific to GPR27. One characteristic of GPR27 is that it is expressed mostly in the central nervous system and is highly conserved among vertebrates. Furthermore, it has been discovered lately to have a connection to insulin secretion [1].

CID 1375606 is a surrogate agonist specific to GPR27. One characteristic of GPR27 is that it is expressed mostly in the central nervous system and is highly conserved among vertebrates. Furthermore, it has been discovered lately to have a connection to insulin secretion [1].

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CID1375606 concentration-dependently activated the chimeric receptor GPR27V2 and recruited β-arrestin 2 in a firefly luciferase complementation assay, with a pEC50 of 6.34 ± 0.13. It showed no activity on the unrelated receptor GPR151 used as a mock control[1]
CID1375606 selectively activated GPR27V2 over the closely related family members GPR85V2 and GPR173V2 in the firefly luciferase complementation assay[1]
CID1375606 induced β-arrestin 2 recruitment to GPR27V2 in an orthogonal NanoLuc Binary Technology (NanoBiT®) assay, with an EC50 of 912 nM. The signal was competed away by co-transfection of untagged β-arrestin 2[1]
CID1375606 induced translocation of β-arrestin 2-GFP from the cytoplasm to the membrane/endosomes in HEK293 cells transiently expressing GPR27V2, as observed by confocal microscopy. No translocation was observed in cells expressing GPR85V2[1]
CID1375606 recruited β-arrestin 2 to the wild-type GPR27 (unmodified C-terminus) in the firefly luciferase complementation assay, with a pEC50 of 5.10 ± 0.16, but with lower efficacy compared to the GPR27V2 chimera. It did not recruit β-arrestin 1[1]
CID1375606 concentration-dependently increased β-arrestin 2 recruitment to wild-type GPR27 in a PathHunter® β-galactosidase complementation assay using CHO-K1 cells[1]
CID1375606 (at 10 µM) did not induce intracellular calcium mobilization in HEK293 cells expressing GPR27, even in the presence of various chimeric/promiscuous Gα subunits (Gαq/s, Gαq/i1, Gαq/i3, Gαq/o, Gαq/z, Gαq/12, Gαq/13, Gα16)[1]
CID1375606 (at 10 µM) did not modulate cAMP levels in HEK293 cells stably expressing GPR27 and the cAMP GloSensor™[1]
CID1375606 (at 10 µM) did not induce TGFα shedding in HEK293 cells expressing GPR27 and an alkaline phosphatase (AP)-TGFα reporter, either in the absence or presence of various chimeric/promiscuous Gα subunits[1]
CID1375606 (at 10 µM) did not induce phosphorylation of ERK1/2 above background levels in HEK293 cells transiently transfected with GPR27[1]

For the primary screening firefly luciferase complementation assay, HEK293 cells stably expressing β-arrestin 2 fused to the N-terminal part of firefly luciferase (FnLArrb2) and the chimeric receptor GPR27V2 fused to the C-terminal part (LFc) were seeded in white 96-well plates. After overnight incubation, cells were stimulated for 10 minutes at room temperature by adding compound stock solutions in DMSO to a final concentration of approximately 15.5 µM. Luminescence was read after the addition of D-luciferin[1]
For concentration-response curves in the firefly luciferase complementation assay, cells were resuspended in HBSS, seeded, and stimulated with ligands at indicated concentrations before luminescence reading[1]
For the NanoBiT® assay, HEK293 cells were co-transfected with plasmids encoding GPR27V2-SmBiT and LgBiT-β-arrestin 2. Twenty-four hours post-transfection, cells were detached, incubated in HBSS with substrate, seeded in a white plate, and stimulated by adding ligand solutions. Luminescence was recorded for 40 minutes[1]
For the PathHunter® β-arrestin assay, CHO-K1 cells stably expressing β-arrestin 2-enzyme acceptor and GPR27-ProLink™ donor were seeded 48 hours before the experiment. Cells were stimulated by adding compound solutions, incubated for 90 minutes at 37°C, followed by addition of detection reagent and chemiluminescence reading[1]
For the β-arrestin 2-GFP translocation assay, HEK293 cells were co-transfected with GPR27V2 and β-arrestin 2-GFP plasmids. Cells were seeded into compartmented dishes, and before imaging, medium was replaced with HBSS. Cells were stimulated with compound (50 µM) and imaged over time on a confocal microscope at room temperature[1]
For G protein pathway assays (calcium, cAMP, TGFα shedding), HEK293 cells were transiently transfected with GPR27 (and relevant chimeric Gα subunits or reporters where applicable). After appropriate incubation (24-48 hours), cells were stimulated with CID1375606 (10 µM) under conditions specific for each assay (e.g., in assay buffer with coelenterazine h for calcium, in GloSensor™ buffer for cAMP, in HBSS for TGFα shedding). Responses were measured via luminescence, fluorescence, or absorbance[1]
For ERK1/2 phosphorylation assays, HEK293 cells transiently transfected with GPR27 were serum-starved overnight, stimulated with CID1375606 (10 µM) for 10 minutes at 37°C, and immediately lysed. Lysates were analyzed by SDS-PAGE and immunoblotting using phospho-ERK1/2 and total ERK1/2 antibodies[1]

[1]. Activation of the Orphan G Protein-Coupled Receptor GPR27 by Surrogate Ligands Promotes β-Arrestin 2 Recruitment. Mol Pharmacol. 2017 Jun;91(6):595-608.

CID1375606 is a synthetic small molecule identified as an alternative agonist of the orphan receptor GPR27[1]
It shares the N-phenyl-2,4-dichlorobenzamide skeleton with another lead compound (ChemBridge ID 5217941)[1]
It is the first reported selective agonist of GPR27 that is inactive against closely related SREB family members GPR85 and GPR173[1]
CID1375606 activates GPR27 by promoting the recruitment of β-arrestin 2, but does not activate the classical G protein-mediated pathways (Gs, Gi, Gq) in the tested experiments, suggesting that it may be a biased agonist or that GPR27 itself may be primarily signaled via β-arrestin[1]
This compound can serve as a pharmacological tool for studying the function and signal transduction of GPR27. Orphan receptor GPR27[1]

C20H14CL2N2O2

385.24

384.043

313493-80-0

1375606

White to off-white solid powder

5.3

2

2

4

26

488

0

C1C=CC(NC(C2C=CC(NC(=O)C3C=CC(=CC=3Cl)Cl)=CC=2)=O)=CC=1

PFHDXBXPRBQVAV-UHFFFAOYSA-N

InChI=1S/C20H14Cl2N2O2/c21-14-8-11-17(18(22)12-14)20(26)24-16-9-6-13(7-10-16)19(25)23-15-4-2-1-3-5-15/h1-12H,(H,23,25)(H,24,26)

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: 2.67 mg/mL (6.93 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 26.7 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.67 mg/mL (6.93 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 26.7 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)