GPR55

CID16020046 inhibited agonist-induced receptor activation in yeast cells expressing human GPR55. The compound exhibited antagonistic effects on extracellular signal-regulated kinases activation and LPI-mediated Ca2+ release in human embryonic kidney (HEK293) cells that were stable in their expression of human GPR55. CID16020046 reduced the activation of serum response element, nuclear factor κ of activated B cells (NF-κB), nuclear factor of activated T cells (NFAT), and lysophosphatidylinositol (LPI) in a concentration-dependent manner. It also prevented GPR55 internalization and the translocation of NFAT and NF-κB.

CID16020046 decreases proinflammatory cytokines and ameliorates intestinal inflammation in model organisms. In healthy mice, CID16020046 has no effect on locomotor activity or anxiety levels[1].

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Daily application of CID16020046 (20 mg kg−1) significantly reduced inflammation scores and myeloperoxidase (MPO) activity. In the DSS colitis model, levels of TNF-α and IL-1β, and the expression of cyclooxygenase (Cox)-2 and STAT-3 were reduced in colon tissues while in TNBS-induced colitis, levels of Cox-2, IL-1β and IL-6 were significantly lowered. Evaluation of leukocyte recruitment by flow cytometry indicated reduced presence of lymphocytes and macrophages in the colon following GPR55 inhibition in DSS-induced colitis. In J774A.1 mouse macrophages, inhibition of GPR55 revealed reduced migration of macrophages and decreased CD11b expression, suggesting that direct effects of CID16020046 on macrophages may have contributed to the improvement of colitis. GPR55−/− knockout mice showed reduced inflammation scores as compared to wild type mice in the DSS model suggesting a proinflammatory role in intestinal inflammation[1].

Compound CID16020046 ((4-[4-(3-hydroxyphenyl)-3-(4-methylphenyl)-6-oxo-1H,4H,5H,6H-pyrrolo[3,4-c]pyrazol-5-yl] benzoic acid) is a selective GPR55 antagonist. In yeast cells expressing human GPR55, CID16020046 antagonized agonist-induced receptor activation. In human embryonic kidney (HEK293) cells stably expressing human GPR55, the compound behaved as an antagonist on LPI-mediated Ca²⁺ release and extracellular signal-regulated kinases activation, but not in HEK293 cells expressing cannabinoid receptor 1 or 2 (CB₁ or CB₂). CID16020046 concentration dependently inhibited LPI-induced activation of nuclear factor of activated T-cells (NFAT), nuclear factor κ of activated B cells (NF-κB) and serum response element, translocation of NFAT and NF-κB, and GPR55 internalization. It reduced LPI-induced wound healing in primary human lung microvascular endothelial cells and reversed LPI-inhibited platelet aggregation, suggesting a novel role for GPR55 in platelet and endothelial cell function. CID16020046 is therefore a valuable tool to study GPR55-mediated mechanisms in primary cells and tissues[2].

After being reconstituted in 500 μL PBS, 2 × 10⁶ cells were preincubated with 1, 5, and 10 μM CID16020046 or DMSO for 30 minutes. The cells were then stimulated for an additional half-hour at 37°C using 1 nM of monocyte chemotactic protein 1 (MCP-1). Fifteen minutes prior to the incubation period's conclusion, Alexa Fluor 647 anti-mouse CD11b was added. Cells were counted on an FACSCalibur flow cytometer following the addition of the fixative solution. Data were reported as a percentage change to the vehicle treatment, and experiments were run in triplicate.

CID16020046 (or vehicle) was injected subcutaneously (s.c.) 30 min prior to onset of the colitis models at a dose of 20 mg kg−1 and given once daily for 7 days in the DSS or for 3 days in the TNBS model. [1]
Colitis was induced by either 2.5% dextran sulfate sodium (DSS) supplemented in the drinking water of C57BL/6 mice or by a single intrarectal application of trinitrobenzene sulfonic acid (TNBS).[1]

[1]. Neurogastroenterol Motil. 2015 Oct; 27(10): 1432–1445.

[2]. J Pharmacol Exp Ther . 2013 Jul;346(1):54-66.

Background G protein-coupled receptor 55 (GPR55) is a lysophospholipid receptor responsive to certain cannabinoids. The role of GPR55 in inflammatory processes of the gut is largely unknown. Using the recently characterized GPR55 inhibitor CID16020046, we determined the role of GPR55 in experimental intestinal inflammation and explored possible mechanisms of action. Conclusions and inferences Pharmacological blockade of GPR55 reduces experimental intestinal inflammation by reducing leukocyte migration and activation, in particular that of macrophages. Therefore, CID16020046 represents a possible drug for the treatment of bowel inflammation.[1]

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G protein-coupled receptor 55 (GPR55) possesses pro-oncogenic activity and its function can be competitively inhibited with (R,R')-4'-methoxy-1-naphthylfenoterol (MNF) through poorly defined signaling pathways. Here, the anti-tumorigenic effect of MNF was investigated in the human pancreatic cancer cell line, PANC-1, by focusing on the expression of known cancer biomarkers and the expression and function of multidrug resistance (MDR) exporters such as P-glycoprotein (Pgp) and breast cancer resistance protein (BCRP). Incubation of PANC1 cells with MNF (1μM) for 24h significantly decreased EGF receptor, pyruvate kinase M2 (PKM2), and β-catenin protein levels and was accompanied by significant reduction in nuclear accumulation of HIF-1α and the phospho-active forms of PKM2 and β-catenin. Inhibition of GPR55 with either MNF or the GPR55 antagonist CID 16020046 lowered the amount of MDR proteins in total cellular extracts while diminishing the nuclear expression of Pgp and BCRP. There was significant nuclear accumulation of doxorubicin in PANC-1 cells treated with MNF and the pre-incubation with MNF increased the cytotoxicity of doxorubicin and gemcitabine in these cells. Potentiation of doxorubicin cytotoxicity by MNF was also observed in MDA-MB-231 breast cancer cells and U87MG glioblastoma cells, which express high levels of GPR55. The data suggest that inhibition of GPR55 activity produces antitumor effects via attenuation of the MEK/ERK and PI3K-AKT pathways leading to a reduction in the expression and function of MDR proteins. Pharmacol Res. 2016 Sep:111:757-766.

C25H19N3O4

425.436065912247

425.138

C, 70.58; H, 4.50; N, 9.88; O, 15.04

834903-43-4

16020046

White to off-white solid powder

4.603

3

5

4

32

701

0

O=C(C1C=CC(N2C(C3C=C(O)C=CC=3)C3=C(NN=C3C3C=CC(C)=CC=3)C2=O)=CC=1)O

VGUQVYZXABOXCX-UHFFFAOYSA-N

InChI=1S/C25H19N3O4/c1-14-5-7-15(8-6-14)21-20-22(27-26-21)24(30)28(18-11-9-16(10-12-18)25(31)32)23(20)17-3-2-4-19(29)13-17/h2-13,23,29H,1H3,(H,26,27)(H,31,32)

4-[4-(3-hydroxyphenyl)-3-(4-methylphenyl)-6-oxo-1,4-dihydropyrrolo[3,4-c]pyrazol-5-yl]benzoic acid

CID-16020046; CID16020046; 834903-43-4; CID-16020046; CID16020046; 4-[4-(3-hydroxyphenyl)-3-(4-methylphenyl)-6-oxo-1,4-dihydropyrrolo[3,4-d]pyrazol-5-yl]benzoic acid; 5AUY4Y2UPU; MLS000675307; SMR000314029; CID 16020046

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)

DMSO: ~100 mg/mL (~235.1 mM)

Solubility in Formulation 1: 25 mg/mL (58.76 mM) in 20%PEG300 80% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
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: ≥ 2.5 mg/mL (5.88 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (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 25.0 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 3: ≥ 2.5 mg/mL (5.88 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (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 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 4: ≥ 2.5 mg/mL (5.88 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 25.0 mg/mL clear DMSO stock solution to 900 μL corn oil and mix evenly.

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