GPR55
G protein-coupled receptor 55 (GPR55) antagonist [1]

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.

---

In J774A.1 mouse macrophages, CID16020046 concentration-dependently reduced C5a (5 nM)-induced cell migration. Incubation with 1 and 5 μM CID16020046 significantly decreased migration compared to vehicle control.
In the same J774A.1 cell line, MCP-1 (1 nM)-induced expression of the activation marker CD11b was concentration-dependently decreased by CID16020046 (1, 5, 10 μM), almost back to control levels.
CID16020046 (1 and 2.5 μM) strongly diminished the migration of isolated human neutrophils induced by the chemoattractant fMLP (100 nM).
The concentrations of CID16020046 used in these in vitro assays (1, 5, 10 μM) did not change cell viability.

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].

---

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].

---

In the DSS-induced colitis model in C57BL/6 mice, daily subcutaneous administration of CID16020046 (20 mg/kg) for 7 days significantly reduced macroscopic inflammation scores and myeloperoxidase (MPO) activity in the colon compared to vehicle-treated animals. Colon length shortening was also improved.
In the TNBS-induced colitis model, daily subcutaneous administration of CID16020046 (20 mg/kg) for 3 days also significantly reduced macroscopic inflammation scores and MPO activity.
Histological examination of colon sections from both DSS and TNBS models showed that CID16020046 treatment resulted in more preserved crypt architecture and reduced leukocyte infiltration into the lamina propria compared to vehicle-treated animals.
In the DSS model, CID16020046 treatment led to reduced levels of the pro-inflammatory cytokines TNF-α and IL-1β in colon tissue, while IL-6 levels were unchanged.
In the TNBS model, CID16020046 treatment significantly lowered levels of IL-1β and IL-6 in colon tissue, while TNF-α was also lowered but not significantly.
CID16020046 treatment significantly decreased cyclooxygenase-2 (Cox-2) protein expression in colon tissue from both DSS and TNBS models.
Phosphorylation of STAT3 (pSTAT3) was differentially regulated: it was decreased in the DSS model but increased in the TNBS model after CID16020046 treatment.
Flow cytometric analysis of leukocyte recruitment in the DSS model showed that CID16020046 treatment led to an almost 50% decrease in the number of infiltrated macrophages and lymphocytes in the colonic lamina propria, while the number of monocytes and eosinophils did not change significantly. The number of neutrophils was increased.
Immunohistochemical staining for CD3 (T lymphocytes) and F4/80 (macrophages) in colon sections from the DSS model confirmed reduced immunoreactivity in the submucosa after CID16020046 treatment.
GPR55 knockout mice subjected to DSS-induced colitis exhibited significantly reduced inflammatory scores and MPO activity compared to wild-type littermates, supporting a pro-inflammatory role for GPR55.
In an open field test with healthy C57BL/6 mice, daily subcutaneous administration of CID16020046 (20 mg/kg) for 6 days did not alter locomotor activity (total distance travelled) or anxiety-like behavior (time spent in the center) compared to vehicle-treated animals. Body weight and temperature were also unaffected.

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.

---

Migration Assay (J774A.1 macrophages): J774A.1 mouse macrophages were starved overnight in medium containing 0.5% FBS. Cells were then incubated with CID16020046 (1, 5 μM) or vehicle. A suspension of 3 x 10^5 cells was placed in the upper compartment of a Transwell plate with an 8 μm pore membrane. The chemoattractant C5a (5 nM) was added to the medium in the lower compartment. Cells were allowed to migrate for 2 hours at 37°C in a humidified incubator with 5% CO2. After migration, non-migrated cells on the upper side of the filter were removed. The filter was dried, fixed in formaldehyde, and mounted. Migrated cell nuclei were counted under a fluorescent microscope.
CD11b Expression Assay (J774A.1 macrophages): J774A.1 cells (2 x 10^6) were transferred into PBS and preincubated for 30 minutes with CID16020046 (1, 5, 10 μM) or vehicle. Cells were then stimulated with MCP-1 (1 nM) for another 30 minutes at 37°C. Alexa Fluor 647-conjugated anti-mouse CD11b antibody was added 15 minutes before the end of the incubation. After adding a fixative solution, cells were analyzed on a flow cytometer for CD11b expression.
Migration Assay (Human Neutrophils): Human neutrophils were isolated from healthy donor blood. Cells were resuspended in PBS at a density of 2 x 10^6 cells/mL. The chemoattractant fMLP (100 nM) was placed in the bottom wells of a 48-well micro-Boyden chamber with a 5 μm polycarbonate membrane. The neutrophil suspension, along with different concentrations of CID16020046 (1, 2.5 μM) or vehicle, was placed in the upper wells. The chamber was incubated at 37°C for 1 hour. After incubation, cells remaining in the upper compartment were removed. Cells that migrated to the lower compartment were transferred to tubes, fixed, and analyzed by flow cytometry.

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]

---

DSS-induced Colitis Model: Male C57BL/6N mice (5-6 weeks old) received 2.5% dextran sulfate sodium (DSS) in their drinking water for 5 days to induce colitis. CID16020046 (20 mg/kg) or vehicle (DMSO) was administered subcutaneously once daily, starting 30 minutes before DSS exposure and continuing for a total of 7 days. Mice were euthanized on day 7, and colons were collected for analysis.
TNBS-induced Colitis Model: Male C57BL/6N mice were lightly anesthetized. Trinitrobenzene sulfonic acid (TNBS; 4 mg in 100 μL of 30% ethanol) was administered intrarectally via a gavage needle to induce colitis. CID16020046 (20 mg/kg) or vehicle (DMSO) was administered subcutaneously once daily, starting 30 minutes before TNBS administration and continuing for a total of 3 days. Mice were euthanized on day 3, and colons were collected for analysis.
GPR55 Knockout Mouse Model: GPR55 knockout mice and wild-type littermates were subjected to the DSS-induced colitis protocol as described above. Inflammation was assessed macroscopically and by MPO activity.
Open Field Test (Behavioral Study): Healthy C57BL/6N mice received daily subcutaneous injections of CID16020046 (20 mg/kg) or vehicle (DMSO) for 6 consecutive days. On day 6, 30 minutes after the last injection, each mouse was placed in the center of an open field arena. Their behavior was recorded for 5 minutes using a video tracking system to assess locomotor activity and anxiety-like behavior.

In open field experiments on healthy mice, subcutaneous injection of CID16020046 (20 mg/kg) daily for 6 consecutive days did not induce any disease symptoms, altered motor activity, or anxiety-like behavioral changes. Compared to the vector control group, mouse body weight and body temperature remained unchanged. The concentration of CID16020046 used in in vitro experiments (up to 10 μM) did not affect cell viability.

[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 that responds to certain cannabinoids. The role of GPR55 in the process of intestinal inflammation is unclear. We used the recently identified GPR55 inhibitor CID16020046 to determine the role of GPR55 in experimental intestinal inflammation and to explore its possible mechanism of action.

Conclusions and inferences
Pharmacological blocking of GPR55 can alleviate experimental intestinal inflammation by reducing leukocyte migration and activation, especially macrophage migration and activation. Therefore, CID16020046 is a potential therapeutic agent for intestinal inflammation. [1]

---

G protein-coupled receptor 55 (GPR55) has pro-cancer activity and its function can be competitively inhibited by (R,R')-4'-methoxy-1-naphthol benznotol (MNF) through some signaling pathways that are not yet clear. This study used the human pancreatic cancer cell line PANC-1 as a model to focus on the expression of known cancer biomarkers and the expression and function of multidrug resistance (MDR) efflux proteins (such as P-glycoprotein (Pgp) and breast cancer resistance protein (BCRP)) to explore the antitumor effect of MNF. After incubating PANC1 cells with MNF (1 μM) for 24 hours, the levels of EGF receptor, pyruvate kinase M2 (PKM2), and β-catenin proteins were significantly reduced, while the nuclear accumulation of HIF-1α, as well as phosphorylated active forms of PKM2 and β-catenin, was also significantly reduced. Inhibition of GPR55 using MNF or the GPR55 antagonist CID 16020046 reduced the content of MDR proteins in the total cell extract, while also reducing the nuclear expression of Pgp and BCRP. MNF treatment significantly increased the nuclear accumulation of doxorubicin in PANC-1 cells, and MNF pre-incubation enhanced the cytotoxicity of doxorubicin and gemcitabine to these cells. Enhancing effects of MNF on doxorubicin cytotoxicity were also observed in MDA-MB-231 breast cancer cells and U87MG glioblastoma cells expressing high levels of GPR55. Data suggest that inhibiting GPR55 activity can exert antitumor effects by attenuating the MEK/ERK and PI3K-AKT pathways, thereby reducing the expression and function of MDR proteins. Pharmacol Res. 2016 Sep 111:757-766.

---

CID16020046 is a novel, highly selective G protein-coupled receptor 55 (GPR55) antagonist.
GPR55 is an atypical cannabinoid receptor that is activated by lysophosphatidylinositol (LPI) and regulated by certain cannabinoids.
This study demonstrates that pharmacological inhibition of GPR55 using CID16020046 protects mice from intestinal inflammation in colitis models (DSS and TNBS). This protective effect was associated with reduced recruitment of colonic leukocytes (especially macrophages and lymphocytes), decreased levels of pro-inflammatory cytokines, and reduced Cox-2 expression. GPR55 gene knockout also reduced susceptibility to DSS-induced colitis, confirming the pro-inflammatory role of this receptor in intestinal inflammation. CID16020046 has a direct effect on macrophages, inhibiting the expression of their migration and activation marker (CD11b) in vitro. At the tested dose, this antagonist did not exhibit central nervous system activity or induce disease behavior in healthy mice, indicating good safety in these aspects. CID16020046 is a potential therapeutic agent for inflammatory bowel disease.

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.

---

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.

---

View More

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.

---

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.

---

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