ERK2/1; PKCβII

With IC50 values of 0.70 μM and 0.29 μM, respectively, ML192 and 1 μM ML186 both inhibit β-arrestin transport when triggered by 10 μM L-α lysophospholipid phosphoinositol (LPI) or 1 μM ML186 [1]. In GPRSS-expressing U2OS cells, ML192 strongly suppresses ERK1/ML192 (0, 10, 30 and 100 µM), while in wild-type GPR55 intercept cells, ML192 disrupts PKCβII translocation [1]. [1] two phosphorylation.

Chemical Library Screening[1]
A β-arrestin (see methods below), high-throughput, high-content screen (HCS) of 300,000 compounds was used here to identify potent GPR55 selective antagonists. This work was performed in collaboration with the Molecular Libraries Probe Production Centers Network program. For more details about this library of compounds, see http://mli.nih.gov/mli/compound-repository/mlsmr-compounds/. Compounds were screened for antagonism (PubChem AID 2026) at GPR55 (using LPI as the agonist), as well as for both agonism and antagonism at GPR35 (PubChem AIDs 2809, 2815), CB1 (PubChem AIDs 2814, 2835) and CB2 (PubChem AIDs 2822, 2836). A cell line permanently expressing a β-arrestin GFP biosensor and an enhanced receptor of interest (i.e., GPR55, GPR35, CB1 or CB2) were employed in the high content imaging assay. Assay protocol descriptions (according to AID number) are accessible at the PubChem website (http://pubchem.ncbi.nlm.nih.gov/). Potent GPR55 antagonist compounds that lacked agonism or antagonism at GPR35, CB1 or CB2, were further evaluated for inhibition of pERK activation and PKCβII translocation produced by the GPR55 agonists, LPI or ML186 (see methods below). A set of novel GPR55 antagonist molecular scaffolds were selected from the screen ML191 (CID23612552), ML192 (CID1434953) and M193(CID1261822), and the binding of each compound was explored using a computer model of the GPR55 inactive state.

Cell-based assays[1]
Compounds LPI, ML191, ML192, ML193 and ML186 (MolPort) were dissolved in DMSO to 10mM. 10mM stock solutions were dissolved in Hanks’ balanced salt solution (HBSS) to working concentrations.

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β-Arrestin Translocation[1]
U2OS cells permanently expressing HA-GPR55E and βarr2-GFP have been previously described. Cells were seeded onto glass coverslips at 80–85% confluence and placed in 24-well plates (BD Falcon ™). Cells were maintained at 37°C in 5% CO2 overnight. Cells were washed briefly with HBSS before drug application. Experiments were performed using HBSS as the assay buffer. Agonist-stimulated redistribution of βarr2-GFP was assessed following 40 min drug treatment at room temperature (RT). Cells were then fixed with 4% paraformaldehyde for 25 min at room temperature followed by three washes with PBS and one wash with double distilled water. The antagonism protocol included 15 min of pre-incubation with the antagonist, followed by a 40 min co-application with the agonist.

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PKCβII TranslocationAssay of GPR55 Activation [1]
HEK 293 cells plated in 35-mm glass well Matek plastic dishes were transiently transfected with 175 μl of solution containing 1.5 μg/ml PKCβII-GFP cDNA or the PKC plasmid and 5 μg/ml human GPR55 cDNA in pCMV-Sport6 using a standard calcium phosphate protocol. Cells expressing GPR55 and PKCβII-GFP were utilized 24 h after transfection. Cells were washed with warm MEM and maintained at 37°C in 5% CO2 for 30–45 min after drug application. Inhibition of agonist -stimulated redistribution of PKCβII-GFP was assessed after drug treatment at room temperature.

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Extracellular signal-regulated kinase 1/2 assays [1]
Extracellular signal-regulated kinase 1/2 phosphorylation was measured by immunoblotting, as previously described. GPR55E-expressing U2OS cells were grown to sub-confluence in 60-mm plates and serum-starved overnight before assay. Cells were rinsed once with HBSS and antagonists compounds were applied for 30 min prior to agonist application (LPI 10 μM, 10 min). Following drug treatment the cells were disrupted in a lysis buffer (50 mM Hepes, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 10% glycerol, 1% Triton X-100, 10 μM MgCl2, 20mM p-nitrophenyl phosphate, 1 mM Na3VO4, 25mM NaF, and a protease inhibitor mixture (1:25, pH 7.5)). Lysates were immediately placed on ice for 10 min and then centrifuged at 16,000 × g for 30 min at 4°C. Supernatants, corresponding to the cytosolic fraction, were collected, and protein concentrations were determined by the Bradford assay using bovine serum albumin as a standard. Cytosolic fractions (20 μg) were separated on a 10% gel by SDS-PAGE followed by immunoblotting. Antibodies against doubly phosphorylated ERK1/2 (1:5000) were detected using a LI-COR Odyssey IR Imager. A polyclonal antibody against total ERK1/2 (1:1000) was used to confirm equal protein loading. Densitometric analysis was performed using LI-COR Odyssey IR Imager. The value obtained for both ERK1 and ERK2 was normalized to total ERK1/2 levels. The data were normalized to response achieved by 10 μM LPI, and presented as percentage inhibition.

[1]. Identification of the GPR55 antagonist binding site using a novel set of high-potency GPR55 selective ligands. Biochemistry. 2013 Dec 31;52(52):9456-69.

2-furanyl-[4-(2-methyl-5,6,7,8-tetrahydro-[1]benzothiolo[2,3-d]pyrimidin-4-yl)-1-piperazinyl]methanone is a N-arylpiperazine.

C20H22N4O2S

382.482

382.146

C, 62.81; H, 5.80; N, 14.65; O, 8.37; S, 8.38

460331-61-7

460331-61-7;

1434953

White to off-white solid powder

1.3±0.1 g/cm3

1.664

3.07

0

6

2

27

556

0

O1C=CC=C1C(N1CCN(C2C3C4CCCCC=4SC=3N=C(C)N=2)CC1)=O

GDPDARVUXXOYAJ-UHFFFAOYSA-N

InChI=1S/C20H22N4O2S/c1-13-21-18(17-14-5-2-3-7-16(14)27-19(17)22-13)23-8-10-24(11-9-23)20(25)15-6-4-12-26-15/h4,6,12H,2-3,5,7-11H2,1H3

furan-2-yl-[4-(2-methyl-5,6,7,8-tetrahydro-[1]benzothiolo[2,3-d]pyrimidin-4-yl)piperazin-1-yl]methanone

MLS000526305; ML192; CID1434953; ML 192; MLS-000526305; ML-192; 460331-61-7; MLS000526305; 2-furanyl[4-(5,6,7,8-tetrahydro-2-methyl[1]benzothieno[2,3-d]pyrimidin-4-yl)-1-piperazinyl]-methanone; furan-2-yl(4-(2-methyl-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-yl)piperazin-1-yl)methanone; furan-2-yl-[4-(2-methyl-5,6,7,8-tetrahydro-[1]benzothiolo[2,3-d]pyrimidin-4-yl)piperazin-1-yl]methanone; SMR000116779; 2-furanyl-[4-(2-methyl-5,6,7,8-tetrahydro-[1]benzothiolo[2,3-d]pyrimidin-4-yl)-1-piperazinyl]methanone; CID-1434953; ML192

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 (~261.45 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.54 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 2: ≥ 2.5 mg/mL (6.54 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 3: ≥ 2.5 mg/mL (6.54 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 of corn oil and mix evenly.

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