GPR55 ( IC50 = 221 nM )

ML-193 (0.01-100 μM; pretreated for 15 min) has an IC50 of 0.22 μM, while ML186 (1 μM) inhibits β-arrestin trafficking induced by L-α-lysophophosphatidylinositol (LPI, 10 μM)[2].
ML-193 (0.01-10 μM; pretreated for 30 min) reduces the LPI-mediated ERK1/2 phosphorylation, with an IC50 of 0.2 μM in U2OS cells[2].
ML-193 (5 μM; pretreated for 30 min) reduces the increases in hNSC proliferation rates that GPR55 agonists cause[4].
ML-193 (5 μM; 10 d) reduces the increases in hNSC differentiation brought on by ML184[4].

ML193 (at 1 and 5 µg/rat; intrastriatal at a rate of 1 μL/min) improves motor coordination in PD rats and attenuates sensorimotor deficits and slip steps[3].

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6-OHDA(6-hydroxydopamine)-lesioned rats had impaired behaviours in all tests. Intra-striatal administration of LPI in 6-OHDA-lesioned rats increased time on the rotarod, decreased latency to remove the label, with no significant effect on slip steps, and locomotor activity. Intra-striatal administration of ML193 also increased time on the rotarod, decreased latency to remove the label and slip steps in 6-OHDA-lesioned rats mostly at the dose of 1 µg/rat. Conclusions: This study suggests that the striatal GPR55 is involved in the control of motor functions. However, considering the similar effects of GPR55 agonist and antagonist, it may be concluded that this receptor has a modulatory role in the control of motor deficits in an experimental model of Parkinson.[3]

Primary Screen[1]
The read-out needed to be independent of the orphan status of GPR55. Therefore the well-characterized pathway of β-arrestin mediated internalization following receptor activation was chosen. In the particular embodiment, a β-arrestin biosensor comprised of GFP fused to β-arrestin protein was stably expressed in an engineered stable cell line and GPR55E was cloned into this cell line. GPR55E, a C-tail modified variant of GPR55, with greater β-arrestin responsiveness and similar pharmacology was used for the primary assay and will be known throughout the probe report as GPR55 Kapur et al. This image-based high-content screen (HCS) is based then on the fluorescence redistribution of GFP-β-arrestin complex bound receptors from a homogeneous distribution in the cytoplasm via the plasma membrane into clathrin-coated intracellular pit, then into vesicles during the process of receptor internalization (See Figure 2 on right). Upon activation by ligand binding (lysophosphatidyinositol derivatives), the GPR55 receptor undergoes deactivation or “desensitization” by binding of the β-arrestin protein to the activated receptor. The GPCR-β-arrestin complex internalizes, the ligand is removed and the receptor is recycled back to the cell membrane. Localization of the fluorescently labeled β-arrestin can be monitored by image analysis. Dr. Abood found that transient transfections with unmodified GPR55 resulted in similar functional redistribution (data not shown). The primary screen assay is designed to identify compounds inhibiting GPR55 signaling activated by EC80 concentration of the GPR55 agonist lysophosphatidylinositol (LPI). It is important to note that at an EC80 agonist concentration, the calculated IC50’s of tested compounds are probably 5–8 fold higher than the actual binding affinities Ki of the compounds.

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Confirmation assays
Initial hit confirmation of compound solutions resupplied by the MLSMR was done at a single compound concentration (10 μM) in duplicates using the primary screen assay to confirm activity of the hit compounds. Compounds with confirmed activity at 10 μM were tested in 7-point dose responses (0.5 to 32 μM) to evaluate potency. Potent compounds (IC50 <5 μM) were clustered into scaffolds and 10-point dose responses (0.06 to 32 μM) were performed for dry powder compounds selected from hits and their commercially available analogs.

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Secondary Assays: Counterscreen / Selectivity assays
Since GPR55 is thought to be a putative cannabinoid receptor, selectivity against the other cannabinoid receptors (CB1 and CB2) was established using the same image-based high-content assay technology. Additionally, selectivity against the GPR35 receptor was evaluated, since GPR35 and GPR55 share ~30% identity. In addition to providing a selectivity panel, these assays eliminated false positives caused by assay artifacts, since all selectivity assays utilized the same assay technology. Selectivity Assays were performed in 10-point dose responses (0.06 to 32 μM) for dry powder compounds.

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Post Probe Characterization Tertiary Assays (Assay Provider) The identified GPR55 antagonist probes and selected analogs are characterized further by two assays performed in the assay provider’s and her collaborator’s labs. The pERK assay evaluates downstream activity in the GPR55 signaling pathway (which can represent either G-protein dependent signaling, or G-protein independent signaling), while the PKC β II translocation assay evaluates a G-protein dependent signaling pathway

To investigate the effects of GPR55 activation on hNSC proliferation, cells were plated on laminin‐coated 6‐well plates. Cells were allowed to adhere overnight and then treated with LPI (1 μM), the endogenous ligand for GPR55, or synthetic agonists, O‐1602 (1 μM) or ML184 (1 μM), in a reduced growth factor media (5% growth factor). Reduced growth factor medium was utilized to better mimic a less proliferative phenotype while still maintaining a ‘stemness’ state. Analysis by flow cytometry showed no significant reduction of nestin+ or Sox2+ populations after 48 h (data not shown). Cells treated with the selective GPR55 antagonist ML193 (5 μM) were pretreated for 30 min prior to addition of agonist. Vehicle‐treated cells received 0.1% DMSO in 5% growth factor media. For differentiation studies, cells were treated with either vehicle, ML184 (1 μM), ML193 (5 μM), or a combination of ML184 (1 μM) and ML193 (5 μM) in ReNcell medium that did not contain growth factors.[4]

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PKCβII TranslocationAssay of GPR55 Activation 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 (e.g. ML193). Inhibition of agonist -stimulated redistribution of PKCβII-GFP was assessed after drug treatment at room temperature.[2]

Male Wistar rats (200-250 g) were induced experimental Parkinson by 6-hydroxydopamine (6-OHDA, 10 µg/rat)[3]
1 and 5 µg/rat
Injected into the right striatum at a rate of 1 μL/min[3]

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Experimental Parkinson was induced by unilateral intra-striatal administration of 6-hydroxydopamine (6-OHDA, 10 µg/rat). L-α-lysophosphatidylinositol (LPI, 1 and 5 µg/rat), an endogenous GPR55 agonist, and ML193 (1 and 5 µg/rat), a selective GPR55 antagonist, were injected into the striatum of 6-OHDA-lesioned rats. Motor performance and balance skills were evaluated using the accelerating rotating rod and the ledged beam tests. The sensorimotor function of the forelimbs and locomotor activity were assessed by the adhesive removal and open field tests, respectively.[3]

[1]. Screening for Selective Ligands for GPR55-Antagonists. Probe Reports from the NIH Molecular Libraries Program. 2010 Oct 30.

[2]. 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.

[3]. The effect of intra-striatal administration of GPR55 agonist (LPI) and antagonist (ML193) on sensorimotor and motor functions in a Parkinson's disease rat model. Acta Neuropsychiatr. 2021 Feb;33(1):15-21.

[4]. Activation of GPR55 increases neural stem cell proliferation and promotes early adult hippocampal neurogenesis. Br J Pharmacol. 2018 Aug;175(16):3407-3421.

N-[4-[(3,4-dimethyl-5-isoxazolyl)sulfamoyl]phenyl]-6,8-dimethyl-2-(2-pyridinyl)-4-quinolinecarboxamide is an aromatic amide.

C28H25N5O4S

527.599

527.162

C, 63.74; H, 4.78; N, 13.27; O, 12.13; S, 6.08

713121-80-3

1261822

White to light yellow solid powder

1.4±0.1 g/cm3

1.670

4.63

2

8

6

38

921

0

C1=CC=C(C2=NC3C(=CC(=CC=3C(C(NC3=CC=C(S(NC4ON=C(C)C=4C)(=O)=O)C=C3)=O)=C2)C)C)N=C1

HTSLEZOTMYUPLU-UHFFFAOYSA-N

InChI=1S/C28H25N5O4S/c1-16-13-17(2)26-22(14-16)23(15-25(31-26)24-7-5-6-12-29-24)27(34)30-20-8-10-21(11-9-20)38(35,36)33-28-18(3)19(4)32-37-28/h5-15,33H,1-4H3,(H,30,34)

N-[4-[(3,4-dimethyl-1,2-oxazol-5-yl)sulfamoyl]phenyl]-6,8-dimethyl-2-pyridin-2-ylquinoline-4-carboxamide

CID 1261822; ML193; CID-1261822; CID1261822; MLS000862518; N-(4-(N-(3,4-Dimethylisoxazol-5-yl)sulfamoyl)phenyl)-6,8-dimethyl-2-(pyridin-2-yl)quinoline-4-carboxamide; N-[4-[(3,4-dimethyl-1,2-oxazol-5-yl)sulfamoyl]phenyl]-6,8-dimethyl-2-pyridin-2-ylquinoline-4-carboxamide; SMR000300559; ML 193; ML-193

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: ~33.3 mg/mL (~63.2 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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