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Screening for Selective Ligands for GPR55 - Antagonists

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Received: ; Last Update: May 26, 2011.

The orphan G protein coupled receptor, GPR55, has gained notoriety because of its putative identification as a cannabinoid receptor subtype. The significance of this assignment should not be underestimated given the importance of cannabinoids to drug abuse and the potential utility of cannabinoid ligands in treating behavioral disorders leading to conditions such as obesity. The validity of assigning GPR55 to the cannabinoid family is problematic based upon the discovery that endogenous compounds not related to cannabinoid receptor ligands also bind and signal through GPR55. As a consequence, it is important to identify GPR55-selective ligands that can precisely define GPR55’s role in regulating addictive behaviors. Cannabidiol has been reported as a GPR55 antagonist in the literature to GPR55 activation by O1062, but this could not be confirmed our laboratory (Dr. Abood & Dr. Barak). There are no other known small molecule antagonists of GPR55 reported to date, only the CB1 inverse agonist/antagonists SR141716A (3.9 μM) and AM251 (9.6 μM). We have identified 3 potent and selective antagonists for GPR55, that represent 3 different chemical core scaffolds: 1) a quinoline aryl sulfonamide, ML193 (CID1261822) with a 221 nM potency for GPR55 and >145-fold, >27-fold and >145-fold antagonist selectivity against GPR35, CB1 and CB2, respectively and >145-fold agonist selectivity against all of these counter-receptors; 2) a thienopyrimidine, ML192 (CID1434953) with 1080 nM potency for GPR55 and >45-fold antagonist and agonist selectivity against GPR35, CB1 and CB2; and 3) a piperadinyloxadiazolone, ML191 (23612552) with 160 nM potency for GPR55 and >100-fold selectivity against GPR35, CB1 and CB2. All three probes also are active in inhibiting the downstream responses of ERK phosphorylation and PKC β II translocation.

Probe project: Selective GPR55 Antagonists: Part 2

Assigned Assay Grant #: 1X01 DA026205-01 (revised on transfer to NIDA) - previously 1X01 MH084187-01

Screening Center Name & PI: Sanford-Burnham Center for Chemical Genomics & John C. Reed

Chemistry Center Name & PI: same as above

Assay Submitter & Institution: Mary E. Abood, California Pacific Medical Center Research Institute (currently at Temple University, Center for Substance Abuse Research).

Collaborating PI: Lawrence S. Barak, Duke University

PubChem Summary Bioassay Identifier (AID): 2026

Probe Structure & Characteristics

This Center Probe Report provides the first description of 3 novel chemical probes that represent three chemical series for GPR55, an orphan GPCR receptor:

  • ML193 (quinoline aryl sulfonamide),
  • ML192 (thienopyrimidine), and
  • ML191 (piperadinyloxadiazolone).


CID 1261822



CID 1434953



CID 23612552

Target NameIC50/EC50 (nM) [SID, AID]Anti-target Name(s)IC50/EC50 (μM) [SID, AID]SelectivitySecondary Assay(s) Name: IC50/EC50 (nM) [SID, AID]


quinoline aryl sulfonamide
221 nM IC50 [SID87225762, AID2820]GPR35 Agonist>32 μM EC50 [SID87225762, AID2809]> 145XPKC β II
Active @ 10μM [SID87225762, AID488947]

65 nM [SID87225762, AID Pending]
GPR35 Antagonist>32 μM IC50 [SID87225762, AID2815]> 145X
CB1 Agonist>32 μM EC50 [SID87225762, AID2814]> 145X
CB1 Antagonist24.6 μM IC50 [SID87225762, AID2835]27.1X
CB2 Agonist>32 μM EC50 [SID87225762, AID2822]> 145X
CB2 Antagonist>32 μM IC50 [SID87225762, AID2836]> 145X


702 nM IC50 [SID87225747, AID2820]GPR35 Agonist>32 μM EC50 [SID87225747, AID2809]> 45XPKC β II
Active @ 100μM [SID87225747, AID488947]

827 μM [SID87225762, AID Pending]
GPR35 Antagonist>32 μM IC50 [SID87225747, AID2815]> 45X
CB1 Agonist>32 μM EC50 [SID87225747, AID2814]> 45X
CB1 Antagonist>32 μM IC50 [SID87225747, AID2822]> 45X
CB2 Agonist>32 μM EC50 [SID87225747, AID2835]> 45X
CB2 Antagonist>32 μM IC50 [SID87225747, AID2836]> 45X


1,076 nM EC50 [SID87225750, AID2820]GPR35 Agonist>32 μM EC50 [SID87225750, AID2809]> 29XPKC β II
Active @ 30μM [SID87225750, AID488947]

328 nM [SID87225762, AID Pending]
GPR35 Antagonist>32 μM IC50 [SID87225750, AID2815]> 29X
CB1 Agonist>32 μM EC50 [SID87225750, AID2814]> 29X
CB1 Antagonist17.7 μM IC50 [SID87225750, AID2822]16.4X
CB2 Agonist>32 μM EC50 [SID87225750, AID2835]> 29X
CB2 Antagonist>32 μM IC50 [SID87225750, AID2836]> 29X

Recommendations for the scientific use of this probe

This proposal will provide tools for delineating the biochemistry of GPR55, potentially provide compounds for targeted therapeutics of pathways underlying addictive behaviors, and clarify our understanding of the molecular basis of addiction in general. In addition, recent studies suggest a role for GPR55 in inflammatory and neuropathic pain, bone development and cancer (1–8). The availability of high potency antagonists will facilitate the study of the biological roles of this receptor. Antagonists from this project provide novel tool compounds in which to evaluate GPR55 signaling in cells and in the future in animals. All other current putative tool compounds, were selected on the basis of being cannabinoid ligands, whereas new scaffolds will have unblemished pedigrees in that area.

1. Scientific Rationale for Project

Specific Aims (from the originating grant)

The specific aims of the originating proposal were: 1) To identify small molecule agonists of GPR55 by high content screening of MLSCN (Molecular Library Screening Center Network) libraries (covered in this probe report) and 2) To identify small molecule antagonists of GPR55 by high content screening of MLSCN libraries We propose to provide the MLSCN with cell lines expressing the receptors and β-arrestin fluorescent protein biosensors. These studies will provide tools for delineating the biochemistry of GPR55 to enable an understanding of the molecular basis of cannabinoid addiction and treatment.

Background and Significance

Cannabidiol has been reported as a GPR55 antagonist in the literature (9,10) to GPR55 activation by O1062, but could not be confirmed as such in the assay provider’s laboratory. There are no other known small molecule antagonists of GPR55 reported to date, so this screen did not have any positive controls other than the absence of agonist. The only previously reported GPR55 agonists were lysophosphatidylinositol (LPI) and the CB1 inverse agonist/antagonists SR141716A and AM251 (see Figure 1 below). These compounds had the following reported GPR55 potencies: SR141716A GPR55 EC50=3.9 μM; AM251 GPR55 EC50=9.6 μM; and LPI GPR55 EC50=1.2 μM. However, the two small molecule, drug-like ligands SR14176A and AM251 are not selective.

Figure 1. Previously reported GPR55 agonists.

Figure 1

Previously reported GPR55 agonists.

Marijuana is the most widely abused illegal drug and its spectrum of effects suggests that several receptors are responsible for its activity. Two cannabinoid receptor subtypes, CB1 and CB2, have been identified; however, in support of the notion that other cannabinoid receptors remain to be identified, the complex pharmacological properties of exogenous cannabinoids and endocannabinoids are not fully explained by CB1 and CB2 signal transduction. Recently the orphan G protein coupled receptor, GPR55, was declared as one of the missing cannabinoid receptor subtypes. However, the validity of this assignment remains in question based upon the identification of another class of GPR55 ligands. As a consequence of the identification of GPR55 as a target for cannabinoid binding, it becomes important to identify GPR55-selective ligands that can define its role in addiction. The primary high throughput assays proposed for identifying GPR55 active compounds are based upon the ability to monitor the activation states of ligand-bound GPR receptors using high content imaging employing β-arrestin green fluorescent protein biosensors. The pharmacology of GPR55 remains complex, with different laboratories reporting distinct ligand selectivity. However, it has become clear that GPR55 does share some ligands with the known cannabinoid receptors. The availability of selective high potency antagonists will facilitate the study of this receptor.

a. Describe the original goal for probe characteristics as identified in the CPDP

These studies will provide tools for delineating the biochemistry of GPR55 to enable an understanding of the molecular basis of cannabinoid addiction and treatment as well as to delineate the pharmacology of this receptor. The probe criteria are potency of better than 1–5μM and better than 10-fold selectivity against CB1, CB2, and GPR35.

b. For each assay implemented and screening run please provide

i. PubChem Bioassay Name(s), AID(s), Assay-Type (Primary, DR, Counterscreen, Secondary)

Table 1Summary of Assays and AIDs

PubChemBioAssay NameAIDsProbe TypeAssay TypeAssay FormatAssay Detection & well format
Summary of Image-based HTS for Selective Antagonists of GPR55 [Summary]2026AntagonistSummaryN/AN/A
Image-based HTS for Selective Antagonists of GPR55 [Confirmatory]2013AntagonistPrimaryCell-basedHCS – TransFluor & 384
SAR analysis of selective Antagonists of GPR55 using an Image-Based Assay [Confirmatory]2820
AntagonistSARCell-basedHCS – TransFluor & 384
SAR analysis of Agonists of the GPR35 Receptor using an Image-Based Assay2809
AgonistSelectivityCell-basedHCS – TransFluor & 384
SAR analysis of Antagonists of the GPR35 Receptor using an Image-Based Assay2815
AntagonistSelectivityCell-basedHCS – TransFluor & 384
SAR analysis of agonists of the Cannabinoid Receptor 1 using an Image-Based Assay2814
AgonistSelectivityCell-basedHCS – TransFluor & 384
SAR analysis of antagonists of the Cannabinoid Receptor 1 using an Image-Based Assay2822
AntagonistSelectivityCell-basedHCS – TransFluor & 384
SAR analysis of agonists of the Cannabinoid Receptor 2 using an Image-Based Assay2835
AgonistSelectivityCell-basedHCS – TransFluor & 384
SAR analysis of antagonists of the Cannabinoid Receptor 2 using an Image-Based Assay2836
AntagonistSelectivityCell-basedHCS – TransFluor & 384
ii. Assay Rationale & Description (when describing primary screen it would be useful to see standard metrics like, Z′, S:B for the optimized assay). Table of reagents and source
Primary Screen

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. (11). 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). This assay technology is commercially marketed as Transfluor® assay by Molecular Devices). 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 (7). 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.

Figure 2. GPR55 Example Images of Positive and Negative Controls.

Figure 2

GPR55 Example Images of Positive and Negative Controls. Effect of 25 μM agonist LPI (left panel) compared to DMSO control (right panel)

The primary screening protocol is described below.

Assay materials

Table 2Reagents used for the uHTS experiments

U2OS (Human Osteosarcoma) cell line stably expressing GFP-tagged β-arrestin and over-expressing the GPR55 receptorCells from AP, scaled-up by SBCCG
MEM with L-glutamine, Pen-strep, 10% Fetal Bovine Serum and selection antibiotics - 100ug/ml G418 and 50ug/ml ZeocinInvitrogen
384-well plates, black with clear bottomGreiner
Agonist LysophosphatidylinositolAvanti Polar
Paraformaldehyde - FixativeACROS Organics
DAPI – Nuclei StainInvitrogen
The following uHTS protocol was implemented

Day 1:

  1. 45ul of cell suspension (200,000 cells/ml in culture medium) was dispensed in each well of the assay plates using a Wellmate bulk dispenser.
  2. Plates are incubated overnight or approximately 20 hours at 37 degree C and 5% CO2.


  1. Serum is removed by media aspiration and replacing with 45ul serum-free MEM prior to addition of compounds.
  2. Compound addition was done on a Biomek FX with 384-head dispenser (Beckman):
    1. 5ul of 100μM compound solution was added to columns 3 through 24 of the assay plates for a final assay compound concentration of 10μM and 0.25% DMSO.
    2. 5ul of DMSO control (2.5% DMSO) working solution was added to column 1 and 2.
  3. Plates were preincubated with compounds for 30 minutes at 37 degrees C and 5% CO2.
  4. 5ul of agonist (50μM LPI) working solution was added to the entire plate except for column 1 for a final compound concentration of 5 μM (~EC80).
  5. Plates were incubated for 75 minutes at 37 degrees C and 5% CO2.
  6. Media was aspirated leaving 20ul liquid in each well using a Titertek plate washer.
  7. 40ul of fixative working solution was added to each well using a Wellmate bulk dispenser (Matrix) for a final concentration of 4% PFA.
  8. Plates were incubated for 40 minutes at room temperature.
  9. Fixative was aspirated and plates were washed twice with 50ul PBS leaving 20ul liquid in each well using a Titertek plate washer.
  10. 40ul of DAPI working solution was added using a Wellmate bulk dispenser for a final DAPI concentration of 100ng/ml. Aluminum plate sealers were applied to each plate.

Imaging and Analysis:

  1. Image acquisition was performed on an Opera QEHS (Perkin Elmer) with 45 plate capacity loader/stacker and the following settings:
    • 20x 0.45 NA air objective
    • Acquisition camera set to 2-by-2 binning for an image size of 688 by 512 pixels
    • 2 channels acquired sequentially: Exp1Cam1 = β-arrestin GFP using 488 nm laser excitation and 540/70 nm emission filters, Exp2Cam2 = DAPI (nuclei) using 365 nm Xenon lamp excitation and 450/50 nm emission filters
    • 2 fields per well for Primary screen, 4 fields per well for Hit Confirmation
  2. Image analysis was performed using the Acapella Spot Detection Algorithm and the following analysis settings:
    1. Nuclei Detection
      • Threshold Adjustment: 1.5
      • Nuclear Splitting Adjustment: 7
      • Minimum Nuclear Area: 70
      • Minimum Nuclear Contrast: 0.1
    2. Cytoplasm Detection
      • Cytoplasm Threshold Adjustment: 0.45
      • Cytoplasm Individual Threshold Adjustment: 0.15
      • Spot Detection
      • Spot Minimum Distance 3
      • Spot Peak Radius 0
      • Spot Reference Radius 2
      • Spot Minimum Contrast 0.25
      • Spot Minimum to Cell Intensity 1
  3. Metrics were calculated from
    1. Nuclei Images:
      • Cell Count ("NumberofCellsAnalyzed")
      • Nuclei Area ("AreaoftheNucleus")
      • Integrated Intensity of the Nuclei ("TotalIntegratedIntensityoftheNucleus")
      • Average Intensity of the Nuclei ("AverageIntensityoftheNucleus")
    2. GFP Images:
      • Integrated Intensity of the Cytoplasm ("TotalCytoplasmIntensity")
      • Integrated Intensity of the Detected Spots ("TotalSpotIntensity")
      • Ratio of the Integrated Spot to Integrated Cytoplasm Intensities ("RatioofSpotIntensitytoCytoplasmintensity")
      • Number of Spots per Cell ("AverageSpotsPerCell")
      • Percentage of Cells Positive for Spot Formation ("PercentagePositiveCells")
  4. Actives from the primary screen were determined using CBIS software (ChemInnovations) by calculating the % Activation of the "AverageSpotsPerCell" metric and using a hit criteria of Activity > 60% and "NumberofCellsAnalyzed" > 100 and “TotalCytoplasmIntensity” < 500,000. Wells with cell counts lower than 100 in the 2 acquired images were flagged "cytotoxic / low cell count". Wells with a very high total GFP intensity (“TotalCytioplasmIntensity” > 500,000) were flagged artifacts due to autofluorescence. All flagged wells were excluded from hit selection.

The primary screen was performed at a compound concentration of 10μM in 384-well format. The average Z′ for the screen was 0.78, the average Signal-To-Background (S/B) was 32.4, Signal-To-Noise was 228, and Signal Window was 12.3 using the average number of spots per cell (“AverageSpotsPerCell”) as the primary assay read-out.

Rationale for confirmatory, counter and selectivity assay See flowchart below:

Critical Path Flowchart for GPR55 Antagonist Project.

Critical Path Flowchart for GPR55 Antagonist Project

Assay artifacts – Assay technology: The primary and confirmatory assay is an image-based HCS, which is based on translocation of GFP-labeled β-arrestin from the cytosolic compartment via the plasma membrane to intracellular pits/vesicles upon stimulation. These fluorescent pits/vesicles are detected in the images as small fluorescent aggregates or spots (14). This assay read-out is highly specific, and the only assay artifact of concern was high autofluorescence of some test compounds. However, as this is an image based assay, the redistribution features could still be visualized for the most part even with high fluorescence backgrounds, which are expected to be isotropic and homogenous. Unlike a plate reader based assay, the algorithms of the HCS device allow rejection of data if not located and registered to a “cell” during image segmentation. Additionally, one of the HCS parameters quantifies the Total (Integrated) Intensity of the Cytoplasm, which is equivalent to an increased intensity in the GFP channel. Since the GFP intensity in this GFP redistribution assay is approximately the same for all cells independent of signaling activation, a significant increase in the total GFP intensity can be used as rejection criterion if there is potentially uncompensatable compound autofluorescence (i.e. TotalCytoplasmIntensity > 500,000 units). Additionally, the image-based high-content nature of the assay allows for monitoring of the number of cells imaged per well to flag wells with low cell counts due to toxicity or liquid handling.

Assay artifacts – Receptor independent signaling: The formal possibility exits where the compounds may act as antagonist, independent of the GPR55 receptor, i.e. acting directly on components downstream of the GPR55 receptor, e.g. at the GFP-β-arrestin trafficking directly. As shown above in our Critical Path Workflow, any such compounds would be rejected as such a mechanism would also activate the other related GPCR receptors e.g. GPR35, CB1, and CB2. These assays are also performed as image-based HCS assays using the same Transfluor™ HCS redistribution assays. These compounds would then be regarded as “promiscuous” and non-selective for GPR55.

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.

The following are confirmation assays for this project:

  • Assay 1: Image-based HTS for Selective Antagonists of GPR55 (AID 2013)
  • Assay 2: SAR analysis of selective Antagonists of GPR55 using an Image-Based Assay (AID 2820, 485287)

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 (13). Additionally, selectivity against the GPR35 receptor was evaluated, since GPR35 and GPR55 share ~30% identity (8–10,12). 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.

The following are selectivity assays for this project:

  • Assay 1: SAR analysis of Agonists of the GPR35 Receptor using an Image-Based Assay (AID 2809, 485286)
  • Assay 2: SAR analysis of Antagonists of the GPR35 Receptor using an Image-Based Assay (AID 2815, 485283)
  • Assay 3: SAR analysis of Agonists of the Cannabinoid Receptor 1 using an Image-Based Assay (AID 2814, 485282)
  • Assay 4: SAR analysis of Antagonists of the Cannabinoid Receptor 1 using an Image-Based Assay (AID 2822, 485285)
  • Assay 5: SAR analysis of Agonists of the Cannabinoid Receptor 2 using an Image-Based Assay (AID 2835, 485277)
  • Assay 6: SAR analysis of Antagonists of the Cannabinoid Receptor 2 using an Image-Based Assay (AID 2836, 485278)

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

The following are secondary assays for this project:

  • Assay 1: SAR Analysis for the identification of Selective Antagonists of GPR55 using an Image-Based Screen [PKC β II translocation] (AID488947)

2. Center Summary of Results

The following flowchart summarizes the compound triage and decision tree for advancement of compounds:

291,669 compounds from the NIH MLSMR collection (this was the full size of the library at the time of this HCS) were tested in the image-based GPR55 HCS β-Arrestin primary screen. The primary screen resulted in 805 compounds that were considered as hits using the hit criterion of >60% activity as compared to the negative control LPI (lysophosphatidylinositol) and the positive control of the positive control of vehicle (DMSO) only. Additionally, hit compounds had to meet the criterion of >100 cells in the imaged area of the well and total cytoplasmic intensity of < 500,000 relative intensity units. Cherry picks of the identified 805 compounds were ordered from DPI for hit confirmation.

Liquid reorders of 705 compounds were received from DPI in 100% DMSO. A single-concentration (10 μM) confirmatory antagonist assay was performed in duplicate. 273 compounds reconfirmed and were evaluated for potency using a 7-point dose response ranging from 32 μM to 500 nM, in duplicate, on 2 different days. In the dose response experiments, 39 compounds resulted in an IC50 of >1 μM and 228 compounds had an IC50 of 1–10 μM. The maximum-common-substructure-based clustering analysis of 273 compounds resulted in 52 scaffolds and 54 singletons. By analyzing assay data in terms of scaffolds, 44 compounds from 17 scaffolds were selected for follow-up studies. Only 38 of these 44 compounds were readily available as dry powders.

The reordered 38 compounds as well as 67 subsequently ordered analog-by-catalog (ABC) compounds were run in the GPR55 antagonist assay using 10-point dose responses ranging from 32μM to 62nM. 23 of the compounds showed inhibition with I IC50 < 1 μM. The dry powder compounds were also evaluated for selectivity in the following selectivity assays: CB1 agonist, CB1 antagonist, CB2 agonist, CB2 antagonist, GPR35 agonist, and GPR35 antagonist assays. 20 of the compounds with GPR55 IC50 < 5μM were not completely selective: 2 compounds showed less than 10-fold selectivity against GPR35 agonist, and 18 had less than 10-fold selectivity against CB1 antagonist. Out of these 18 compounds 3 were also not selective against CB2 antagonist. All other potent compounds were > 10-fold selective against the tested panel.

GPR55 Antagonist Compound Flowchart.

GPR55 Antagonist Compound Flowchart

3. Probe Optimization

i. Describe SAR & chemistry strategy (including structure and data) that led to the probe

The SAR for probe CID1261822 can be seen in Table 3. This probe series consists of a quinoline core attached to an aryl sulfonamide via an amide linkage. The preferred groups at R1 are 5- or 6-membered heterocycles. At R2, both aryl and heteroaryl substitutents are tolerated. Phenyl substituents at this position can be substituted at either the ortho or para positions. R3 can tolerate either methyl or hydrogen substitution. As can be seen in the table, the preferred heterocycle at R1 is an isoxazole. The presence of this moiety at R1 resulted in the 3 most potent GPR55 antagonists, CIDs 1261822, 1261290, and 1261872. All 3 of these compounds have GPR55 antagonist IC50 values less than 1 μM and are completely selective over the cannabinoid receptors 1 and 2. The isoxazole subclass is also represented in 2 less potent representatives, CIDs 2333197 and 1011349, the latter of which had essentially no inhibitory activity. It can be noted in these 2 examples the R3 substituent was H, whereas in the very potent isoxazoles, R3 is methyl substituted. This underscores the importance of the R3 substitution in this subclass. Some of the other tolerated heterocycles at R1 include thiazoles and pyrimidines. However, both of these groups resulted in analogues with significantly less GPR55 antagonist potency. Finally, it was noted that R2 substitution preferred the 2- and 3-pyridyl substitution. Deviation from these preferred substituents, including the 4-pyridyl as in CID2385808, resulted in a dramatic loss of activity.

Table 3. SAR of GPR55 Antagonist Probe ML193 (#1): CID1261822.

Table 3

SAR of GPR55 Antagonist Probe ML193 (#1): CID1261822.

The SAR for probe CID1434953 can be seen in Table 4. This series consists of a substituted thienopyrimidine core containing the R1 and R2 substituents and an acylated piperazine containing R3. It is clear from the SAR table that the preferred R1 group is a methyl. However, there are limited examples of a small cycloalkane being tolerated at this position, as can be seen in CID655864. Preference for R2 being furan is also clear from the data in the SAR table. When this group is substituted with aromatic or alkyl groups as in CIDs 1434956 or 1434957, respectively, a dramatic loss in GPR55 antagonist potency is seen. Another interesting observation is that when the furan ring is saturated to a tetrahydrofuran, as in CID4877555, activity is abolished as well. Finally, substitution on the core thienopyrimidine ring was explored. While majority of the derivatives possessed an unsubstituted fused tetrahydropyranyl ring, methyl substitution of this ring, as in CIDs 3193014 and 3193022, was also tolerated, resulting in potent, selective antagonists.

Table 4. SAR of GPR55 Antagonist Probe ML192 (#2): CID1434953.

Table 4

SAR of GPR55 Antagonist Probe ML192 (#2): CID1434953.

The SAR for probe molecule CID23612552 is seen in Table 5. This class consists of piperadinyloxadiazolone core substituted with various phenylacetyl groups on a piperdine ring. The core molecule with a phenyl group in the 5-position of the oxadiazolone ring is conserved throughout the derivatives, indicating a tight yet tractable SAR in this series. The piperidine portion of this probe series tolerates a variety of groups having a specific motif consisting of a substituted phenylacetyl group. The benzylic methylene of this group can be sequentially substituted up to a quaternary center, with these derivatives yielding the most potent analogues, including the probe molecule. Of the derivatives possessing this quaternary substitution at the benzylic position, the cyclopropyl analogues clearly afford the most potent derivatives. This is most likely due to the bond angle difference that is seen in divergence from the tetrahedral geometry as in the hydrogen or monosubstituted species, both of which are less potent (e.g. CID23612541). This may also be due to secondary orbital interactions with the adjacent aromatic groups as cyclopropyl bonds have more p character. Substitution of the aromatic portion of the phenylacetyl group is also important. Comparison of the probe CID23612552 to a similar compound that lacks the tolyl substitution, such as CID23612554, leads to a dramatic loss of antagonist potency. Finally, it can be noted that various derivatives show CB1 antagonist activity, with CID23612543 being a potent CB1 antagonist. It can be noted that this molecule bears a spirocyclopentyl group at the benzylic position. The probe molecule, however, is devoid of any CB activity. It appears that the cyclopropyl group lends additional selectivity for GPR55 over the CB receptors.

Table 5. SAR of GPR55 Antagonist Probe ML191 (#3): CID23612552.

Table 5

SAR of GPR55 Antagonist Probe ML191 (#3): CID23612552.

4. Probe

a. Chemical name of probe compounds (from PubChem)

ML193 - CID1261822 is N-(4-(N-(3,4-dimethylisoxazol-5yl)sulfamoyl)-phenyl)-6,8-dimethyl-2-(pyridin-2-yl)quinoline-4-carboxamide

ML192 - CID1434953 is furan-2-yl(4-(2-methyl-5,6,7,8-tetrahydro-benzo[4,5]thieno[2,3-d]pyrimidin-4-yl)piperazin-1-yl)methanone

ML191 - CID23612552 is 5-phenyl-3-(1-(1-(p-tolyl)cyclopropane-carbonyl)piperidin-4-yl)-1,3,4-oxadiazol-2(3H)-one

b. Probe chemical structure including stereochemistry if known

ML 193.

ML 193

CID 1261822

ML 192.

ML 192

CID 1434953

ML 191.

ML 191

CID 23612552

c. Structural Verification Information of probe SID

Probe ML193 - CID1261822 corresponds to SID 87225762

Purity: >98% (HPLC)

Mass Spec: HRMS (ESI) m/z calcd for C28H25N5O4S ([M+H]+), 528, found 528.

Image ml193fu62
Image ml193fu63


PROBE ML192 - CID1434953. The probe SID is 87225747

Purity: >98% (HPLC)

Mass Spec: HRMS (ESI) m/z calcd for C20H22N4O2S ([M+H]+), 383, found 383.

Image ml193fu65
Image ml193fu66


PROBE ML191 - CID23612552. The probe SID is 87225750

Purity: >98% (HPLC)

Mass Spec: HRMS (ESI) m/z calcd for C24H25N3O3 ([M+H]+), 404, found 404.

Image ml193fu68
Image ml193fu69


d. If available from a vendor, please provide details

PubChem CID1261822 is available from Chembridge # 7924036

PubChem CID1434953 is available from Asinex # ASN 04888279

PubChem CID23612552 is available from ChemDiv # D327-0013

e. Provide MLS# that verifies the submission of probe molecule and five related samples that were submitted to the SMR collection

Table 6Probe Submission for GPR55 Antagonist Probes

Probe/AnalogDPI MLS#*BCCG MLS-#CIDSIDSource (vendor or BCCG syn)Amt (mg)Date* Submitted
Probe 1 – ML193: CID1261822
Probe 1
[ ML193]
Analog 1MLS0031831040269911126129087225761ChemBridge2011/11/10
Analog 2MLS0031831000098578126187287225763ChemBridge2011/11/10
Analog 3MLS0031831030257792233285399303185Enamine2011/11/10
Analog 4MLS0031831000206672158268799303168InterBioScreen2011/11/10
Analog 5MLS0031831020226099230820399303183Enamine2011/11/10
Probe 2 – ML192: CID1434953
Probe 2
Analog 1MLS0031831060285143319301487225742Asinex2010/20/10
Analog 2MLS0031831100437136143496099303132Asinex2010/20/10
Analog 3MLS0031831070354783143495699303131Asinex2010/20/10
Analog 4MLS0031831080437131143495799303126Asinex2010/20/10
Analog 5MLS0031831090437135319302299303130Asinex2010/20/10
Probe 3- ML191: CID23612552
Probe 3
Analog 1MLS00318311203590582361256787225751ChemDiv2010/20/10
Analog 2MLS00318311403677052361255099303152ChemDiv2010/20/10
Analog 3MLS00318311504371512361254399303149ChemDiv2010/20/10
Analog 4MLS00318311303591282361255499303154ChemDiv2010/20/10
Analog 5MLS00318311604371532361254899303151ChemDiv2010/20/10

Compounds ordered on 10/20/10, but inordinate wait to receive, also experienced submission delays on vial requests

f. Describe mode of action for biological activity of probe

The identified GPR55 antagonists appear to selectively inhibit GPR55 receptor signaling, but do not inhibit the related cannabinoid receptors 1 and 2 nor the related GPR35 orphan receptor. Since the selectivity assays used the same β-arrestin HCS assay technology and the probes did not inhibit non-selectively, they are not non-specifically inhibiting signaling at or down-stream of the β-arrestin pathway. Since the HCS assay technology measures β-arrestin-GPCR complex internalization into clathrin-coated pits within the cell, the identified antagonist compounds have to inhibit pathway signaling at the β-arrestin-GPCR complex internalization or any process upstream. In tertiary studies by Drs. Abood (still ongoing) and Barak, these probes did show inhibition of ERK1/2 phosphorylation as well as inhibition of PKCβII translocation, confirming the downstream biological response of the compounds inhibitory activities.

Dose response for 3 GPR55 antagonist probes

Dose response curves for the 3 probes for the GPR55 antagonist assay and all selectivity assays.

Dose response curves for the 3 probes for the GPR55 antagonist assay and all selectivity assays

% Efficacy is reported representing either % Inhibition (antagonist assays) or % Activation (agonist assays) as compared to control compounds specific to each assay (refer to PubChem AIDs for details)

This project is judged as highly successful, since the antagonists described in and derived from this project provide “clean” tool compounds with which to evaluate GPR55 signaling in cells and in the future in animals. A recent, March 30, 2011 SciFinder search by Dr. Dahl did not find any reports of antagonists for GPR55.

g. Detailed synthetic pathway for making probe

Scheme 1 (ac). Synthetic routes for the three (3) probe molecules are shown below:

Synthesis of ML193 (Scheme 1a): To a solution of tert-butyl (4-(chlorosulfonyl)phenyl)carbamate (145 mg, 0.5 mmol) and N,N-diisopropylethylamine (1.5 eq.) in dichloromethane (5 mL) was added 3,4-dimethylisoxazol-5-amine. This mixture was stirred at rt overnight. After evaporation of solvent and purification by flash chromatography, the residue was suspended in a 20% solution of trifluoroacetic acid in dichloromethane. Removal of solvent and excess acid afforded the sulfonamide intermediate which was treated with 6,8-dimethyl-2-(pyridin-2-yl)quinoline-4-carbonyl chloride (148 mg, 0.5 mmol) in the presence of N,N-diisopropylethylamine (2 eq.) in dichloromethane. After 3 h at rt, the solvent was removed by rotary evaporation and ML193 was isolated by reverse-phase preparative HPLC as a white solid.

Synthesis of ML192 (Scheme 1a): N-Boc-piperazine (93 mg, 0.5 mmol) was treated with furan-2-carbonyl chloride (65 mg, 0.5 mmol) and N,N-diisopropylethylamine (1.5 eq.) in dichloromethane. After stirring for 1 h, the acylated boc-piperazine intermediate was isolated by silica gel flash chromatography. Removal of the boc group with TFA (20% in dichloromethane, 10 min) followed by evaporation and treatment with 4-chloro-2-methyl-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidine (119 mg, 0.5 mmol) and N,N-diisopropylethylamine (2 eq.) in dichloromethane for 5 h afforded ML 192 which was isolated as a white solid by reverse-phase preparative HPLC.

Synthesis of ML191 (Scheme 1a): Benzohydrazide (136 mg, 1.0 mmol) was treated with bis(trichloromethyl) carbonate (2 eq.) in pyridine at 0 °C. After slow warm to rt, the mixture was stirred for an additional 20 h. After evaporation of solvent, the oxadiazolone product was suspended in a 1% acetic acid in dimethylformamide solution and treated with N-boc-piperidinone (1.2 eq.) and sodium cyanoborohydride (2 eq.). This mixture was stirred at rt for 16 h after which the residue was diluted with water and extracted (ethyl acetate). Evaporation of the organic layer and re-suspension in a 20% solution of trifluoroacetic acid in THF afforded the free amine which, after evaporation of excess acid and solvent, was treated with 1-(p-tolyl)cyclopropanecarbonyl chloride (1.5 eq.) and N,N-diisopropylethylamine (2 eq.) in dichloromethane for 5 h to afford ML 191 which was isolated by reverse-phase preparative HPLC.

h. Center summary of probe properties (solubility, absorbance/fluorescence, reactivity, toxicity, etc

These probes have been profiled for their in vitro ADME/T properties (see Table 7). They all display low to moderate aqueous solubility at various pH levels and lower solubility in PBS versus phosphate-free buffer. The apparent stability of the probe in PBS was initially thought to be poor, however, it was ascertained that their low aqueous solubility confounded this evaluation (data not shown). ML193, ML192 and ML191 were completely stable for up to 48 hrs in 1:1 acetonitrile:PBS, as seen in figures below.

Table 7. Summary of in vitro ADMET/PK Properties of GP55 Agonist Probe(s).

Table 7

Summary of in vitro ADMET/PK Properties of GP55 Agonist Probe(s).

ML193 Stability in 1:1 PBS/ACN.

ML193 Stability in 1:1 PBS/ACN

Time (hr)Remaining
ML192 Stability in 1:1 PBS/ACN.

ML192 Stability in 1:1 PBS/ACN

Time (hr)Remaining
ML191 Stability in 1:1 PBS/ACN.

ML191 Stability in 1:1 PBS/ACN

Time (hr)Remaining

One of the favorable factors is that the permeability of all three scaffolds is high, indicative of being readily absorbed. While all three probe compounds are stable in plasma, ML193 and ML192 are degraded by liver enzymes, though ML193 is less sensitive with 50–60% remaining after 1 hr. While this may be an issue for oral dosing, other routes of administration may provide significant systemic levels. Finally, ML192 and ML191 showed no significant hepatocyte toxicity in vitro, while ML193 had some low hepatic toxicity.

The oxadiazolone carbonyl of ML191 may be reactive to nucleophiles and thus might be an acylating agent. However, we have determined that is NOT reactive to glutathione (see below). The chemical structures of ML193 and ML192, in contrast, do not possess any potentially reactive chemical moieties.

ML191 Glutathione conjugation assay. To assess the potential of the ML191 for covalent modification, the glutathione transferase activity of the probe compound in the rat hepatic S9 fraction was measured. The S9-based GSH transferase assay has been shown to be a reliable means of identifying compounds known to react with GSH. Briefly, compounds were incubated at 10 μM in PBS buffer with 2 mg/ml rat hepatic S9 fraction and 10 mM glutathione at 37°C for 1.5 hours. The incubation was stopped by protein precipitation using acetonitrile. After drying down of the supernatant, the residues were reconstituted and analyzed by positive-ion electrospray LCMS and analyzed for the presence of any glutathione conjugates. The LCMS data after 1.5 hours showed 96% of unchanged probe compound, indicating a low propensity for covalent modification. In addition, none of the expected GSH conjugate masses were detected. The compounds were tested in duplicate along with the positive control diclofenac, a known GSH conjugator, which showed the expected conjugate masses.

Image ml193fu75

ML 191 Glutathione Reactivity Assessment: ML191 was incubated with glutathione and rat S9 fraction for 1.5 hours. (b) HPLC-MS analysis showed 96.0% of the parent compound remained after 1.5 hours. Also, no GSH adduct masses were detecte

These probe molecules have demonstrated potent and selective antagonist activity in the image-based primary assay for inhibition of β-arrestin mediated signaling of the GPR55 orphan receptor, with selectivity over the GPR35, cannabinoid CB1 and CB2 receptors.

i. A tabular presentation summarizing known probe properties (computed properties, must match from PubChem)

Calculated PropertyParameter Value
Molecular Weight [g/mol]527.5942382.47928403.4736
Molecular FormulaC28H25N5O4SC20H22N4O2SC24H25N3O3
H-Bond Donor200
H-Bond Acceptor854
Rotatable Bond Count624
Tautomer Count33
Exact Mass527.162725382.146347403.189592
MonoIsotopic Mass527.162725382.146347403.189592
Topological Polar Surface Area13690.762.2
Heavy Atom Count382730
Formal Charge000
Isotope Atom Count000
Defined Atom StereoCenter Count000
Undefined Atom StereoCenter Count000
Defined Bond StereoCenter Count000
Undefined Bond StereoCenter Count000
Covalently-Bonded Unit Count111

5. Comparative data showing probe specificity for target in biologically relevant assays

Further follow-up studies in the assay provider’s laboratories (Dr. Abood and collaborator Dr. Barak) aimed to validate the biological activities in the signaling pathway downstream of β-arrestin are ongoing. These downstream assays include probe characterization for ERK1/2 activity in GPR55-overexpressing cells and PKC βII translocation in cells with wild-type GPR55 receptor and the assays are described below. The SAR data for the PKC βII translocation assay as well as preliminary data for the pERK assay are summarized for the 3 probes (Table 9).

Table 9. Downstream assays by Assay Provider.

Table 9

Downstream assays by Assay Provider.

Assay for ERK1/2 Inhibition in GPR55-Overexpressing Cells (AID Pending): This assay was performed by the assay provider to characterize inhibition of the downstream ERK1/2 phosphorylation activity of the probe compounds. It is a Western Blot assay that utilizes a cell line permanently expressing a β-arrestin GFP biosensor and human GPR55 receptor. Western blots were necessary because the known agonist, LPI, stimulates ERK only modestly, thus “In-Cell” in situ Westerns were not feasible in this case Upon agonist-mediated GPCR activation, ERK1/2 phosphorylation occurs as measured by pERK1/2 antibodies. The inhibition ability of the probe compound was measured by quantifying the ERK1/2 phosphorylation activity in the presence of agonist. Because of the low-throughput of the manual Western Blot assay, only probe candidates were tested. In the preliminary data, all probes are showing measureable downstream activities for ERK1/2 phosphorylation.

Assay for PKC βII Translocation (AID 488947) in cells with wild-type GPR55 receptor: This imaging assay utilizes HEK293 transiently expressing a PKC βII GFP biosensor and a wild type GPR55 receptor. Upon agonist treatment, the GPR55-mediated G-protein signaling is measured by PKC βII GFP recruitment to the plasma membrane and formation of widespread plasma membrane remodeling. This PKC recruitment is shown as increased fluorescence on plasma membrane and development of blebs. The inhibitory effect of the probe compounds was determined by quantifying inhibition of the PKC βII GFP recruitment to the plasma membrane and formation of widespread plasma membrane remodeling in the presence of agonist. Because of the manual image analysis, results were binned into 5 categories (none, ~1%, ~10%, ~30%, ~60% GFP membrane recruitment/blebs formation) and only probe candidates were tested at 4 concentrations. All probes showed significant inhibition of membrane recruitment/bleb formation indicating inhibition of activity in the signaling pathway.

Future Studies

GPR55 has been implicated in behavior, inflammatory disease, neuropathic pain, osteoporosis and cancer. All these avenues will also be pursued using these novel and potent antagonists. Initially, we plan to use these probes to explore the role of GPR55 though inhibition of its function in a number of in vitro systems where endogenous GPR55 is expressed, such as primary neuronal and glial cultures, neuronal cell lines, and endothelial and bone cells (where solubility is less of an issue). Additionally, GPR55 is expressed in immune cells and this is another area of interest. . For in vivo modeling, we are preparing to assess some of the novel GPR55 agonists in standard behavioral paradigms used in addiction studies, and this will also require assessment of their ability to maintain adequate levels in the central nervous system. CNS active GPR55 antagonists resulting from these studies would have a prominent role in validating the selective behavioral effects of the recently identified GPR55 agonists as well as confirming the off target signaling of CB1 and 2 ligands.


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Compounds ordered on 10/20/10, but inordinate wait to receive, also experienced submission delays on vial requests

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