This was presented in Cape Town in July 2014 as part of World Pharma. The presentation focusses on the cannabinoid receptor-related receptors, GPR18, GPR55 and GPR119; considers reports for their de-orphanisation and the issues associated with their putative endogenous ligands.

1. Role of cannabinoid-related receptors (GPR55, GPR18 and GPR119) in inflammation, satiety and obesity
Steve Alexander
Pharmacology Group, Life Sciences, University of Nottingham
ENGLAND
WorldPharma 2014
Track 6 - Orphan G protein-coupled receptors-
What are the new ligand and new drug targets?

2. Plan
•Cannabinoid receptors
•Cannabinoid receptor-related receptors
–GPR18, GPR119, GPR55
–Pharmacology
•Endogenous ligands
–Opportunistic (off-target) actions
•Synthetic ligands
–Therapeutic potential
2

3. Publications on the cannabinoid receptor-related receptors
In 2013, there were 574 and 287 publications on CB1 and CB2 cannabinoid receptors, respectively.
Source: PubMed
July 2014
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4. CANNABINOID RECEPTORS
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5. Canonical Cannabinoid Receptors
• GPCR
– CB1 ‘CNS’ receptors
• The most abundant GPCR in the CNS
– CB2 ‘immune’ receptors
• Activated by the major psychoactive
component of the Cannabis plant, THC
THC
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6. Endogenous cannabinoids
Anandamide, AEA
Isolated from pig brain
Devane, Science, 1992
2-Arachidonoylglycerol, 2AG
Isolated from dog gut
Mechoulam, Biochem
Pharmacol, 1995
6

7. Endocannabinoid turnover
AEA
2AG
Precursor
N-Arachidonoyl phosphatidylethanolamine
Diacylglycerol
Synthetic enzymes
NAPE-PLD
DGLa,
DGLb
Hydrolytic products
Arachidonic acid and ethanolamine
Arachidonic acid and glycerol
Hydrolytic enzymes
FAAH, FAAH2, NAAA
MGL,
ABHD6,
ABHD12
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•Parallel and independent metabolism
•However, also substrates for COX-2, LOX activities

8. Opportunistic actions of ECs
AEA effect
2AG effect
TRPV1
Agonist (Zygmunt, Nature, 1999)
Agonist (Zygmunt, PLOS One, 2013)
PPARa
Agonist (Sun, BJP, 2007)
Agonist (Kozak, J Biol Chem, 2002)
PPARg
Agonist (Sun, BJP, 2007)
Agonist (Rockwell, Mol Pharmacol, 2006)
GABAA-b2
Positive allosteric modulator (Sigel, PNAS, 2011)
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•Complicates the interpretation of the use of endocannabinoids and enzyme inhibitors

9. ‘Selective’ antagonists
CB1: AM251 CB2: SR144528
Identified in the 1990s, both have been described as
‘inverse agonists’
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10. Primary sequence alignment
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11. GPR18
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12. GPR18: Cloning and initial deorphanization
•Cloned from a human T-cell line in a search for novel chemokine-like receptors
(Kohno, BBRC, 2006)
•N-Arachidonoylglycine (NAGly) as an agonist
–A rapid, transient [Ca2+]i elevation @ 10 μM
–Concentration-dependent, pertussis toxin- sensitive inhibition of cAMP (IC50 value of 20 nM)
(Kohno, BBRC, 2006)
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13. GPR18: Endocannabinoid-like
molecules
2AG
AEA
NAGly
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14. GPR18: NAGly turnover
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NAGly
Possible precursors
AEA
Arachidonic acid and glycine
Possible synthetic enzymes
Cytochrome c
alcohol dehydrogenase
(McCue, BBRC, 2008; Bradshaw, BMC Biochem, 2009)
FAAH (Bradshaw, BMC Biochem, 2009)
Hydrolysis
?

15. GPR18: Opportunistic actions of NAGly
NAGly effect
SLC6A5/GlyT2 transporters
Inhibition (Wiles, J Neurochem, 2006)
SLC8A/NCX sodium/calcium exchangers
Inhibition
(Bondarenko, BJP, 2013)
T-type voltage-gated calcium channels
Inhibition (Barbara, J Neurosci, 2009)
BKca potassium channels
Inhibition
(Parmar, BJP, 2010)
GABAA-b2
Positive allosteric modulator
(Baur, PeerJ, 2013)
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•Ineffective as an agonist at either CB1 or CB2 cannabinoid receptors
(Sheskin, J Med Chem, 1997; Huang, J Biol Chem, 2001)

16. GPR18: other ligands
• Cannabidiol
(weak partial agonist)
(McHugh, BJP, 2012)
• AM251
(very weak partial agonist)
(McHugh, BJP, 2012)
• N-Arachidonoylserine
(antagonist)
(Console-Bram, BJP, 2014)
CBD
AM251
NASer
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17. GPR18: A NAGly receptor or a CB3 cannabinoid receptor?
•NAGly as an agonist
(Kohno, BBRC, 2006; McHugh, BMC Neurosci, 2010; Takenouchi, BBRC, 2012; Console-Bram, BJP, 2014)
•THC as an agonist
–EC50 1 μM, ~1 μM
(McHugh, BJP, 2012; Console-Bram, BJP, 2014)
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18. GPR18: Agonist bias?
•HEK293/GPR18 cells
–Concentration-dependent increases in [Ca2+]i and ERK1/2 phosphorylation by
•NAGly, abn-CBD, O1602 and THC
•PathHunter® CHO-K1 GPR18 cells
–Only THC exhibited recruitment of β-arrestin
(Console-Bram, BJP, 2014)
•“The pairing of N-arachidonoylglycine with GPR18 was not replicated in two studies based on β-arrestin assays”
(Southern, J Biomol Screen, 2013; Yin, J Biol Chem, 2009)
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19. GPR18: Therapeutic potential
•NAGly levels altered in region-specific manner in female rats with mating behaviour
(Bradshaw, AJPRICP, 2006; Stuart, Int J Endocrinol, 2013)
•Agonists effective in models of:
–CNS and peripheral inflammation
(McHugh, BJP, 2012; Takenouchi, BBRC, 2012)
–Glaucoma
(Caldwell, BJP, 2013)
–RVLM regulation of blood pressure
(Penumarti, JPET, 2014)
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20. GPR119
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21. GPR119: Cloning and initial deorphanization
•Identified by mass screening methods
(Takeda, FEBS Letts, 2002)
•In recombinant expression:
–N-Oleoylethanolamine (OEA) as an agonist
–EC50 value of 3 μM for cAMP formation
(Overton, Cell Metab, 2006)
–OEA and 2-oleoylglycerol (2OG) as agonists
–EC50 values of 0.2 and 3 μM
(Hansen, JECM, 2011)
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22. GPR119: Endocannabinoid-like
molecules
2AG
AEA OEA
2OG
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23. GPR119: OEA, 2OG turnover
•Parallel and independent metabolism
•Identical to AEA and 2AG, except:
–Not substrates for COX-2, LOX activities
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24. GPR119: Opportunistic actions of OEA
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OEA effect
TRPV1
Agonist (Movahed, J Biol Chem, 2005)
PPARa
Agonist
(Fu, Nature, 2003; Sun, BJP, 2007)
PPARb/d
Agonist (Fu, Nature, 2003)
•Ineffective as an agonist at either CB1 or CB2 cannabinoid receptors
(Lin, J Med Chem, 1998)

25. PSN632408
GPR119: Other ligands
• PSN632408
(Agonist)
(Overton, Cell Metab, 2006)
• AR231453
(Agonist)
(Chu, Endocrinology, 2007)
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AR231453

26. GPR119: Therapeutic potential
•OEA levels altered in gut in response to fasting/feeding
(Fu, Nature, 2007)
•Agonists effective in models of:
–Satiety/feeding
(Overton, Cell Metab, 2006)
–Type 2 diabetes
(Chu, Endocrinology, 2007; Brocklehurst, BMCL, 2011; Semple, BMCL, 2011; Xia, BMCL, 2011; Sakairi, BMCL, 2012; Kim, J Diabetes Res, 2013; Alper, BMCL, 2014; Wang, BMCL, 2014)
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27. GPR55
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28. GPR55: Cloning and initial deorphanization
•Cloned from a human brain cDNA library
(Sawzdargo, Mol Brain Res, 1999)
•In 2007, three papers gave contrasting pharmacology
–2AG, PEA, AEA, THC, AM251, O1602, abn-CBD as agonists
–CBD as antagonist
(Ryberg, BJP, 2007)
–O1602, abn-CBD as agonists
–2AG, PEA, AEA, THC, AM251, CBD untested
(Johns, BJP, 2007)
–2AG, PEA, AEA, THC, abn-CBD ineffective
–Lysophosphatidylinositol as agonist
(Oka, BBRC, 2007)
•Suggested to couple via G12/13
(Ryberg, BJP, 2007)
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29. GPR55: Endocannabinoid-like
molecules
2AG
AEA
2AGPI (LPI)
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30. GPR55: LPI turnover
LPI
Possible precursor
Phosphatidylinositol
Possible synthetic enzymes
PLA2
DDHD1
Possible hydrolytic products
Lysophosphatidic acid and inositol
2-Acylglycerol and inositol monophosphate
Possible hydrolytic enzymes
Lysophospholipase D Lysophospholipase C
Possible acylation product
Phosphatidylinositol
Possible acylation enzyme
LPI:acyltransferase
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31. GPR55: Opportunistic actions of LPI
LPI effects
Intracellular calcium release
Stimulation (Baran, Endocrinology, 1988)
IKCa potassium channels
Activation (Bondarenko, Pflugers Archiv, 2011)
BKCa potassium channels
Bidirectional modulation (Bondarenko, Pflugers Archiv, 2011)
TRPM8 channels
Activation
(Andersson, J Neurosci, 2007)
TRPV2 channels
Activation
(Monet, BBA, 2009)
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32. GPR55: pharmacology
• AM251
(agonist)
(Ryberg, BJP, 2007)
• Cannabidiol
(weak partial agonist)
(McHugh, BJP, 2012)
• CID16020046
(antagonist)
(Heynen-Genel, NIH Probes, 2010;
Kargl, JPET, 2013; Console-Bram, BJP, 2014)
CBD
AM251
CID16020046
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33. GPR55: Therapeutic potential
•Circulating plasma LPI elevated in obesity
(Moreno-Navarrete, Diabetes, 2012)
•SNPs associated with anorexia nervosa
(Ishiguro, Synapse, 2011)
•Agonists effective in models of:
–Bone turnover
(Whyte, PNAS, 2009)
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34. CONCLUDING REMARKS
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35. Further complications
•CB1:GPR55 heteromers in the striatum
(Martinez-Pinilla, Exp Neurol, 2014)
•CB2:GPR55 heteromers in cancer cells
(Moreno, J Biol Chem, 2014)
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36. Conclusions
•GPR55, GPR18 and GPR119
–“Interesting” (overlapping) pharmacology
–Therapeutic potential
•Cannabinoid receptors or cannabinoid receptor- related receptors?
–Should they remain orphans?
–The cannabinoid receptor community treat them as foster children
–At least until further research allows a more definitive decision to be made
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37. Endocannabinoid-like molecules
CB1/2: 2AG
CB1/2: AEA
GPR55: 2AGPI (LPI)
GPR119: OEA
GPR119: 2OG
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GPR18: NAGly