wcp2014 track 6 alexander v6

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?

Plan

• Cannabinoid receptors

• Cannabinoid receptor-related receptors

– GPR18, GPR119, GPR55

– Pharmacology

• Endogenous ligands – Opportunistic (off-target) actions

• Synthetic ligands

– Therapeutic potential

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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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CANNABINOID RECEPTORS

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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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Endogenous cannabinoids

Anandamide, AEA Isolated from pig brain Devane, Science, 1992

2-Arachidonoylglycerol, 2AG Isolated from dog gut Mechoulam, Biochem Pharmacol, 1995

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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

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

‘Selective’ antagonists

CB1: AM251 CB2: SR144528

Identified in the 1990s, both have been described as ‘inverse agonists’

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Primary sequence alignment

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GPR18

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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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GPR18: Endocannabinoid-like molecules

2AG

AEA

NAGly

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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 ?

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)

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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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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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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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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GPR119

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• 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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2AG

AEA OEA

2OG

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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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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)

PSN632408

GPR119: Other ligands

• PSN632408 (Agonist) (Overton, Cell Metab, 2006)

• AR231453 (Agonist) (Chu, Endocrinology, 2007)

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AR231453


• 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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GPR55

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• 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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2AG

AEA

2AGPI (LPI)

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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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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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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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• 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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CONCLUDING REMARKS

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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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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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Endocannabinoid-like molecules

CB1/2: 2AG

CB1/2: AEA

GPR55: 2AGPI (LPI)

GPR119: OEA

GPR119: 2OG

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GPR18: NAGly