Tamema Choudhury Vincent Li David McNee

NEW CANNABINOID THERAPEUTICS

The endocannabinoid system

• Established cannabinoid receptors: CB1 and CB2.

• Other orphan receptors (e.g. GPR55) as potential targets.

• Endogenous ligands include Anandamide (AEA) and 2-arachidonoyl glycerol (2-AG).

• Variety of potential clinical uses including pain management, obesity and schizophrenia.

Orthosteric site

Pain & inflammation Multiple sclerosis

Suicide & depression

Euphoria, Memory loss, Hallucination

ORTHOSTERIC BLOCKERS

ORTHOSTERIC ACTIVATORS

Obesity & metabolic syndrome Nicotine addiction Mental illness Drug abuse

CB1 Receptor

Signals which control neuronal

excitability

α β γ

Endocannabinoids • Anandamide • 2-AG

Phytocannabinoids • Δ9THC

Synthetic small molecules • Agonists • Inverse Agonists

Orthosteric binding site

CB1 Receptor

Signals which control neuronal

excitability

Pain & inflammation Depression

α β γ

Endocannabinoids

Orthosteric binding site FAAH INHIBITORS

FAAH Inactive Metabolites

Fatty acid amino hydrolase (FAAH)

No side effects?

Effe

ct

Effe

ct

Time

Endogenous Agonist

Time

Allosteric inhibitor

Allosteric site

CB1 Receptor

Signals which control neuronal

excitability

α β γ

Orthosteric binding site

allosteric Novel small molecules

Endocannabinoids • Anandamide • 2AG

GTPγS

Method • Mouse Brain Membrane Preparation

• Homogenised and centrifuged several times with different speeds at 4°C. • Protein concentration determined using spectrophotometry.

• [35S]GTPγS Functional Assay • Measures the activity of G-protein

through the accumulation of membrane-bound Gα[35S]GTPγS.

• Beta particles emitted are detected with a liquid scintillation counter.

• Equilibrium Binding Assay (Affinity) • Measures displacement of [3H]CP55940 at the CB1 receptors orthosteric site. • Beta particles emitted are detected with a liquid scintillation counter.

α β γ

GDP

GPCR

Agonist

Results – JK263-2

-10 -9 -8 -7 -6 -5 -4

-20

0

20

40

60

80

100 DMSO1µm JK263-2

CP55940 log concentration (M)

% S

timul

atio

n [35

S]G

TPγS

Bin

ding

-11 -10 -9 -8 -7 -6 -5 -4

-100

-80

-60

-40

-20

0

20

40

60

80

100

120JK-263-2CP55940

log concentration (M)

% d

ispl

acem

ent o

f [3 H

]CP5

5940

-10 -9 -8 -7 -6 -5 -4

-20

0

20

40

60

80

100

120

140 DMSO100nM JK_263-2

AEA log concentration (M)

% S

timul

atio

n [35

S]G

TPγS

Bin

ding

Results – JK263-2 (cont.)

Results ORG-27569

-10 -9 -8 -7 -6 -5 -4

-20

0

20

40

60 DMSO1µm ORG27569

CP55940 log concentration (M)

% S

timul

atio

n [35

S]G

TPγS

Bin

ding

-10 -9 -8 -7 -6 -5 -4

-100

-80

-60

-40

-20

0

20

40

60

80

100

120ORG27569CP55940

log concentration (M)

% d

ispl

acem

ent o

f [3 H

]CP5

5940

Key findings • JK263-2: Increases both affinity and efficacy. Potential allosteric enhancer.

• ORG-27569: Increases affinity whilst inhibiting efficacy. Potential allosteric inhibitor.

Summary

• Cannabinoid compounds are an important area of pharmacological discovery. The compounds tested may be worthy of further investigation to produce novel clinical therapeutics. • JK263-2 as a treatment for chronic pain. • ORG27569 to assist combating obesity.

• Special thanks to Gemma Baillie, Professor Ross and others in the Cannabinoid Group.