Bijlage

Bijlage 3 Onderzoeksvoorstel

Research proposal “Molecular mechanisms of CB1 cannabinoid receptor activation;
studies using the novel peptide antagonist, hemopressin”.

Introduction
The best-known ligands for the cannabinoid receptor are cannabinoid drugs derived from Cannabis sativa such as Δ9-tetrahydrocannabinol (Δ9-THC, Figure 1). At present two cannabinoid receptors have been identified, named CB1 and CB2 cannabinoid receptor. Both belong to the superfamily of G protein-coupled receptors of the Gi/0 type. Activation of the receptor leads to an inhibitory response, thereby inhibiting adenylyl cyclase and activating MAP kinases. The CB1 cannabinoid receptor is primarily found in brain tissue and the CB2 cannabinoid receptor in immune cells. In recent years endogenous ligands for the cannabinoid receptors have been identified, all of which are metabolites of arachidonic acid. The prototypical ligand is arachidonylethanolamide, anandamine for short. The first antagonist that was reported for the CB1 Figure 1 Structure of Δ9-
receptor was Rimonabant (SR141716). One of the major tetrahydrocannabinol. problems with drugs of abuse is craving. Rimonabant can block craving for food and drugs. Therefore it is potentially an interesting therapeutic in obesity, smoking cessation and alcohol abuse and currently clinical trials are conducted to establish this. The endogenous ligands and the cannabinoid receptors form the endocannabinoid system, which is thought to play an important role in many physiological and pathophysiological processes. Therefore a lot of research is done to gather insight in this system, as to broaden the therapeutic possibilities for its manipulation. The endocannabinoid system is involved in a number of diseases, like Alzheimer’s disease, Parkinson’s disease, major depression, inflammation, neuropathic pain and obesity. The development of drugs based on compounds that modulate the cannabinoid receptors could be a step towards a better treatment of these diseases.1,2,3
Hemopressin as a novel inverse agonist for CB1 cannabinoid receptor
Recent studies by L.A.Devi and co-workers have resulted in the discovery of a novel ligand for the cannabinoid CB1 receptor. In contrast to all known ligands for this receptor this ligand is a peptide, hemopressin, consisting of nine amino acids: PVNFKFLSH. (Figure 2) Hemopressin is a derivative of the α1-chain of haemoglobin and it has been identified as a ligand for CB1 cannabinoid receptors in a screening assay with conformation-state sensitive antibodies.4,5 17 peptides were tested that were isolated from rodent tissue and only hemopressin was found to modulate antibody binding. This result was used to explore the selectivity and potency of hemopressin. Using the secreted alkaline phosphatase (SeAP) assay, which is used to indirectly measure the level of intracellular cAMP, it was found that hemopressin acts as an antagonist with high selectivity for the CB1 receptor. Displacement studies using [3H]SR141716 showed that hemopressin binds with subnanomolar affinity, which indicates that it binds to the same extent as Rimonabant. Tests were done both on striatal membranes that are known to contain a relatively pure population of CB1 receptors and on heterologously expressed recombinant CB1 receptors. In this way it was made sure that the effects were indeed caused by binding to the CB1 receptor. Studies comparing the selectivity of hemopressin for CB1 and CB2 receptors showed that this ligand is capable of blocking the constitutive activity of CB1 but not of CB2 cannabinoid receptors. Besides, hemopressin decreases neuritogenesis in Neuro 2A cells in the absence of agonist. Normally, activation of the CB1 cannabinoid receptor with an agonist results in neurite outgrowth in those cells. The fact that hemopressin is able to decrease this process to an extent where also the constitutive activity is lost indicates that hemopressin acts as an inverse agonist. Figure 2 Structure of Hemopressin: PVNFKFLSH
Lately, there has been more interest in the potential role of the cannabinoid system in pain perception. Cannabinoid receptors are distributed throughout regions that are involved in nociceptive processing. In order to look at the antinociceptive properties of hemopressin, in vivo tests were done on male Wistar rats. The nociceptive threshold was measured by the paw pressure test. Pain was induced by administration of carrageenan or acetic acid. It was found that in both cases hemopressin had an antinociceptive effect, when administered intraplantar, intrathecally and orally. Tests also showed that hemopressin did not alter pentobarbital induced sleeping time or motor activity, thereby excluding these influences on pain perception. So hemopressin has shown to be an inverse agonist with high selectivity for the CB1 cannabinoid receptor. Furthermore, it has antinociceptive properties, independent of the route of administration.5

Goal of the present study - Structure activity relationship (SAR) studies
Devi et al have performed some tests in order to know more about the structure activity relationship of hemopressin. It has been found that the N-terminal seven amino acids are highly conserved among species, thereby suggesting that hemopressin has a conserved biological function. Furthermore, peptides were tested that were hemopressin derivatives with truncated C-termini and this resulted in the loss of CB1 receptor recognition only when 5 amino acids were deleted. So it can be concluded that five N-terminal amino acids are required for receptor recognition.5 However, it is very interesting to know which amino acids in the sequence are essential for receptor recognition and antagonism. The goal of the current project is to gain more insight in the structure activity relationship. Hopefully this will lead to the identification of a stable hemopressin analogue, which can be used as an orally active antinociceptive drug. A dozen of peptide analogues and peptidomimetic compounds are going to be tested during this internship. They will be tested in the various assays (ligand binding, GTPγS binding, adenylate cyclase activity, phosporylation of MAP kinase, calcium release and neurite extension) that are available in the research facilities of Prof. dr. Devi at Mount Sinai School of Medicine (New York, USA). The internship has a duration of 6 months, from May 1st 2008, till References
1. Howlett, A.C., Barth, F., Bonner, T.I., Cabral, G., Casellas, P., Devane, W.A., Felder, C.C., Herkenham, M., Mackie, K., Martin, B.R., Mechoulam, R., Pertwee, R.G., International Union of Pharmacology. XXVII. Classification of Cannabinoid Receptors, Pharmacological Reviews, 54, 161-202, 2002. 2. Howlett, A.C., Breivogel, C.S., Childers, S.R., Deadwyler, S.A., Hampson, R.E., Porrino, L.J., Cannabinoid physiology and pharmacology: 30 years of progress, Neuropharmacology, 47, 3. Mackie, K., Cannabinoid receptors as therapeutic targets, Annu. Rev. Pharmacol. Toxicol., 46, 4. Gupta, A., Décaillot, F.M., Gomes, I., Tkalych, O., Heimann, A.S., Ferro, E.S., Devi, L.A., Conformation state-sensitive antibodies to G-protein-coupled receptors, Journal of biological 5. Heimann, A.S., Gomes, I., Dale, C.S., Pagano, R.L., Gupta, A., de Souza, L.L., Luchessi, A.D., Castro, L.M., Giorgi, R., Rioli, V., Ferro, E.S., Devi, L.A., Hemopressin is a novel inverse agonist of CB1 cannabinoid receptors, accepted for publication in PNAS.

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