This program project application represents a comprehensive collaborative effort the ultimate goal of which is to develop novel drug analogs which produce their therapeutic effects by acting on the newly discovered cannabinoid receptor(s). A central hypothesis of this program is that the recent availability of such receptor(s) offers the opportunity to rationally design analogs with a high degree of selectivity for inducing certain actions of cannabinoids including analgesia, inhibition of vomiting and reduction of intraocular pressure without their undesirable psychoactive effects. Similarly, there will be an opportunity for developing novel ligands which can successfully block the actions of cannabinoids. Such a process will require detailed knowledge of the molecular, biochemical and anatomical features of this receptor and its subtypes which are associated with cannabinoid activity. The receptor active site(s) could thus be used as template(s) for the successful design of these novel analogs. Obtaining intimate knowledge of the receptor's structure and function will require an interdisciplinary approach which could be accomplished through concerted collaborative efforts between several laboratories with a commitment for cannabinoid research and/or the high degree of technical expertise required for an effective approach to this problem. Strong collaborative interactions will be emphasized with the following major specific aims: (1) the development of high affinity ligands for the receptor(s) which will be used for obtaining molecular information on the cannabinoid site(s) of action. The group of ligands to be developed will encompass all four classes of molecules which are associated with cannabimimetic activity including classical cannabinoids (CCs), non- classical cannabinoids (NCCs), aminoalkylindoles (AAIs) and arachidonic acid amides (AAAs). (2) The expression isolation, purification and reconstitution of the cannabinoid receptor(s) and its mutants in viruses and bacteria. (3) Studying the conformational properties of the novel ligands in solution and in the membrane. (4) Obtaining direct information on the structure of the receptor active site(s) with the help of high affinity covalent receptor ligands and by determining the amino acid residues with which the ligands reacted. (5) The testing of the novel analogs on the cannabinoid receptor/G/i protein/adenylate cyclase system, in isolated tissue (mouse vas deferens, guinea pig ileum), and the intact animal (hypokinesia, antinociception, ring test, hypothermia).
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