Purification and characterization of anandamide amidase and development of novel inhibitors

Date of Completion

January 1998

Keywords

Chemistry, Analytical|Chemistry, Biochemistry|Chemistry, Pharmaceutical

Degree

Ph.D.

Abstract

Cannabinoid drugs have a range of therapeutically useful properties. The target areas include analgesics, antiglaucoma agents, antiemetic and antispasmodic agents. However, cannabinoids are plagued by their pyschoactive properties. Anandamide is an endogenous CB1 receptor ligand. Anandamide and its analogs can produce the therapeutically beneficial effects of cannabimimetics without the psychoactive side effects. Modulation of anandamide amidase is one potential pathway for exploiting cannabinoid effects of anandamide analogs.^ In this project, two new, non-radioactive, sensitive assays were developed for measuring the enzyme activity. Using these methods, substrate specificity for anandamide amidase was investigated. It was found that the interaction between the head group of anandamide and the enzyme was probably through hydrogen binding. $(R)$-$\alpha$-$(R)$-$1\sp\prime$-methyl-substitution of anandamide was found to be the best anandamide analog in terms of high enzymatic stability and high binding affinity for cannabinoid receptor. Reversible (arachidonyl aldehyde) and irreversible (sulfonyl flourides) inhibitors of the enzyme were also studied. These inhibitors can provide useful tools for the future investigation on the enzyme active site and catalytic mechanism of the enzyme. Moreover, this enzyme was purified by an immobilized-ligand affinity column and the purified enzyme was identified and characterized by 2D gel electrophoresis and enzyme activity assays. The purification of the enzyme can facilitate the future structural and kinetic studies. ^

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