CIM-Immobilized Butyrylcholinesterase in the characterization of new inhibitors that could ease Alzheimer’s Disease

Alzheimer’s Disease (AD) is associated with a progressive decline in the neurotransmitter acetylcholine (ACh) in the forebrain of afflicted subjects. In healthy human brain, butyrylcholinesterase (BChE, EC 3.1.1.8) has prevalently a supportive role in the hydrolysis of ACh, whereas acetylcholinesterase (AChE) activity predominates. Nevertheless in AD brain, since AChE levels rapidly decrease, the role of BChE rises. It has been demostrated that selectively inhibiting the action of BChE in the brain results in an increase of cerebral acetylcholine levels and improves learning in rats (1). These findings support BChE as key target in AD and may translate into new therapeutics for AD. The goal of the present work is the development and characterization of a new enzyme reactor containing immobilized human recombinant BChE (hrBChE) for the on-line evaluation of the kinetics of new specific, pseudo-irreversible and brain-targeted BChE inhibitors developed by N. Greig and coworkers. For this purpose, human recombinant BChE was covalently immobilized onto an EDA CIM® disk and on-line studies were performed by inserting the IMER into a HPLC system. A specific chromogenic reagent coupled with UV detection was used to follow product formation and to determine both activity and inhibition. Carbamoylation and decarbamoylation constants were determined for a series of physostigmine analogues. Results allowed us to elucidate inhibition duration and structure activity relationships. Moreover, both kinetic constants could be determined in a single experiment. (1)Nigel H. Greig et al. PNAS 2005, 102:17213-8