ON-LINE CHARACTERIZATION OF CHOLINESTERASE INHIBITORS BY IMMOBILIZED ENZYME REACTORS

In the search for new therapeutics for Alzheimer’s Disease (AD), the investigation of the mechanism of action of new cholinesterase inhibitors represents a key aspect for new lead selection and subsequent optimization and development. However, the in vitro characterization of drug candidates on isolated target enzymes often requires a long operating time and a large amount of disposable expensive material. The recent development and aplication of stable immobilized enzyme reactors (IMERs) can ameliorate these concerns, and such in-solution methods should be potentially utilized in automated procedures to obtain efficient and effective higher throughput screening. In this context, the focus of this work was the development and characterization of a new IMER containing immobilized human recombinant butyrylcholinesterase (BChE) for the on-line kinetic characterization of specific, pseudo-irreversible and brain-targeted BChE inhibitors as potential drug candidates for AD. The selected phenserine and cymserine analogues were synthesized, characterized by classical ex vivo binding assays [1], and assessed by BChE-IMER. For a pseudo-irreversible inhibitor, the kinetics constants describe the mode and duration of enzyme inhibition that will, in turn, influence the duration of the inhibitors’s in vivo pharmacological actions. The kinetic constants were determined for selected phenserine and cymserine analogues using a purposely-designed on-line procedure. Specifically, agents were inserted on to the BChE-IMER that fed directly into a HPLC system connected to a UV-Vis detector, BChE-IMER activity was determined on the basis of the Ellman reaction [2]. The carbamoylation and decarbamoylation phases at different inhibitor concentrations were then monitored continuously over time. Results allowed elucidation of the inhibition duration, mode of action and structure-activity relationships of the inhibitors of interest, which were compared to available values deriving from classical assessment. (1) N.H. Greig, T. Utsuki, D.K. Ingram, Y. Wang, G. Pepeu, C. Scali, Q.S. Yu, I. Mamczarz, H.W. Holloway, T. Giordano, D. Chen, K. Furukawa, K. Sambamurti, A. Brossi, D.K. Lahiri: Selective butyrylcholinesterase inhibition elevates brain acetylcholine, augments learning and lowers Alzheimer beta-amyloid peptide in rodent. Proc Natl Acad Sci USA 2005, 102: 17213-18. (2) G.L. Ellman, K.D. Courtney, V. Jr. Andres, R.M. Featherstone. A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem Pharmacol 1961, 7: 88-95.