FAST vs IN-DEPTH CHARACTERIZATION OF NEW CHOLINESTERASES INHIBITORS BY IMMOBILIZED ENZYME REACTORS

The cholinergic enzymes acetyl- and butyrylcholinesterase (AChE and BuChE, respectively) represent well-known targets for Alzheimer’s Disease (AD) treatment. In particular, the role of AChE on amyloid misfolding and aggregation has been further investigated by transgenic mice overexpressing human acetylcholinesterase and the Swedish amyloid precursor protein mutation [1]. In the search for new cholinesterase inhibitors, high throughput screening techniques are required for hit identification in the early drug discovery process. In this context, immobilized enzyme reactors (IMERs) containing key target enzymes may fulfill pharma industry requirements. Fast and universal screening procedures for natural compounds or new synthetic products would reduce the number of false negative responses and allow a reliable selection of new potential hits. In this work, monolithic immobilized enzyme reactors (IMERs) containing covalently bound AChE or BuChE have been developed, characterized after insertion into HPLC systems, and applied to the screening of known and new inhibitors [2,3]. A specific chromogenic reagent was used to follow product formation and to determine both activity and inhibition. Since screening purpose might differ along drug discovery process and be either focused on fast data output (low information content per compound) or on a deeper investigation (high information content per compound), different operative procedures have been optimized to allow either (i) fast evaluation of different classes of new chemical entities or (ii) a better characterization of the selective active ones, i.e., mechamism of action of reversible and pseudo-irreversible inhibitors [3,4]. Besides, to reduce false negative results, particular attention has been devoted to non specific interaction managment. Even if each IMER has its own requirements for optimal performances, both AChE- and BuChE-IMER showed an increased data output, reliability and stability, which directly translate into cost reduction. Moreover, in the optimized conditions, fast screening of compounds with different lipophilicity could be performed. 1] Hedberg MM, Svedberg MM, Mustafiz T, Yu W-F, Mousavi M, Guan Z-Z, Unger C, Nordberg A. Neuroscience, 2008, 152, 223-233 [2] Bartolini M, Cavrini V, Andrisano V. J. Chromatogr. A, 2005, 1065: 135-144 [3] Bartolini M, Greig NH, Yu QS., Andrisano V. J. Chromatogr. A, 2009, 1216: 2730-2738 [4] Bartolini M, Cavrini V, Andrisano V. J. Chromatogr. A. 2007, 1144: 102-110