Fast, accurate and reproducible analyses are required in new lead selection and drug discovery. Lately, immobilization of target proteins and their insertion in flow-through systems have been quite spread out, as ligand high throughput screening methodologies [1,2]. However, in a second stage following the screening step, selected hits need to be further characterized in terms of mechanism of action and kinetic parameters. In this context, the understanding of selected potent cholinesterase (ChE) inhibitors mechanism of action is a key information to rationally design new compounds, as drug candidates for the treatment of Alzheimer’s disease. In particular, potent reversible mixed mode AChE inhibitors are searched in view of the role of AChE peripheral binding site in inducing beta-amyloid aggregation and senile plaques formation. Besides representing a valid tool to screen new inhibitors (IC50 or single dose inhibition) [3], ChE immobilized reactors (ChE-IMERs) are now used to characterize both reversible and pseudo-irreversible inhibitors, by on-line HPLC automated assays. To this purpose, cholinesterase immobilized reactors (ChE-IMERs) were validated for the determination of the mechanism of action and inhibitory constants of new leads in a highly reliable and automated mode. After the approval in 2000 of Rivastigmine, carbamates regained interest for the treatment of Alzheimer’s disease. In this view, the online assay for the characterization of carbamoylation and decarbamoylation steps of pseudo-irreversible ChE inhibitors has been developed. ChE-IMER inhibition and regeneration are followed in a single experiment in a continuous mode. By the application of this methodology, new leads as the prototype of new classes of compounds for AD treatment were discovered. [1] I.W. Wainer, R. Kaliszan, T.A. Noctor, J. Pharm. Pharmacol. 45 (1993) 367. [2] R.J. Hodgson, T.R. Besanger, M.A. Brook, J.D. Brennan, Anal. Chem. 77 (2005) 7512. [3] M. Bartolini, V. Cavrini, V. Andrisano, J. Chromatogr. A 1031 (2004) 27. Acknowledgments: The research was supported by grants (FIRB and PRIN) from MIUR (Rome, Italy)
Characterization of Cholinesterase inhibitors by means of immobilized enzyme reactor / M. Bartolini; M.L. Bolognesi; A. Cavalli; C. Melchiorre; A. Rampa; M. Recanatini; V. Andrisano. - STAMPA. - (2007), pp. 69-69. (Intervento presentato al convegno IXth International Meeting on Cholinesterases tenutosi a Suzhou, China nel 6-10 Maggio 2007).
Characterization of Cholinesterase inhibitors by means of immobilized enzyme reactor
BARTOLINI, MANUELA;BOLOGNESI, MARIA LAURA;CAVALLI, ANDREA;MELCHIORRE, CARLO;RAMPA, ANGELA;RECANATINI, MAURIZIO;ANDRISANO, VINCENZA
2007
Abstract
Fast, accurate and reproducible analyses are required in new lead selection and drug discovery. Lately, immobilization of target proteins and their insertion in flow-through systems have been quite spread out, as ligand high throughput screening methodologies [1,2]. However, in a second stage following the screening step, selected hits need to be further characterized in terms of mechanism of action and kinetic parameters. In this context, the understanding of selected potent cholinesterase (ChE) inhibitors mechanism of action is a key information to rationally design new compounds, as drug candidates for the treatment of Alzheimer’s disease. In particular, potent reversible mixed mode AChE inhibitors are searched in view of the role of AChE peripheral binding site in inducing beta-amyloid aggregation and senile plaques formation. Besides representing a valid tool to screen new inhibitors (IC50 or single dose inhibition) [3], ChE immobilized reactors (ChE-IMERs) are now used to characterize both reversible and pseudo-irreversible inhibitors, by on-line HPLC automated assays. To this purpose, cholinesterase immobilized reactors (ChE-IMERs) were validated for the determination of the mechanism of action and inhibitory constants of new leads in a highly reliable and automated mode. After the approval in 2000 of Rivastigmine, carbamates regained interest for the treatment of Alzheimer’s disease. In this view, the online assay for the characterization of carbamoylation and decarbamoylation steps of pseudo-irreversible ChE inhibitors has been developed. ChE-IMER inhibition and regeneration are followed in a single experiment in a continuous mode. By the application of this methodology, new leads as the prototype of new classes of compounds for AD treatment were discovered. [1] I.W. Wainer, R. Kaliszan, T.A. Noctor, J. Pharm. Pharmacol. 45 (1993) 367. [2] R.J. Hodgson, T.R. Besanger, M.A. Brook, J.D. Brennan, Anal. Chem. 77 (2005) 7512. [3] M. Bartolini, V. Cavrini, V. Andrisano, J. Chromatogr. A 1031 (2004) 27. Acknowledgments: The research was supported by grants (FIRB and PRIN) from MIUR (Rome, Italy)I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
