The development and characterization of a human recombinant acetylcholinesterase (AChE) micro-IMER (immobilized enzyme reactor), prepared by using an in situ immobilization procedure was previously reported (1). 0.22 Units of AChE were immobilized onto a monolithic disk (12 mm in diameter and 3 mm in thickness) based on polymeric macroporous material and available under the trademark CIMÒ (Convective Interaction MediaÒ). Well-known inhibitors whose potency was distributed over four orders of magnitude were analyzed by using the AChE-IMER. Very short analysis time (2 min), absence of backpressure, low aspecific matrix interactions and immediate recovery of enzyme activity were the best characteristic of this micro-IMER. In this study the micro reactor was used to evaluate the inhibitory potency of a class of new synthesized inhibitors characterized by a polyamine backbone. The IC50 were assessed by injecting increasing concentrations of the tested inhibitor and saturating concentration of substrate. The reduction of the product area was correlated to the inhibition of the enzymatic activity by the tested inhibitor. Inhibition curves were obtained for each compound by plotting the % inhibition versus the logarithm of injected inhibitor concentration. The IC50 values obtained by micro-IMER were compared with the IC50 values calculated on the free enzyme by the Ellman’s assay and a linear correlation was found (r2 = 0.9984). The results indicate that the AChE-micro-IMER could be used for high throughput screening for new classes of inhibitors in the search for new leads. If we consider that the development of new inhibitors require a large number of compounds to be tested, the short time of analysis and very low aspecific interaction showed by this micro-IMER will allow to rapidly process hundreds of potential inhibitors.
Evaluation of the inhibitory potency of a new series of AChE-inhibitors by using a monolithic AChE-micro-IMER
BARTOLINI, MANUELA;TUMIATTI, VINCENZO;ANDRISANO, VINCENZA
2004
Abstract
The development and characterization of a human recombinant acetylcholinesterase (AChE) micro-IMER (immobilized enzyme reactor), prepared by using an in situ immobilization procedure was previously reported (1). 0.22 Units of AChE were immobilized onto a monolithic disk (12 mm in diameter and 3 mm in thickness) based on polymeric macroporous material and available under the trademark CIMÒ (Convective Interaction MediaÒ). Well-known inhibitors whose potency was distributed over four orders of magnitude were analyzed by using the AChE-IMER. Very short analysis time (2 min), absence of backpressure, low aspecific matrix interactions and immediate recovery of enzyme activity were the best characteristic of this micro-IMER. In this study the micro reactor was used to evaluate the inhibitory potency of a class of new synthesized inhibitors characterized by a polyamine backbone. The IC50 were assessed by injecting increasing concentrations of the tested inhibitor and saturating concentration of substrate. The reduction of the product area was correlated to the inhibition of the enzymatic activity by the tested inhibitor. Inhibition curves were obtained for each compound by plotting the % inhibition versus the logarithm of injected inhibitor concentration. The IC50 values obtained by micro-IMER were compared with the IC50 values calculated on the free enzyme by the Ellman’s assay and a linear correlation was found (r2 = 0.9984). The results indicate that the AChE-micro-IMER could be used for high throughput screening for new classes of inhibitors in the search for new leads. If we consider that the development of new inhibitors require a large number of compounds to be tested, the short time of analysis and very low aspecific interaction showed by this micro-IMER will allow to rapidly process hundreds of potential inhibitors.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
