Human acetylcholinesterase (AChE) is a widely studied target enzyme in the development of therapeutic agents for the treatment of Alzheimer’s disease (AD). AChE inhibitors have been for a long time the only drugs available on the market for the symptomatic treatment of this widespread pathology and still represent 75% (3 out of 4) of marketed drugs. The interest on AChE inhibitors has evolved in last two decades after AChE’s peripheral binding site (PAS) was hypothesised to promote the deposition of the neurotoxic -amyloid peptide (A).1 Therefore, in the light of this non cholinergic activity, new AChE inhibitors, able to prevent the interaction between AChE’s PAS and A, have been designed and investigated. Miniaturization and automation of the screening system will greatly implement the drug discovery process for AD, in which a large number of new chemical entities needs to be screened for affinity towards a specific target, such as AChE. On the light of these premises, in this talk, two different approaches to the development of screening tools for the investigation of compounds able to bind to the CAS (catalytic anionic site) and PAS of human AChE will be presented. In particular, first, the development and application of AChE-based enzyme reactors for the automated and rapid screening of inhibitors at the CAS will be described. These bioreactors have been obtained by the covalent immobilization of the target enzyme on a suitable monolithic chromatographic material, inserted into a HPLC system and used for the evaluation of the inhibitory activity and mechanism of action of new ChE inhibitors.2, 3 Then, the initial development of a new AChE-based fluorescence sensing surface for the identification of PAS binders through propidium displacement studies will be presented. The initial selection of the suitable multilayered material, and of the optimal pattern thickness required to maximize fluorescence signal and maintain chemical stability will be also discussed. Then, the selective immobilization of recombinant human AChE on the SiO2 architectures with optimal geometry and chemistry will be shown. Thanks to the combined use of atomic force microscopy and CLSM it was demonstrated that the enzyme distribution selectively matched with the initial SiO2 features (independently from their shapes and dimensions). In the optimal design, the AChE-based biosensing surface showed an efficient fluorescence emission after labelling with propidium, a selective fluorescent probe of the peripheral binding site of the AChE.4 1. Inestrosa, N. C.; Alvarez, A.; Perez, C. A.; Moreno, R. D.; Vicente, M.; Linker, C.; Casanueva, O. I.; Soto, C.; Garrido, J. Neuron 1996, 16, 881-91. 2. Bartolini, M.; Cavrini, V.; Andrisano, V. J Chromatogr A 2007, 1144, 102-10. 3. Bartolini, M.; Greig, N. H.; Yu, Q. S.; Andrisano, V. J Chromatogr A 2009, 1216, 2730-8. 4. Bartolini, M.; Naldi, M.; Nicolau, D. V.; van Delft, F. C.; van Zijl, J.; Snijder, J.; van den Heuvel, E. F.; Naburgh, E. P.; Calonghi, N.; Andrisano, V. Anal Bioanal Chem 2012.

ACETYLCHOLINESTERASE-BASED ANALYTICAL TOOLS FOR DRUG SCREENING

BARTOLINI, MANUELA;NALDI, MARINA;ANDRISANO, VINCENZA
2012

Abstract

Human acetylcholinesterase (AChE) is a widely studied target enzyme in the development of therapeutic agents for the treatment of Alzheimer’s disease (AD). AChE inhibitors have been for a long time the only drugs available on the market for the symptomatic treatment of this widespread pathology and still represent 75% (3 out of 4) of marketed drugs. The interest on AChE inhibitors has evolved in last two decades after AChE’s peripheral binding site (PAS) was hypothesised to promote the deposition of the neurotoxic -amyloid peptide (A).1 Therefore, in the light of this non cholinergic activity, new AChE inhibitors, able to prevent the interaction between AChE’s PAS and A, have been designed and investigated. Miniaturization and automation of the screening system will greatly implement the drug discovery process for AD, in which a large number of new chemical entities needs to be screened for affinity towards a specific target, such as AChE. On the light of these premises, in this talk, two different approaches to the development of screening tools for the investigation of compounds able to bind to the CAS (catalytic anionic site) and PAS of human AChE will be presented. In particular, first, the development and application of AChE-based enzyme reactors for the automated and rapid screening of inhibitors at the CAS will be described. These bioreactors have been obtained by the covalent immobilization of the target enzyme on a suitable monolithic chromatographic material, inserted into a HPLC system and used for the evaluation of the inhibitory activity and mechanism of action of new ChE inhibitors.2, 3 Then, the initial development of a new AChE-based fluorescence sensing surface for the identification of PAS binders through propidium displacement studies will be presented. The initial selection of the suitable multilayered material, and of the optimal pattern thickness required to maximize fluorescence signal and maintain chemical stability will be also discussed. Then, the selective immobilization of recombinant human AChE on the SiO2 architectures with optimal geometry and chemistry will be shown. Thanks to the combined use of atomic force microscopy and CLSM it was demonstrated that the enzyme distribution selectively matched with the initial SiO2 features (independently from their shapes and dimensions). In the optimal design, the AChE-based biosensing surface showed an efficient fluorescence emission after labelling with propidium, a selective fluorescent probe of the peripheral binding site of the AChE.4 1. Inestrosa, N. C.; Alvarez, A.; Perez, C. A.; Moreno, R. D.; Vicente, M.; Linker, C.; Casanueva, O. I.; Soto, C.; Garrido, J. Neuron 1996, 16, 881-91. 2. Bartolini, M.; Cavrini, V.; Andrisano, V. J Chromatogr A 2007, 1144, 102-10. 3. Bartolini, M.; Greig, N. H.; Yu, Q. S.; Andrisano, V. J Chromatogr A 2009, 1216, 2730-8. 4. Bartolini, M.; Naldi, M.; Nicolau, D. V.; van Delft, F. C.; van Zijl, J.; Snijder, J.; van den Heuvel, E. F.; Naburgh, E. P.; Calonghi, N.; Andrisano, V. Anal Bioanal Chem 2012.
Vth Conversatory on Medicinal Chemistry
L-5
L-5
M. Bartolini; M. Naldi; D.V. Nicolau; F.C.M.J.M. van Delft; J. van Zijl; J. Snijder; F.C. van den Heuvel; V. Andrisano
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11585/130317
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