The research efforts in drug discovery field are based on the knowledge of the molecular aspects of the disease and on the development of new techniques necessary to investigate the biological systems at molecular level. The selection of new leads is therefore a challenging task and involve various essential steps, the first being the identification/validation of new targets, then the selection of molecules able to bind to the target(s), and finally the study of the effects of hitting the target at molecular, cellular and whole animal level. In the case of Alzheimer’s disease (AD), the most common form of dementia in adults, acetylcholinesterase (AChE) has been the first target for the development of new drugs since the discovery of the cholinergic deficit in the central nervous system. However, basic research showed that cognitive impairment could be due not only to a cholinergic deficit but also to a cascade of biochemical events leading to the accumulation in the brain of proteins such as ß-amyloid (Ab) and hyper-phosphorylated tau protein. Important targets are amyloid fibrillogenesis, beta-secretase (BACE1), one of the enzymes which cleave APP (amyloid precursor protein) and GSK3b, a tau protein phosphorylating kinase. On the other hand, other non cholinergic role of AChE in the AD has been discovered: some evidences suggest that AChE peripheral binding site may play a key role in the development of senile plaques, accelerating Ab deposition. Once the disease targets have been selected, the determination of the activity of the new compounds must be carried out quickly and in a way that allows the verification of the design hypothesis. Drug activity is in fact mediated by different types of interactions with specific biological targets and the esteem of these interactions may elucidate the mechanism of action. To this aim, in a first instance, high throughput screening methods (HTS) of a large number of compounds for the selection of few lead compounds are required. Secondly, specific methods, which elucidate the selected compound mechanism of action, must be employed, before the ultimate and most advanced tools, transgenic animal models of the disease, can be used to study the effects of single compounds on the disease phenotype. Here we report the development of purposely designed integrated methodologies to define the multifunctional activity profile for new AD drug discovery. With regard to the assessment of the activity of chemical libraries, the affinity chromatography on HPLC immobilized-enzyme column (or immobilized enzyme reactors, IMER) is one of most promising methodologies for HTS applications. Human recombinant AChE and BACE1 monolithic micro-IMERs (immobilized enzyme reactor) have been developed for on-line automated HT HPLC inhibition studies (IC50 and mechanism of inhibition); secondly, fluorometric, circular dichroism, mass spectrometry, AFM methods were optimised for monitoring the inhibition of AChE-induced Ab fibril formation and the inhibition of spontaneous Ab aggregation, elucidating at which intermediate level of the Ab aggregation cascade the inhibitors halt the process (monomer, soluble oligomers, protofibrils, fibrils). Finally, mass spectrometry combined with UHPLC was applied to investigate the mechanism of action of GSK3b inhibitors. By the application of these integrated approaches, new leads as the prototype of new classes of multifunctional compounds for AD treatment were discovered.

Analytical approaches in Alzheimer’s disease Drug Discovery

DE SIMONE, ANGELA;BARTOLINI, MANUELA;FIORI, JESSICA;D'URZO, ANNALISA;NALDI, MARINA;ANDRISANO, VINCENZA
2015

Abstract

The research efforts in drug discovery field are based on the knowledge of the molecular aspects of the disease and on the development of new techniques necessary to investigate the biological systems at molecular level. The selection of new leads is therefore a challenging task and involve various essential steps, the first being the identification/validation of new targets, then the selection of molecules able to bind to the target(s), and finally the study of the effects of hitting the target at molecular, cellular and whole animal level. In the case of Alzheimer’s disease (AD), the most common form of dementia in adults, acetylcholinesterase (AChE) has been the first target for the development of new drugs since the discovery of the cholinergic deficit in the central nervous system. However, basic research showed that cognitive impairment could be due not only to a cholinergic deficit but also to a cascade of biochemical events leading to the accumulation in the brain of proteins such as ß-amyloid (Ab) and hyper-phosphorylated tau protein. Important targets are amyloid fibrillogenesis, beta-secretase (BACE1), one of the enzymes which cleave APP (amyloid precursor protein) and GSK3b, a tau protein phosphorylating kinase. On the other hand, other non cholinergic role of AChE in the AD has been discovered: some evidences suggest that AChE peripheral binding site may play a key role in the development of senile plaques, accelerating Ab deposition. Once the disease targets have been selected, the determination of the activity of the new compounds must be carried out quickly and in a way that allows the verification of the design hypothesis. Drug activity is in fact mediated by different types of interactions with specific biological targets and the esteem of these interactions may elucidate the mechanism of action. To this aim, in a first instance, high throughput screening methods (HTS) of a large number of compounds for the selection of few lead compounds are required. Secondly, specific methods, which elucidate the selected compound mechanism of action, must be employed, before the ultimate and most advanced tools, transgenic animal models of the disease, can be used to study the effects of single compounds on the disease phenotype. Here we report the development of purposely designed integrated methodologies to define the multifunctional activity profile for new AD drug discovery. With regard to the assessment of the activity of chemical libraries, the affinity chromatography on HPLC immobilized-enzyme column (or immobilized enzyme reactors, IMER) is one of most promising methodologies for HTS applications. Human recombinant AChE and BACE1 monolithic micro-IMERs (immobilized enzyme reactor) have been developed for on-line automated HT HPLC inhibition studies (IC50 and mechanism of inhibition); secondly, fluorometric, circular dichroism, mass spectrometry, AFM methods were optimised for monitoring the inhibition of AChE-induced Ab fibril formation and the inhibition of spontaneous Ab aggregation, elucidating at which intermediate level of the Ab aggregation cascade the inhibitors halt the process (monomer, soluble oligomers, protofibrils, fibrils). Finally, mass spectrometry combined with UHPLC was applied to investigate the mechanism of action of GSK3b inhibitors. By the application of these integrated approaches, new leads as the prototype of new classes of multifunctional compounds for AD treatment were discovered.
SIMCC 2015 - Spanish-Italian Medicinal Chemistry Congress - Book of Abstracts
35
35
De Simone, Angela; Bartolini, Manuela; Fiori, Jessica; D’Urzo, Annalisa; Naldi, Marina.; Andrisano, Vincenza
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11585/550205
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