IMMOBILIZED ENZYME REACTORS INTO THE DRUG DISCOVERY PROCESS: THE ALZHEIMER’S DISEASE CASE

In the search for new therapeutics, the high throughput screening (HTS) of ligands for key target proteins represent the principal hit identification tool for early drug discovery. Important aspects that have to be considered are cost-based or amount-based limitation of target availability, need of speed and automation and easy coupling of the assay to separation systems and appropriate detectors. In this context, affinity chromatography based on immobilized target proteins may match these issues and solve problems related to expensive protein consumption. Numerous targets in drug discovery are represented by enzymes and several drugs are available whose mechanism of action is related to enzyme inhibition. Therefore, in the field of drug discovery, monolithic immobilized enzyme reactors (IMERs) can represent valid tools in the different phases of the drug discovery pathway, starting with active compounds (hit) identification, through drug development and lead optimization, early ADMET (absorption, distribution, metabolism, excretion, toxicity) studies and quality control of protein drugs. In this work the IMER approach was applied to drug discovery in Alzheimer’s disease (AD), a multifactorial pathology that represents the main cause of dementia in elderly people. Among other aspects, AD is characterized by a cholinergic deficit associated to anomalous proteins misfolding and aggregation, and neuronal death. Therefore, the cholinergic enzymes acetyl- and butyrylcholinesterase (AChE and BuChE, respectively) represent well-known targets for new anti-Alzheimer drugs. Monolithic IMERs containing covalently bound AChE or BuChE have been developed, characterized after insertion on HPLC systems, and applied to the screening of known and new inhibitors.[1,2] In the optimization phase, further insights into the mechamism of action must be evaluated. Mechanism of action of reversible and peuso-irreversible inhibitors could be investigated by IMERs through simple operative procedures.[3] Moreover, since metabolic stability against liver enzymes and drug-drug interactions (DDI) are key aspects for lead development, the immobilization of cytochrome P450s would also offer advantages in fast drug profile assessment. IMERs based on 2D6 and 3A4 isoforms of cytochrome P450 were designed and developed with this purpose.[4] Even if each IMER has its own requirements for optimal performances, they generally showed an increased data output, reliability and stability, which directly translate into cost reduction. All these aspects represent important features for potential applications in pharma industry.