Numerous targets in drug discovery are enzymes and several drugs elicit their pharmacological action through enzyme inhibition. Thus, new screening methodologies capable of identifying new enzyme inhibitors in a faster, more reproducible and automated way have been investigated over the last decades. In this context, chromatographic columns containing immobilized enzymes (IMERs - immobilized enzyme reactors) represent a new technological platform to rapidly screen enzyme inhibitors, alternative to in-solution bioassays. Enzyme immobilization offers advantages in terms of minimizing costs, increasing enzyme stability and data reproducibility. However, limitations are also present, e.g. unwanted interactions between screened compounds and the chromatographic support can affect the quality of data output. In this talk, different operative procedures involving an acetylcholinesterase (AChE)-based IMER will be presented. AChE is a well-known target enzyme in drug discovery for Alzheimer’s disease and AChE inhibitors constitute the largest class of compounds in the market for the symptomatic treatment of this pathology. The development of AChE-based monolithic IMERs, their combination with HPLC-UV and the design of experimental set-ups, which were tailored to overcome screening limitations and to enlarge retrievable information on the mechanisms of action, will be presented. In particular, different experimental set-up were optimized to make AChE-IMER providing (i) fast evaluation and ranking of different classes of compounds, independently from their interaction with the chromatographic support (ii) determination of inhibitory potency (IC50 values) and (ii) information regarding the mode of enzyme inhibition not only in terms of affinity (Ki value) but also in terms of kinetics (koff).
TAILORED USE OF ACETYLCHOLINESTERASE IMMOBILIZED REACTORS
BARTOLINI, MANUELA;ANDRISANO, VINCENZA
2014
Abstract
Numerous targets in drug discovery are enzymes and several drugs elicit their pharmacological action through enzyme inhibition. Thus, new screening methodologies capable of identifying new enzyme inhibitors in a faster, more reproducible and automated way have been investigated over the last decades. In this context, chromatographic columns containing immobilized enzymes (IMERs - immobilized enzyme reactors) represent a new technological platform to rapidly screen enzyme inhibitors, alternative to in-solution bioassays. Enzyme immobilization offers advantages in terms of minimizing costs, increasing enzyme stability and data reproducibility. However, limitations are also present, e.g. unwanted interactions between screened compounds and the chromatographic support can affect the quality of data output. In this talk, different operative procedures involving an acetylcholinesterase (AChE)-based IMER will be presented. AChE is a well-known target enzyme in drug discovery for Alzheimer’s disease and AChE inhibitors constitute the largest class of compounds in the market for the symptomatic treatment of this pathology. The development of AChE-based monolithic IMERs, their combination with HPLC-UV and the design of experimental set-ups, which were tailored to overcome screening limitations and to enlarge retrievable information on the mechanisms of action, will be presented. In particular, different experimental set-up were optimized to make AChE-IMER providing (i) fast evaluation and ranking of different classes of compounds, independently from their interaction with the chromatographic support (ii) determination of inhibitory potency (IC50 values) and (ii) information regarding the mode of enzyme inhibition not only in terms of affinity (Ki value) but also in terms of kinetics (koff).I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
