High Throughput Screening via Mass Spectrometry: A Case Study Using Acetylcholinesterase

BioTrove, Inc., Woburn, MA, USA.
Assay and Drug Development Technologies (Impact Factor: 1.53). 09/2004; 2(4):373-81. DOI: 10.1089/1540658041850715
Source: PubMed


Mass spectrometry-based screening can be applied to a wide range of targets, including those intractable targets that use substrates such as lipids, fatty acids, phospholipids, steroids, prostaglandins, and other compounds not generally amenable to conventional screening techniques. The major limitation to this approach is throughput, making HTS via mass spectrometry impractical. We present a mass spectrometry-based technique and hardware for lead discovery applications. Mass spectrometry enables the design of label-free assays using biologically native substrates for a wide range of enzymatic targets. This system can be used for the direct quantification of analytes in complex reaction mixtures with typical throughputs of 4-5 s per sample. A mass spectrometry-based assay was developed to identify inhibitors of acetylcholinesterase, an enzyme with clinical importance in Alzheimer's disease. The system was used to screen a small chemical library. Several potent inhibitors were identified, and the IC(50) values of the inhibitors were determined.

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    ABSTRACT: In the current age of polypharmacy, it is increasingly likely that a new chemical entity (NCE) will be prescribed with a second drug that demonstrates a narrow therapeutic index. As a result, one has to consider interactions involving drug-metabolizing enzymes and transporters. NCEs with drug–drug interaction (DDI) liabilities may have limited marketing potential, as they may alter the pharmacokinetic profile of a co-administered drug resulting in either unwanted side effects or loss of pharmacological activity. Within the current competitive landscape, therefore, it is highly desirable to select candidates with reduced potential for DDIs and most pharmaceutical companies spend considerable resources screening and triaging NCEs for induction and inhibition of drug-metabolizing enzymes (e.g., cytochromes P450) and transporters. Thus, the purpose of the present chapter is to provide an industrial perspective on how the existing strategies are utilized to enable the selection of suitable candidates with reduced DDI risk. Additional emphasis will be placed on in vitro tools and the challenges associated with the prediction of DDIs prior to first in man.
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    ABSTRACT: An electrophoretically mediated microanalysis (EMMA) method for screening acetylcholinesterase (AChE) inhibitors in natural extracts is described. In this method, solutions of AChE and the mixture of the substrate and the natural extract were successively injected into the capillary, and mixed electrophoretically by applying a voltage for a short time. Afterwards the voltage was reapplied to separate the product from the unreacted substrate and the natural extract. The measured peak area of the product at UV 230 nm represents the enzyme activity. Since the extract is mixed with the substrate, there is no need to separate the components before testing the inhibition. The inhibitory activity of the natural extract as a whole can be easily found if the peak area of the product is reduced. This makes the present method suitable for screening inhibitors in complex mixtures, such as natural extracts. Compared to the commonly used spectrometric method for screening of AChE inhibitors, the major advantage of the present method is the elimination of Ellman reagent, which is essential for the spectrometric method. This not only simplifies the experimental procedure but also minimizes false-positive results. Moreover, it is an obvious advantage of combining the separation power with the on-column enzyme assay for further investigating which compound(s) is/are responsible for the inhibition. The method was validated using a commercially available AChE inhibitor tacrine and a small chemical library containing four AChE inhibitors and 32 natural extracts. Inhibitors in natural extracts were identified with the present method.
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