High throughput screening via mass spectrometry: a case study using acetylcholinesterase.
ABSTRACT 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: A derivatization method in mass spectrometry for small molecular analysis was developed to solve the problems of volatility of many analytes, difficult ionization of analytes, and undiscriminating isobaric analytes. This derivatization method, oximation of -ketoacid, in the transaminase reaction leads to change of mass difference between amine reactants and ketoacid products. In addition, regardless of the kinds of ketoacid, the linear relationship between the peak intensity and its concentration (from 1 mM to 10 mM) shows that quantitative analysis of the conversion of the reaction can be executed by the analysis of peak intensity of the corresponding oximated ketoacids. Furthermore, this method can be used for identifying transaminase as well as determining its substrate specificityKSBB Journal. 01/2004; 19(5).
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ABSTRACT: This review describes the key steps and methods which are used to develop enzyme assays suitable for high-throughput screening (HTS) applications. The goals of HTS enzyme assays are defined relative to lower-throughput bench top assays and important aspects which go into constructing robust and sensitive enzyme assays are described. Methods that have been applied to common enzyme classes are reviewed and pitfalls related to assay artifacts are discussed. We also suggest a reporting format to describe the steps in HTS enzyme assays.Perspectives in Science. 05/2014; 1(s 1–6):56–73.
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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.01/2010;