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 high-throughput mass spectrometry assay to measure the catalytic activity of UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase, LpxC, is described. This reaction is essential in the biosynthesis of lipopolysaccharide (LPS) of gram-negative bacteria and is an attractive target for the development of new antibacterial agents. The assay uses the RapidFire mass spectrometry platform to measure the native LpxC substrate and the reaction product and thereby generates a ratiometric readout with minimal artifacts due to detection interference. The assay was robust in a high-throughput screen of a library of more than 700,000 compounds arrayed as orthogonal mixtures, with a median Z' factor of 0.74. Selected novel inhibitors from the screening campaign were confirmed as binding to LpxC by biophysical measurements using a thermal stability shift assay. Some inhibitors showed whole-cell antimicrobial activity against a sensitive strain of Escherichia coli with reduced LpxC activity (strain D22; minimum inhibitory concentrations ranging from 0.625-20 microg/mL). The results show that mass spectrometry-based screening is a valuable high-throughput screening tool for detecting inhibitors of enzymatic targets involving difficult to detect reactions.Journal of Biomolecular Screening 12/2009; 15(1):52-61. · 2.21 Impact Factor
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ABSTRACT: Quantification of small molecules using liquid chromatography/tandem mass spectrometry (LC/MS/MS) on a triple quadrupole mass spectrometer has become a common practice in bioanalytical support of in vitro adsorption, distribution, metabolism and excretion (ADME) screening. The bioanalysis process involves primarily three indispensable steps: MS/MS optimization for a large number of new chemical compounds undergoing various screening assays in early drug discovery, high-throughput sample analysis with LC/MS/MS for those chemically diverse compounds using the optimized MS/MS conditions, and post-acquisition data review and reporting. To improve overall efficiency of ADME bioanalysis, an integrated system was proposed featuring an automated and unattended MS/MS optimization, a staggered parallel LC/MS/MS for high-throughput sample analysis, and a sophisticated software tool for LC/MS/MS raw data review as well as biological data calculation and reporting. The integrated platform has been used in bioanalytical support of a serum protein binding screening assay with high speed, high capacity, and good robustness. In this new platform, a unique sample dilution scheme was also introduced. With this dilution design, the total number of analytical samples was reduced; therefore, the total operation time was reduced and the overall throughput was further improved. The performance of the protein binding screening assay was monitored with two controls representing high and low binding properties and an acceptable inter-assay consistency was achieved. This platform has been successfully used for the determination of serum protein binding in multiple species for more than 4000 compounds.Rapid Communications in Mass Spectrometry 12/2010; 24(24):3593-601. · 2.51 Impact Factor
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ABSTRACT: High-throughput in vitro ADMET (absorption, distribution, metabolism, excretion and toxicity) profiling has become an important common practice in the pharmaceutical industry to assess compound liability early in the drug discovery process. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is the bioanalytical method of choice for ADMET profiling assays that require compound-specific detection. However, the in vitro ADMET profiling environment, with its unique bioanalytical requirements of analyzing many samples generated from many discrete compounds in a high-throughput fashion, poses significant challenges for the traditional LC-MS/MS technology and process workflow, which were originally designed and optimized for single-compound bioanalysis. This article reviews advances made during the last several years in both conventional high-throughput LC-MS/MS approaches and a number of promising novel MS-based technologies specifically developed to address the unique challenges in an ADMET environment. The advantages and limitations of each technology are also discussed. In addition, software solutions to enable and integrate these hardware improvements into the high-throughput ADMET workflow are reviewed. The reader will gain an updated knowledge of the state-of-the-art technologies and practices, as well as promising novel MS-based methodologies in the field of ADMET bioanalysis. Recent advances such as automated MS/MS optimization, high-speed and multiplexed LC separation, and integrated software support have significantly increased the speed and quality of ADMET bioanalysis using LC-MS/MS. Emerging novel technologies in front-end sample introduction, ionization and mass analysis are expected to further push the current throughput limit and potentially transform the existing bioanalytical paradigm in the future.Expert Opinion on Drug Metabolism & Toxicology 03/2010; 6(3):321-36. · 2.94 Impact Factor