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Rapid LC-MS Based High-Throughput Screening Method, Affording No False Positives or False Negatives, Identifies a New Inhibitor for Carbonic Anhydrase

Imaduwage, Kasun Prabodha
Lakbub, Jude
Desaire, Heather
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Abstract
Developing effective high-throughput screening (HTS) methods is of paramount importance in the early stage of drug discovery. While rugged and robust assays may be easily developed for certain enzymes, HTS assays designed to identify ligands that block protein binding are much more challenging to develop; attenuating the number of false positives and false negatives under high-throughput screening conditions is particularly difficult. We describe an MS-based HTS workflow that addresses these challenges. The assay mitigates false positives by selectively identifying positive hits exclusively when a ligand at the binding site of interest is displaced; it mitigates false negatives by detecting a reporter compound that ionizes well, not by detecting the ligand binder, which may not ionize. The method was validated by detecting known binders of three proteins, pepsin, maltose binding protein (MBP), and carbonic anhydrase (CA) in the presence of hundreds of non-binders. We also identified a novel CA binder, pifithrin-µ, which could not have been identified by any other MS-based assay because of its poor ionization efficiency. This new method addresses many of the challenges that are currently encountered during high-throughput screening.
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Date
2017-09-04
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Publisher
Nature Research
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Keywords
Chemical biology, Mass spectrometry
Citation
Imaduwage, K. P., Lakub, J., Desaire, H., (2017), Rapid LC-MS Based High-Throughput Screening Method, Affording No False Positives or False Negatives, Identifies a New Inhibitor for Carbonic Anhydrase, Scientific Reports 7, 10324 https://doi.org/10.1038/s41598-017-08602-w
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