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Drug screening of pharmaceutical discovery compounds by micro‐size exclusion chromatography/mass spectrometry
Author(s) -
Wabnitz Paul A.,
Loo Joseph A.
Publication year - 2001
Publication title -
rapid communications in mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.528
H-Index - 136
eISSN - 1097-0231
pISSN - 0951-4198
DOI - 10.1002/rcm.546
Subject(s) - chemistry , size exclusion chromatography , mass spectrometry , chromatography , macromolecule , small molecule , drug discovery , ligand (biochemistry) , binding affinities , affinity chromatography , affinities , drug detection , combinatorial chemistry , stereochemistry , organic chemistry , biochemistry , receptor , enzyme
Micro‐size exclusion chromatography coupled with capillary liquid chromatography (capLC) and mass spectrometry (MS) provides a rapid and simple approach to the preliminary screening of active ligands toward a specific target macromolecule. In this study, the effectiveness of this technique is demonstrated by a number of small molecule ligands with known binding affinities towards the protein target. All ligands were incubated together with a target protein under native conditions. Separation was then achieved by microcentrifugation where the high molecular weight (MW) compounds were selectively passed through the size‐exclusion material. The retained low MW compounds were then recovered and analyzed by capLC/MS. The absence of the ligand indicated strong affinity towards the target, while ligand detection indicated inactivity. This assay demonstrated the drugs that were acting as strong inhibitors of Co‐PDF from those showing to be comparatively inactive. The relative binding rank order of the drugs towards Co‐PDF was also determined. The results were validated by a corresponding set of control experiments in which the target molecules were excluded from the process. In principle, high‐throughput micro‐size exclusion chromatography, coupled with capLC/MS, offers a powerful technique as a preliminary screen in determining both the strong binding affinity and the relative affinity rank ordering of ligands towards a specific target macromolecule, and is complementary with other analytical drug screening techniques. Copyright © 2001 John Wiley & Sons, Ltd.