title: Drug screening of pharmaceutical discovery compounds by micro-size exclusion chromatography/mass spectrometry.
abstract: 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.
authors.affiliation: Department of Pharmacokinetics, Dynamics and Metabolism. Pfizer Global Research & Development, Ann Arbor Laboratories, 2800 Plymouth Road, Ann Arbor, MI 48105, USA. paul.wabnitz@pfizer.com
authors.forename: Paul A
authors.initials: PA
authors.lastname: Wabnitz
authors.forename: Joseph A
authors.initials: JA
authors.lastname: Loo
mesh_terms: D000581:Amidohydrolases; D000626:Aminopeptidases / Q000037:antagonists & inhibitors / Q000378:metabolism; D002850:Chromatography, Gel; D002853:Chromatography, Liquid / Q000379:methods*; D003035:Cobalt; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D015507:Drugs, Investigational / Q000302:isolation & purification* / Q000378:metabolism; D004791:Enzyme Inhibitors; D013058:Mass Spectrometry / Q000379:methods*; D008970:Molecular Weight; D004364:Pharmaceutical Preparations / Q000302:isolation & purification* / Q000378:metabolism; D011485:Protein Binding; D011506:Proteins / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization
chemical_list: D015507:Drugs, Investigational; D004791:Enzyme Inhibitors; D004364:Pharmaceutical Preparations; D011506:Proteins; D003035:Cobalt; D000626:Aminopeptidases; D000581:Amidohydrolases; C084507:peptide deformylase