title: High-throughput 1,536-well fluorescence polarization assays for α(1)-acid glycoprotein and human serum albumin binding.
abstract: Two major plasma proteins in humans are primarily responsible for drug binding, the α(1)-acid-glycoprotein (AGP) and human serum albumin (HSA). The availability of at least a semiquantitative high-throughput assay for assessment of protein binding is expected to aid in bridging the current gap between high-throughput screening and early lead discovery, where cell-based and biochemical assays are deployed routinely to test up to several million compounds rapidly, as opposed to the late-stage candidate drug profiling methods which test at most dozens of compounds at a time. Here, we describe the miniaturization of a pair of assays based on the binding- and displacement-induced changes in fluorescence polarization (FP) of fluorescent small molecule probes known to specifically target the drug-binding sites of these two proteins. A robust and reproducible assay performance was achieved in ≤4 µL assay volume in 1,536-well format. The assays were tested against a validation set of 10 known protein binders, and the results compared favorably with data obtained using protein-coated beads with high-performance liquid chromatography analysis. The miniaturized assays were taken to a high-throughput level in a screen of the LOPAC(1280) collection of 1,280 pharmacologically active compounds. The adaptation of the AGP and HSA FP assays to a 1,536-well format should allow their use in early-stage profiling of large-size compound sets.
authors.affiliation: NIH Chemical Genomics Center, National Center for Advancing Translational Sciences, National Institutes of Health, Bethesda, MD, USA.
authors.forename: Adam
authors.initials: A
authors.lastname: Yasgar
authors.forename: Silviya D
authors.initials: SD
authors.lastname: Furdas
authors.forename: David J
authors.initials: DJ
authors.lastname: Maloney
authors.forename: Ajit
authors.initials: A
authors.lastname: Jadhav
authors.forename: Manfred
authors.initials: M
authors.lastname: Jung
authors.forename: Anton
authors.initials: A
authors.lastname: Simeonov
mesh_terms: D055808:Drug Discovery; D016231:Fluorescence Polarization Immunoassay; D057166:High-Throughput Screening Assays; D009961:Orosomucoid / Q000378:metabolism*; D015203:Reproducibility of Results; D012709:Serum Albumin / Q000378:metabolism*
chemical_list: D009961:Orosomucoid; D012709:Serum Albumin