High-Throughput 1,536-Well Fluorescence Polarization Assays for α1-Acid Glycoprotein and Human Serum Albumin Binding

Abstract

Two major plasma proteins in humans are primarily responsible for drug binding, the α1-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 LOPAC1280 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.

Entrapment of alpha1-acid glycoprotein in high-performance affinity columns for drug-protein binding studies

“…The values from Refs. [52,61] were measured at pH 7.4 and 25 °C. The values of 3.46–4.33 × 10 5 M −1 from Ref.…”

Fluorescence polarization assays in high-throughput screening and drug discovery: a review

“…In an effort to allow assessment of a larger set of compounds from a medicinal chemistry program or to profile drug libraries, Mathias et al developed a FP assay for protein binding, which was subsequently miniaturized to a 1,536-well format [45,46]. Given that FP requires the assessment of a specific molecular interaction, a serum-based binding assay was not feasible, so the investigators developed two separate assays for binding to HSA and AGP.…”

Interpreting physicochemical experimental data sets

“…There are many experimental methods which have been used to determine the extent of plasma protein binding. These include equilibrium dialysis (ED), [59][60][61] microdialysis, [61,62] dynamic dialysis, [63,64] ultrafiltration, [63,65] ultracentrifugation, [63,66,67] charcoal-binding kinetic methods, [68,69] surface plasmon resonance (SPR), [70] albumin PAMPA, [71] albumin-immobilised beads, [72] various chromatographic techniques such as immobilised-albumin support coupled with high performance liquid chromatography, [73,74] high-performance frontal analysis, [63] a multitude of spectroscopic techniques UV spectroscopy, [75] circular dichroism, [75] NMR [75]. However equilibrium dialysis remains the gold standard and the method most frequently used in drug discovery.…”

“…As such numerous methods have utilised albumin alone to determine plasma protein binding. Several techniques have applied human serum albumin (HSA) immobilised onto inert supports including surface plasmon resonance (SPR-BIA-CORE), [70] HSA immobilised on beads, [72] and HSA immobilised gradient HPLC [73,74]. While often giving good comparisons to ED derived data questions about potential for some compounds to bind to the solid support surface and perturbation of the albumin binding site caused by the chemical binding to the support remain a concern.…”