Receptor–Ligand Interactions Studied with Homogeneous Fluorescence-Based Assays Suitable for Miniaturized Screening

Abstract

Cell membrane receptors play a central role in controlling cellular functions, making them the target of drugs for a wide variety of diseases. This report describes how a recently developed method, fluorescence intensity distribution analysis (FIDA), can be used to develop homogeneous, nonradioactive high throughput screening assays for membrane receptors. With FIDA, free ligand and ligand accumulated on receptor-bearing membrane vesicles can be distinguished on the basis of their particle brightness. This allows the concentration of both bound and free ligand to be determined reliably from a single measurement, without any separation. We demonstrate that ligand affinity, receptor expression level, and potency of inhibitors can be determined using the epidermal growth factor and beta(2)-adrenergic receptors as model systems. Highly focused confocal optics enable single-molecule sensitivity, and sample volumes can thus be reduced to 1 microl without affecting the quality of the fluorescence signal. Our results demonstrate that FIDA is an ideal method for membrane receptor assays offering substantial benefits for assay development and high throughput pharmaceutical screening.

Single-Molecule Detection Technologies in Miniaturized High-Throughput Screening: Fluorescence Intensity Distribution Analysis

“…In a screening scenario (and for most of the data presented here), the fitting process would use fixed brightness values of, for example, bound and free ligand, which may be determined individually beforehand, and float the particle number for each of these species. This would not take into account inhomogeneous brightness distributions that occur, for example, when ligands bind to vesicles 20,21 or beads coated with the respective receptor. 22 In this situation, a different fitting algorithm, called inverse transformation with regularization (ITR), can be applied that calculates the concentrations for molecules for a quasi-continuous brightness distribution.…”

“…4A-B, 5C, 9) for excitation. 20,21 For the cFLA measurements (Fig. 4C), a fast-pulsed laser diode (635 nm, PicoQuant, Berlin, Germany) was used for excitation, and the instrument was equipped with a module for time-correlated single-photon counting.…”

“…However, even in the absence of (de)quenching during the binding event, a brightness change can be obtained if the fluorescent ligand binds to particles with multiple binding sites (e.g., membrane vesicles with multiple receptor copies 20,21 ) or beads 28 labeled with the respective binding partner. However, there is a technical difficulty with such kind of assays: for systems in which the analytes to be distinguished differ in mass by several orders of magnitude (e.g., a bead and unbound conjugate), relying on diffusion processes alone to obtain a statistically sufficient number of encounters between the detection volume and both particle populations would result in very long read times and a large bias in the signal quality toward the smaller, more rapidly diffusing species.…”

A New Simple Cell-Based Homogeneous Time-Resolved Fluorescence QRET Technique for Receptor-Ligand Interaction Screening

“…[21][22][23] This study was based on a homogeneous and quantitative assaying of receptor-ligand interaction using whole intact cells. The cells were frozen after culturing and subsequent harvesting and thawed for assaying.…”

NanoStore: A Concept for Logistical Improvements of Compound Handling in High-Throughput Screening

“…The β 2 -AR assay format was as previously described. 11 The drymode low-volume assay was performed as for NEP, using the MkII Evoscreen system, whereas wet-mode low-volume assays were performed on an Evotec Assay Development Station (Insight Reader, MONA and DINA; Evotec Technologies GmbH, Hamburg, Germany).…”