Overcoming Compound Fluorescence in the FLiK Screening Assay with Red-Shifted Fluorophores

Abstract

In the attempt to discover novel chemical scaffolds that can modulate the activity of disease-associated enzymes, such as kinases, biochemical assays are usually deployed in high-throughput screenings. First-line assays, such as activity-based assays, often rely on fluorescent molecules by measuring a change in the total emission intensity, polarization state, or energy transfer to another fluorescent molecule. However, under certain conditions, intrinsic compound fluorescence can lead to difficult data analysis and to false-positive, as well as false-negative, hits. We have reported previously on a powerful direct binding assay called fluorescent labels in kinases ('FLiK'), which enables a sensitive measurement of conformational changes in kinases upon ligand binding. In this assay system, changes in the emission spectrum of the fluorophore acrylodan, induced by the binding of a ligand, are translated into a robust assay readout. However, under the excitation conditions of acrylodan, intrinsic compound fluorescence derived from highly conjugated compounds complicates data analysis. We therefore optimized this method by identifying novel fluorophores that excite in the far red, thereby avoiding compound fluorescence. With this advancement, even rigid compounds with multiple π-conjugated ring systems can now be measured reliably. This study was performed on three different kinase constructs with three different labeling sites, each undergoing distinct conformational changes upon ligand binding. It may therefore serve as a guideline for the establishment of novel fluorescence-based detection assays.

Monitoring Ligand‐Induced Conformational Changes for the Identification of Estrogen Receptor Agonists and Antagonists

“…This assay technology was originally introduced to screen for type II and III kinase inhibitors in high-throughput formats [12] and since then has been successfully refined and transferred to other proteins. [13] We show that this fluorescent labels in nuclear receptors (FLiN) assay is av aluable tool to screen for agonists and antagonists of the human ERb LBD in high-throughput formats and is suitable for distinguishing between the different conformations of helix 12 induced by the ligand.…”

Intramolecular Didehydro-Diels–Alder Reaction and Its Impact on the Structure–Function Properties of Environmentally Sensitive Fluorophores.

“…ACRYLODAN has been employed in direct binding assays as a kinase label to detect ligands that stabilize inactive kinase conformations, 20 while LAURDAN has been utilized to study lateral organization and heterogeneity of lipid membranes by reporting local permeation of water. 21 One drawback to using LAURDAN as a fluorescent probe is its low photostability, which limits its application to two-photon microscopy.…”

How to Translate a Bioassay Into a Screening Assay for Natural Products: General Considerations and Implementation of Antimicrobial Screens

“…Similarly, colored and/or autofluorescent pure compounds can cause artifactual results. Indeed, many natural compounds are rigid and planar and possess multiple conjugated aromatic moieties, which increase the probability of endogenous fluorescence [48]. This is the case in widely distributed natural molecules such as coumarins, anthraquinone derivatives (for instance, hypericin, present in the alcoholic extract of Hypericum perforatum) and pigments such as carothenes, chlorophyll, or chlorophyll breakdown products such as phaeophorbide A [49].…”

“…In these cases, the compound spectral properties cause interferences with the light detection step of the screening assay, and they are mostly predominant in assays that are run in absorbance and fluorescence (FI, FP, and FRET) modes [51]. Such interferences are manifested by a typical increase in the background signal of the assay but also by the participation in unwanted FRET with the assay fluorophore [48]. Given that a vast majority of screening assays is nowadays run with absorbance-or fluorescence-based technologies, these issues cannot be ignored.…”