HTRF: A Technology Tailored for Drug Discovery - A Review of Theoretical Aspects and Recent Applications

Abstract

HTRF (Homogeneous Time Resolved Fluorescence) is the most frequently used generic assay technology to measure analytes in a homogenous format, which is the ideal platform used for drug target studies in high-throughput screening (HTS). This technology combines fluorescence resonance energy transfer technology (FRET) with time-resolved measurement (TR). In TR-FRET assays, a signal is generated through fluorescent resonance energy transfer between a donor and an acceptor molecule when in close proximity to each other. Buffer and media interference is dramatically reduced by dual-wavelength detection, and the final signal is proportional to the extent of product formation. The HTRF assay is usually sensitive and robust that can be miniaturized into the 384 and 1536-well plate formats. This assay technology has been applied to many antibody-based assays including GPCR signaling (cAMP and IP-One), kinases, cytokines and biomarkers, bioprocess (antibody and protein production), as well as the assays for protein-protein, proteinpeptide, and protein-DNA/RNA interactions.Since its introduction to the drug-screening world over ten years ago, researchers have used HTRF to expedite the study of GPCRs, kinases, new biomarkers, protein-protein interactions, and other targets of interest. HTRF has also been utilized as an alternative method for bioprocess monitoring. The first-generation HTRF technology, which uses Europium cryptate as a fluorescence donor to monitor reactions between biomolecules, was extended in 2008 through the introduction of a second-generation donor, Terbium cryptate (Tb), enhancing screening performance. Terbium cryptate possesses different photophysical properties compared to Europium, including increased quantum yield and a higher molar extinction coefficient. In addition to being compatible with the same acceptor fluorophors used with Europium, it can serve as a donor fluorophore to green-emitting fluors because it has multiple emission peaks including one at 490 nm. Moreover, all Terbium HTRF assays can be read on the same HTRF-compatible instruments as Europium HTRF assays.Overall, HTRF is a highly sensitive, robust technology for the detection of molecular interactions in vitro and is widely used for primary and secondary screening phases of drug development. This review addresses the general principles of HTRF and its current applications in drug discovery.

Development of a High-Throughput Screening Paradigm for the Discovery of Small-Molecule Modulators of Adenylyl Cyclase: Identification of an Adenylyl Cyclase 2 Inhibitor

“…It is possible that the active compounds have inherent fluorescence that can be measured in the same wavelengths as those used for cAMP detection, thereby skewing the measured fluorescence and affecting the resulting estimation of the cAMP concentration (Degorce et al, 2009). Thus, we assessed the ability of the best compounds (i.e., those that provided at least 50% inhibition in the confirmation assay) to inhibit AC2 activity in a non-fluorescence-based assay.…”

Evaluation ofAaDOP2 Receptor Antagonists Reveals Antidepressants and Antipsychotics as Novel Lead Molecules for Control of the Yellow Fever Mosquito,Aedes aegypti

“…The HTRF screening platform also provides flexibility, as it can be used to detect modulation of additional downstream GPCR signaling pathways, including extracellular signal-regulated kinase 1/2 and Ca 21 /inositol trisphosphate (Degorce et al, 2009). Other improvements to our established insecticide discovery pipeline for small-molecule modulators of AaDOP2 (Meyer et al, 2012) include the enhanced-throughput larval mosquito bioassay (Table 4) to rapidly assess larval toxicity and the utilization of an injection assay to evaluate toxicity in adult A. aegypti.…”

Overview of Recent Progress in Protein-Expression Technologies for Small-Molecule Screening

“…Affinity tags are also often used in protein-protein interaction screens as handles for the attachment of reagents to detect the interaction between two differentially tagged constructs using homogeneous timeresolved fluorescence-based assays. 55 Affinity tags are also critical for affinity-based screening methodologies that rely on capturing protein-small-molecule complexes to identify hits. 7 One of the most commonly used affinity tags on constructs today is the polyHis tag.…”