Fluorescence resonance energy activation assay

Compounds that interfere with the detection mechanism of the HTS assay wiU, in many cases, be detected as highly potent actives [31]. One example would be compounds that intrinsically emit or absorb light at the wavelengths used in a fluorescence-based assay such as fluorescence resonance energy transfer (FRET). In an HTS screen using a fluorescence-based assay at Wyeth, 1.2% of the samples tested showed not just high fluorescence but the maximum possible initial (time 0) reading on the fluorimeter. 

Since the initial paper by Fields and Song, there have been significant technical improvements in the method. DNA-binding domains and transcription activation domains have been optimized to reduce false positives and increase the transcription read-out. A variety of reporter plasmids have been engineered to detect a broad range of protein-protein interactions. Much more is understood about the nature of false positives and how to rout them out. Moreover, in response to the utility of this approach, several laboratories have begun to develop transcription-based assays that can be carried out in bacteria, or protein-protein interaction assays based on alternate readouts such as enzyme complementation or fluorescence resonance energy transfer (FRET). 

Several methods are available today to test candidate ion channel active drugs (ICADs) electrophysiology (patch clamp), binding assays, radioactive flux assays, membrane potential-sensitive fluorescent dyes, ion-sensitive dyes, and voltage sensing based on fluorescence resonant energy transfer (FRET). 

EUiammer, A. Novel assays for inositol phosphorylceramide synthase activity and for inhibitors using fluorescence resonance energy transfer. U.S. Pat. Appl. Publ. US 2007269844, 2007 Chem. Abstr. 2007, 148, 4443. 

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