Temperature-Dependent Expression of a CFP-YFP FRET Diacylglycerol Sensor Enables Multiple-Read Screening for Compounds That Affect C1 Domains

Abstract

Intramolecular CFP-YFP fluorescence resonance energy transfer (FRET) sensors expressed in cells are powerful research tools but have seen relatively little use in screening. We exploited the discovery that the expression of a CFP-YFP FRET diacylglycerol sensor (DAGR) increases over time when cells are incubated at room temperature to assess requirements for robust measurements using a Molecular Devices Spectramax i3x fluorescence plate reader. Expression levels resulting in YFP fluorescence >10-fold higher than untransfected cells and phorbol ester-stimulated FRET ratio changes of 60% or more were required to consistently give robust Z′ > 0.5. As a means of confirming that these conditions are suitable for screening, we developed a novel multiple-read protocol to assay the NCI’s Mechanistic Set III for agonists and antagonists of C1 domain activation. Sixteen compounds prevented C1 domain translocation. However, none blocked phorbol ester-stimulated protein kinase C (PKC) activity assessed using a phospho-specific antibody—six actually stimulated PKC activity. Cytometry, which produces higher Z′ for a given FRET ratio change, might have been a better approach for discovering antagonists, as it would have allowed lower phorbol ester concentrations to be used. We conclude that CFP-YFP FRET measured in a Spectramax i3x plate reader can be used for screening under the conditions we defined. Our strategy of varying expression level and FRET ratio could be useful to others for determining conditions needed for robust cell-based intramolecular CFP-YFP FRET measurements on their instrumentation.

The National Cancer Institute’s Plated Compound Sets Can Be a Valuable Resource for Academic Researchers

“…We also screened the Mechanistic Set for compounds that act as either agonists or antagonists of C1 domains using a multiple read assay based on expression of a FRET reporter for diacylglycerol. 13 We confirmed that 16 compounds demonstrated antagonist activity in the primary screen, a hit rate again of ~2%. All of these compounds also blocked translocation of the sensor from cytosol to membrane as expected when measured using microscopy, which served as an orthogonal assay, but none demonstrated the expected inhibitory effects on activation of protein kinase C (PKC)—in fact, some activated PKC.…”

Z’ Does Not Need to Be > 0.5

“…Second, and related, the requirement that Z' be > 0.5 may lead researchers to conduct assays under conditions that maximize Z' but hinder detection of useful compounds, as has been noted by Glickman 6 and Bray et al 3 As an example of our experience with this, we recently developed a screen for compounds that act as antagonists of phorbol dibutyrate (PDBU) binding to C1 domains. 12 To achieve Z' > 0.5, we conducted the screen with a very high concentration (100 nM) of the activator PDBU. Since the Kd for PDBU binding is in the low nM range, 13 however, we speculate that using PDBU in excess may have prevented us from finding competitive antagonists.…”

C1 Compound Biosensors: Design, Functional Study, and Applications

“…Given the recent advances in synthetic biology, the transcriptional regulator (transcription factor [TF])-based biosensor has emerged as one of the most powerful tools to increase enzyme activity or to discover new enzymes or strains. Various TF-based biosensors have been developed for enzyme engineering and the discovery of novel biocatalysts, using a fluorescence-activated cell sorting (FACS)-based high-throughput screening system [14,15,16,17,18,19,20,21]. Formaldehyde- and formate-dependent TFs have been reported in the operon of formaldehyde detoxification and the expression system of anaerobic formate dehydrogenase, respectively [22,23].…”