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A coincidence reporter-gene system for high-throughput screening

Nature Methods volume 9, page 937 (2012)Cite this article

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We confirmed the function of a preliminary biocircuit design by stoichiometric coexpression of the unrelated bioluminescent reporters firefly luciferase (FLuc) and Renilla luciferase (RLuc), using 'ribosome skipping' facilitated by the short P2A peptide3 in a HEK293 cell. (Supplementary Fig. 1 and Supplementary Methods). We demonstrated the accurate discrimination of forskolin-activated adenylyl-cyclase signaling through the cAMP-response element from signals mediated by the known FLuc and RLuc stabilizers, PTC124 and BTS, respectively (Fig. 1b,c and Supplementary Fig. 2). Using the LOPAC1280 chemical library, we conducted a quantitative HTS experiment, in which full titrations of each compound are tested4 to identify potentiators of the CREB pathway (Supplementary Figs. 3 and 4 and Supplementary Table 1). The screen revealed, for example, coincident FLuc and RLuc signal outputs for 17 adenosine analog agonists of endogenous purinergic 2Y known to signal through G proteins in this cell type5 (Supplementary Table 2a). We observed excellent correlation between the half-maximal effective concentration (EC50) values calculated from the orthogonal reporter outputs (Fig. 1d). Our experiments also illustrated the phenomenon of reporter-dependent artifacts: we identified five aryl sulfonamides that showed selective agonist activity for RLuc only (Supplementary Table 2b, compounds 20–24). These compounds share a similar core scaffold with two known RLuc inhibitors and selectively inhibit the enzymatic activity of RLuc over that of FLuc (Supplementary Fig. 5), thus tying these particular artifacts to the phenomenon of reporter stabilization4. Cross-section data analysis of the screen (Supplementary Fig. 6) also demonstrates how coincidence detection enhances the testing of compound libraries in single-concentration format.

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Figure 1: Coincidence reporter biocircuit.

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Acknowledgements

We thank P. Dranchak and R. MacArthur for assistance with supplementary data and P. Shinn for compound management. This work was supported by the US National Institutes of Health (NIH) Roadmap for Medical Research.

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Authors and Affiliations

  1. National Center of Advancing Translational Sciences, NIH, Bethesda, Maryland, USA

    Ken C-C Cheng & James Inglese

  2. National Human Genome Research Institute, NIH, Bethesda, Maryland, USA

    James Inglese

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  1. Ken C-C Cheng
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  2. James Inglese
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Correspondence to James Inglese.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–6, Supplementary Table 2 and Supplementary Methods (PDF 603 kb)

Supplementary Table 1

All quantitative HTS data for replicate measurements on FLuc and RLuc. Data is arranged as follows: column A, NCGC sample ID; column B, SMILE; column C, common name; columns D, H, L and P, curve class (CC) for respective reporters and experimental runs; columns E, I, M and Q, logEC50 values as determined from the respective reporter output for a given experimental run; columns F, J, N and R, EC50 values as determined from the respective reporter output for a given experimental run; and columns G, L, O and S, efficacy values as determined from the respective reporter output for a given experimental run. All adenosine receptor agonists are colored green, and muscarinic receptor agonists are colored coded blue. Forskolin is colored red. In this 1,536-well, transient transfection assay format, an activity considered substantial required a curve fit that yielded a CC of positive high quality (i.e., 1.1, 1.2 or 2.1) in either of the reporter outputs. (XLSX 225 kb)

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Cheng, KC., Inglese, J. A coincidence reporter-gene system for high-throughput screening. Nat Methods 9, 937 (2012). https://doi.org/10.1038/nmeth.2170

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