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. 2022 Jun 23;13:3586. doi: 10.1038/s41467-022-31206-6

Fig. 1. Screening for stabilizers of the 14-3-3β/CFTRpS753pS768 complex.

Fig. 1

a Topology model of CFTR showing the two transmembrane domains (TDM), the nucleotide binding domains (NBD), and the regulatory domain (RD). The 14-3-3 binding motif encompassing the phosphorylation sites pS753 and pS768 (CFTRpS753pS768) was used in this study and is shown as peptide sequence. This sequence—CFTRpS753pS768—was used in this study as a synthetic peptide. b Fluorescence polarization (FP) assay of fluorescein isothiocyanate (FITC)-labeled CFTRpS753pS768 peptide (100 nM) with 14-3-3β, in the absence (black) or presence (purple) of 100 μM fusicoccin A (FC-A), n = 3 technical replicates. The 14-3-3β concentration used for the FP screening assay was 10 μM (dashed line). c HTS FP assay of the Cyclenium library. The samples contain 100 nM of FITC-CFTR_pS753pS768 peptide, 10 μM 14-3-3β, and 50 μM compound. FC-A (100 µM) was added as a positive control and DMSO as the negative control. Hit compounds are defined as a polarization increase which is higher than 20% of the response of FC-A. Red squares are compounds that appeared positive in the negative control without 14-3-3. Orange triangles gave a suspicious high or low total photon count and need to be treated with care. d The eight-point dose-response FP follow-up assay of the Cyclenium library hit compounds stabilizing the interaction between 14-3-3 (10 μM) and labeled CFTRpS753pS768 (100 nM) peptide. Background polarization was subtracted from all values, n = 3 technical replicates. Compounds selected for further analysis are indicated with an asterisk. e Polarization in the absence of 14-3-3. Background polarization was subtracted from all values, n = 3 technical replicates. f Representatives from the four chemotypes of active compounds after screening for stabilizers of the 14-3-3β/CFTRpS753pS768 complex. Source data are provided as a Source Data file.