Figure 3

Screening of an FDA-approved drug library with the MeCP2-TBL1 assay and PRKACA-PRKAR2A counter assay. (A) Distribution of hits from a screen of 1981 compounds from the ApexBio FDA-approved Drug Library assayed at 20 µM. The interaction between MeCP2 and TBL1 was monitored via the luminescence signal in a NanoLuc complementation assay (top panel). Also shown is the distribution of hits from a control screen using a NanoLuc complementation assay based on the interaction between PRKACA and PRKAR2A (middle panel). For the top and middle panels the measured luminescence for each compound was normalized to the mean luminescence obtained with DMSO. The y-axis shows the − log₁₀ of these normalized values. The bottom panel shows inhibition in the MeCP2-TBL1 assay after adjusting for activity of compounds in the PRKACA-PRKAR2A assay. The top five hits (e–i) are indicated above the 35% inhibition threshold (red line). (B) Chemical structures of the non-peptide hits from dual screening of the ApexBio library with the MeCP2-TBL1 assay and the control PRKACA-PRKAR2A assay. (C) Individual validation of the top five hits from the ApexBio library screen against both the MeCP2-TBL1 and the PRKACA-PRKAR2A NanoLuc complementation assays. At 20 µM, flunarizine, luminespib, oridonin, and the alpha-1 antitrypsin fragment 235–243 all achieved statistically significantly greater inhibition in the MeCP2-TBL1 assay than in the control assay (n = 7; * p < 0.001 (t-test)). Error bars represent standard deviations. BIX02189 did not have a statistically different effect between the two assays (n = 7; p = 0.47 (t-test)).