Figure 7 | Scientific Reports

Figure 7

From: Discovering cell-active BCL6 inhibitors: effectively combining biochemical HTS with multiple biophysical techniques, X-ray crystallography and cell-based assays

Figure 7

NanoBRET assay sensitivity and activity of benzimidazolone compounds. (A) Effect of the NanoBRET configuration on the assay sensitivity to compound inhibition in HEK293T cells. The blue histograms show the NanoBRET ratio for the C-terminally NanoLuc tagged BCL6 and HaloTag tagged SMRT with and without the BCL6 inhibitor 27. The red histograms show the ratio for the N-terminally HaloTag tagged BCL6 and NanoLuc tagged SMRT with and without 27. We selected the former because of its higher sensitivity to compound inhibition after 6 h treatment with 25 µM 27. (B) Concentration responses for 27 in both NanoBRET configurations. An IC50 value could be determined with C-terminally NanoLuc tagged BCL6 (blue triangles), but not when BCL6 was tagged N-terminally with the Halo-Tag (red diamonds). (C) Schematic representation of the optimal NanoBRET configuration for maximal sensitivity to compound inhibition. (D) Plot showing the correlation (R2 = 0.62, n = 175) between biochemical (TR-FRET IC50 on x-axis) and cellular (NanoBRET IC50 on y-axis) activities of inhibitors from the benzimidazolone series. Data points are above the unity line (black), indicating cellular activities were on average an order of magnitude lower than biochemical potencies.

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