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. 2019 Jun 7;8:e47172. doi: 10.7554/eLife.47172

Figure 3. Differential HDX-MS and activity screening of 38 RORγ modulators reveals structural determinants for activation.

(A) Δ%D (difference from DMSO treated control) values for 38 RORγ modulators are plotted as a heat map according to the color key at the bottom left. Each row represents a compound and each column represents a peptide indicated by the labels at the right and bottom respectively. The locations of each peptide in the crystal structure are labeled along the top. A hierarchical clustering algorithm based on Ward’s method was performed to cluster compounds based on their exchange signatures and the corresponding dendrogram is shown on the left. (B-J) Activity correlation analysis results found that structural dynamics measurements correlate with functional activity. Regions of RORγ that correlate with coactivator peptide recruitment, thermal stability, and activation in cell-based assays are shown in panel D, G, and J, respectively. Lighter shades indicate that the slope of a linear regression was significantly (adjusted p value < 0.05) non-zero, while darker shades indicate an R (Takeda et al., 2012) was greater than 0.4. Representative coactivator peptide recruitment correlation plots for helix 12 and helix four are shown in B and E, respectively. The correlation between thermal stability and the BSR peptide are shown in panel (H). Representative correlation plots cell-based activity are shown for H2, H12 and H7 are shown in panels C, F, and I, respectively.

Figure 3.

Figure 3—figure supplement 1. Summary and reproducibility of biochemical and two timepoint HDX-MS screening data (related to Figures 3 and 4).

Figure 3—figure supplement 1.

38 RORγ modulators were tested in AlphaScreen-based peptide recruitment assays and thermal shift assays in two separate preparations of compounds and protein to assess reproducibility. The run to run comparisons are shown in panel (A and B). The distributions of the compound activities are shown in panels (C and D). AlphaScreen-based peptide recruitment activity correlated with fluorescent polarization-based peptide recruitment EC50 values as shown in panel (E). The Reproducibility of 2 timepoint HDX-MS screening was performed using two batches of protein and the results of the run-to-run correlations are shown in panel (F).