NMR-based platform for fragment-based lead discovery used in screening BRD4-targeted compounds

Abstract

Aim: Fragment-based lead discovery (FBLD) is a complementary approach in drug research and development. In this study, we established an NMR-based FBLD platform that was used to screen novel scaffolds targeting human bromodomain of BRD4, and investigated the binding interactions between hit compounds and the target protein.

Methods: 1D NMR techniques were primarily used to generate the fragment library and to screen compounds. The inhibitory activity of hits on the first bromodomain of BRD4 [BRD4(I)] was examined using fluorescence anisotropy binding assay. 2D NMR and X-ray crystallography were applied to characterize the binding interactions between hit compounds and the target protein.

Results: An NMR-based fragment library containing 539 compounds was established, which were clustered into 56 groups (8-10 compounds in each group). Eight hits with new scaffolds were found to inhibit BRD4(I). Four out of the 8 hits (compounds 1, 2, 8 and 9) had IC50 values of 100-260 μmol/L, demonstrating their potential for further BRD4-targeted hit-to-lead optimization. Analysis of the binding interactions revealed that compounds 1 and 2 shared a common quinazolin core structure and bound to BRD4(I) in a non-acetylated lysine mimetic mode.

Conclusion: An NMR-based platform for FBLD was established and used in discovery of BRD4-targeted compounds. Four potential hit-to-lead optimization candidates have been found, two of them bound to BRD4(I) in a non-acetylated lysine mimetic mode, being selective BRD4(I) inhibitors.

Figure 1

Chemical structure of ten fragments identified as BRD4(I) hit compounds. Of these ten hit compounds, compound 1 and compound 2 share a quinazolin scaffold, and compound 3 and compound 4 share a [1,2,4]triazolo[1,5-a]pyrimidin core structure.

Figure 2

Inhibitory activities of compound 1, compound 6 and compound 8 on BRD4(I) determined with fluorescence anisotropy binding assays. Quantification plots of the fluorescence anisotropy binding assays for hit compounds 1, 6 and 8 are presented in A, B and C, respectively.

Figure 3

NMR-based characterization of the solution structure of BRD4(I). (A) [¹H, ¹⁵N] HSQC spectrum of BRD4(I). Backbone amide resonance assignments are labeled with the one-letter amino acid code and the sequence number. The insert shows an expanded view of a region with cross-peaks partially overlapped. (B) Consensus chemical shift index (Cα) for BRD4(I). The predicted secondary structural elements are shown together with the CSI plot. (C) Ribbon representation of the crystal structure of BRD4(I) (PDB code: 2OSS).

Figure 4

Chemical shift perturbation (CSP) analysis for BRD4(I) upon hit compound binding. Solid and dashed lines indicate the mean and mean±SD values, respectively. Residues with CSP values above the dashed line are labeled. (A) CSP analysis for BRD4(I) after binding of hit compound 1. (B) CSP analysis for BRD4(I) after binding of hit compound 6. (C) CSP analysis for BRD4(I) after binding of hit compound 7. (D) CSP analysis for BRD4(I) after binding of hit compound 8. (E) CSP analysis for BRD4(I) after binding of hit compound 9. (F) CSP analysis for BRD4(I) after binding of hit compound 10.

Figure 5

Expanded view of BRD4(I)-hit compound co-crystal structures and schematic diagrams of BRD4(I)-hit compound interactions. (A) Expanded view of BRD4(I) bound to hit compound 1. (B) Schematic diagram of BRD4(I)-hit compound 1 interactions. Extensive hydrophobic interactions form between BRD4(I) and hit compound 1, and the involved residues in BRD4(I) and the atoms in hit compound 1 are highlighted with spiked lines. (C) Expanded view of BRD4(I) bound to hit compound 6. (D) Schematic diagram of BRD4(I)-hit compound 6 interactions. The residues in BRD4(I) and the atoms in hit compound 6, which are involved in the hydrophobic interaction network of these two molecules, are highlighted with spiked lines. The hydrogen bonds are highlighted with green dashed lines, and the blue spheres represent water molecules. (E) Expanded view of BRD4(I) bound to hit compound 9. (F) Schematic diagram of BRD4(I)-hit compound 9 interactions. The residues in BRD4(I) and the atoms in hit compound 9, which are involved in the hydrophobic interaction network of these two molecules, are highlighted with spiked lines. The hydrogen bonds are highlighted with green dashed lines, and the blue spheres represent water molecules.