Targeting Ligandable Pockets on Plant Homeodomain (PHD) Zinc Finger Domains by a Fragment-Based Approach

Abstract

Plant homeodomain (PHD) zinc fingers are histone reader domains that are often associated with human diseases. Despite this, they constitute a poorly targeted class of readers, suggesting low ligandability. Here, we describe a successful fragment-based campaign targeting PHD fingers from the proteins BAZ2A and BAZ2B as model systems. We validated a pool of in silico fragments both biophysically and structurally and solved the first crystal structures of PHD zinc fingers in complex with fragments bound to an anchoring pocket at the histone binding site. The best-validated hits were found to displace a histone H3 tail peptide in competition assays. This work identifies new chemical scaffolds that provide suitable starting points for future ligand optimization using structure-guided approaches. The demonstrated ligandability of the PHD reader domains could pave the way for the development of chemical probes to drug this family of epigenetic readers.

Figure 1

Druggable pockets on BAZ2A/B PHD and validated fragments. (A) Crystal structure of BAZ2A PHD in complex with ART tripeptide. F_o – F_c electron density map of the peptide is contoured at 3σ. The R2 side chain of the peptide is not visible in the electron density. (B) Close-up view of the interactions. (C) Druggable binding sites in BAZ2A PHD (PDB: 4QF2) identified by FTMap, shown as green mesh. Protein surface is colored according to the electrostatic potential. (D) Overlay of (¹⁵N–¹H) HSQC spectra recorded on the apo form of ¹⁵N-BAZ2B PHD (blue) and after 5 mM fragment addition (red). Arrows represent the shift direction. (E) Chemical structures of the in silico fragments validated by HSQC. Fragments reporting binding by NMR to the histone pocket are shown in red, and fragments reporting binding by NMR to the back pocket are shown in blue.

Figure 2

Biophysical and structural validation of fragment hits. (A) Docking pose of BAZ2B PHD and Fr3 showing a set of residues shifted in HSQC and clustered at the histone pocket. Residues are colored according to the intensity of the shifts: strong shifts in red (Δδ > + 2σ), intermediate shifts in orange (Δδ > + σ) and lower shifts or no shifts in green (Figure S3). (B) Docking pose of BAZ2B PHD and Fr7 with shifts clustered at the back pocket of BAZ2B and close-up view of in silico predicted interactions. (C) Crystal structure of BAZ2A PHD in complex with Fr19 (in sticks, with green carbons). F_o – F_c electron density map is contoured at 3 σ around the bound fragment. The Thr3 methyl hydrophobic pocket is colored in yellow, and the acidic wall is red. (D) Chemical structures of optimized fragments.

Figure 3

Insights on the binding mode of optimized fragments. (A) Crystal structure of BAZ2B PHD in complex with Fr21 bound to the histone pocket. (B) Crystal structure of BAZ2A PHD in complex with Fr23 bound to the histone pocket. F_o – F_c electron density map of the fragments is in gray and contoured at 2.5σ. (C) Superposition of BAZ2 PHDs in complex with Fr23. The black arrow shows the dihedral angle. The red arrow shows the different orientation of Val1713 in BAZ2A and Ile1968 in BAZ2B. (D) Relative torsion energy of surrogate arylamides. The observed dihedral angles of the parent compounds in complex with BAZ2 are highlighted with an arrow. (E) AlphaLISA dose–response curves of Fr23. The error bars represent the standard deviation of each point (see the Supporting Information for more details). (F) Superposition of BAZ2A in complex with Fr23 and ART peptide.