Analysis of the binding of mixed lineage leukemia 1 (MLL1) and histone 3 peptides to WD repeat domain 5 (WDR5) for the design of inhibitors of the MLL1-WDR5 interaction

Abstract

MLL1 is a histone 3 lysine 4 (H3K4) methyltransferase and a promising new cancer therapeutic target. The catalytic activity of MLL1 is regulated by the formation of a core complex consisting of MLL1, WDR5, RbBP5, and Ash2L. The interaction between WDR5 and MLL1 plays an essential role in regulation of the H3K4 methyltransferase activity of MLL1 and targeting this interaction using small molecules may represent an attractive therapeutic strategy. In this study, we have defined the essential elements in MLL1 required for its high-affinity binding to WDR5. Our data showed that the minimal elements crucial for high-affinity binding of MLL1 to WDR5 are -CO-ARA-NH- motif and two intramolecular hydrogen bonds that stabilize the conformation of this motif. Two 3-mer peptides, Ac-ARA-NH(2) and Ac-ART-NH(2), were designed based upon MLL1 and H3 sequences and achieved K(i) values of 120 and 20 nM to WDR5, respectively. Our study provides a concrete basis for the design of potent peptidomimetics and nonpeptidic compounds to inhibit MLL1 activity by targeting the MLL1 and WDR5 interaction.

Figure 1

Binding models for Ac-ARA-NH₂ and H₂N-ARA-NH₂ in complex with WDR5 and key intramolecular hydrogen bonds within these peptides. (a) Hydrogen bonds 1 and 2 and the atoms involved in Ac-ARA-NH₂ peptide. (b) Predicted binding model of Ac-ARA-NH₂ in complex with WDR5. (c) Predicted binding model of H₂N-ARA-NH₂ in complex with WDR5. (b,c) The intramolecular hydrogen bonds are shown as yellow dots. Carbon atoms are shown in cyan in the peptide and gray in WDR5. The nitrogen and oxygen atoms are colored in blue and red, respectively. (d) Probability of the hydrogen bond distance between the nitrogen and oxygen pairs in hydrogen bonds 1 and 2 in Ac-ARA-NH₂ based upon MD simulation. (e) Probability of the hydrogen bond distance between the nitrogen and oxygen pairs in hydrogen bond 2 in NH2-ARA-NH₂ based upon MD simulation.

Figure 2

Saturation binding experiments of tracers with WDR5. (a) Saturation curves of WIN-FAM-1, WIN-FAM-2, 10mer-Thr-FAM, and 10mer-Ala-FAM with WDR5; 0.6 nM of the tracer was used for these experiments. (b) Stability of the saturation binding experiment for 10mer-Thr-FAM tracer over 24 h. mP values were measured at the times indicated.

Figure 3

Competitive binding curves for Ac-10mer, Ac-3mer, WIN, and H₂N-10mer as determined using a fluorescence polarization based assay.