Small-molecule inhibition of MLL activity by disruption of its interaction with WDR5

Abstract

WDR5 (WD40 repeat protein 5) is an essential component of the human trithorax-like family of SET1 [Su(var)3–9 enhancer-of-zeste trithorax 1] methyltransferase complexes that carry out trimethylation of histone 3 Lys4 (H3K4me3), play key roles in development and are abnormally expressed in many cancers. In the present study, we show that the interaction between WDR5 and peptides from the catalytic domain of MLL (mixed-lineage leukaemia protein) (KMT2) can be antagonized with a small molecule. Structural and biophysical analysis show that this antagonist binds in the WDR5 peptide-binding pocket with a Kd of 450 nM and inhibits the catalytic activity of the MLL core complex in vitro. The degree of inhibition was enhanced at lower protein concentrations consistent with a role for WDR5 in directly stabilizing the MLL multiprotein complex. Our data demonstrate inhibition of an important protein–protein interaction and form the basis for further development of inhibitors of WDR5-dependent enzymes implicated in MLL-rearranged leukaemias or other cancers.

Synthesis, Optimization, and Evaluation of Novel Small Molecules as Antagonists of WDR5-MLL Interaction

“…23 In this assay, binding of the fluorescein-labeled peptide to WDR5 (K D = 458 ± 45 nM) increases the FP signal. Binding compounds compete with and displace the labeled peptide, resulting in the reduction of signal.…”

“…As expected, the compound occupies the central cavity of WDR5 and recapitulates interactions previously observed with less potent analogs (PDB codes 3SMR and 3UR4), including direct and water mediated hydrogen bonds with S91 and C261, opening-up of a cavity occupied by the nitro group, and aromatic stacking with F133. 23 The conformation of side chains within 4 Å of the compound is almost perfectly conserved (RMSD = 0.15 Å). The scaffold sits in the pocket, adopting a conformation closer to that observed for the 2-chlorophenyl analog (RMSD = 0.33 Å, PDB code 3SMR) versus that of the 3-methoxyphenyl analog (RMSD = 1.47 Å, PDB code 3UR4).…”

“…25 As part of our ongoing research involving the discovery and development of potent, selective, and cell-permeable small molecule inhibitors (chemical probes) for epigenetic proteins, 26 we recently disclosed several novel compounds that antagonize WDR5−MLL interaction, including WDR5-0102 (2) ( Figure 1). 23 This proof of principle study prompted us to undertake the SAR-driven optimization of prototypic antagonist WDR5-0102 (2) described in this Letter, leading eventually to preparation of the more potent antagonist 47.…”

“…11,12,23 The amide group of 2 forms one direct and one water-mediated (W517) hydrogen bond with the side chain of S91 and the backbone nitrogen of C261, respectively. The N-methylpiperazine moiety, predicted to be significantly protonated at physiological pH, 27 forms a water-mediated (W576) hydrogen bond with the backbone carbonyl of C261, and it occupies the bottom of the pocket.…”

“…23 To better understand the increased potency of compound 47, we solved its structure in complex with WDR5 (PDB code 4IA9) ( Figure 3). As expected, the compound occupies the central cavity of WDR5 and recapitulates interactions previously observed with less potent analogs (PDB codes 3SMR and 3UR4), including direct and water mediated hydrogen bonds with S91 and C261, opening-up of a cavity occupied by the nitro group, and aromatic stacking with F133.…”