Impact of WIN site inhibitor on the WDR5 interactome.

The chromatin-associated protein WDR5 is a promising pharmacological target in cancer, with most drug discovery efforts directed against an arginine-binding cavity in WDR5 called the WIN site. Despite a clear expectation that WIN site inhibitors will alter the repertoire of WDR5 interaction partners, their impact on the WDR5 interactome remains unknown. Here, we use quantitative proteomics to delineate how the WDR5 interactome is changed by WIN site inhibition. We show that the WIN site inhibitor alters the interaction of WDR5 with dozens of proteins, including those linked to phosphatidylinositol 3-kinase (PI3K) signaling. As proof of concept, we demonstrate that the master kinase PDPK1 is a bona fide high-affinity WIN site binding protein that engages WDR5 to modulate transcription of genes expressed in the G2 phase of the cell cycle. This dataset expands our understanding of WDR5 and serves as a resource for deciphering the action of WIN site inhibitors.

Structural Elucidation of Peptide Binding to KLHL-12, a Substrate Specific Adapter Protein in a Cul3-Ring E3 Ligase Complex.

KLHL-12 is a substrate specific adapter protein for a Cul3-Ring ligase complex. It is a member of the Kelch ß-propeller domain subclass of Cullin-Ring substrate recognition domains. This E3 ubiquitin ligase complex has many activities, including acting as a negative regulator of the Wnt signaling pathway by mediating ubiquitination and subsequent proteolysis of Dvl3/Dsh3. KLHL-12 is also known to mediate the polyubiquitination of the dopamine D4 receptor (D4.2), the ubiquitination of KHSRP, a protein that is involved in IRES translation, and also the ubiquitination of Sec31, which is involved in endoplasmic reticulum-Golgi transport by regulating the size of COPII coats. Earlier studies broadly defined the substrate binding regions for D4.2 and Dvl3/Dsh3 to KLHL-12. We tested several peptides from these regions and succeeded in identifying a short peptide that bound to KLHL-12 with low micromolar affinity. To better understand the sequence specificity of this peptide, we used alanine substitutions to map the important residues and obtained an X-ray structure of this peptide bound to KLHL-12. This structure and our peptide affinity measurements suggest a sequence motif for peptides that bind to the top face of KLHL-12. Understanding this binding site on KLHL-12 may contribute to efforts to find small molecule ligands that can either directly inhibit the degradation of substrate proteins or be used in targeted protein degradation strategies using PROTACs.

Discovery and Structure-Based Optimization of Potent and Selective WD Repeat Domain 5 (WDR5) Inhibitors Containing a Dihydroisoquinolinone Bicyclic Core.

WD repeat domain 5 (WDR5) is a member of the WD40-repeat protein family that plays a critical role in multiple chromatin-centric processes. Overexpression of WDR5 correlates with a poor clinical outcome in many human cancers, and WDR5 itself has emerged as an attractive target for therapy. Most drug-discovery efforts center on the WIN site of WDR5 that is responsible for the recruitment of WDR5 to chromatin. Here, we describe discovery of a novel WDR5 WIN site antagonists containing a dihydroisoquinolinone bicyclic core using a structure-based design. These compounds exhibit picomolar binding affinity and selective concentration-dependent antiproliferative activities in sensitive MLL-fusion cell lines. Furthermore, these WDR5 WIN site binders inhibit proliferation in MYC-driven cancer cells and reduce MYC recruitment to chromatin at MYC/WDR5 co-bound genes. Thus, these molecules are useful probes to study the implication of WDR5 inhibition in cancers and serve as a potential starting point toward the discovery of anti-WDR5 therapeutics.

Size-exclusion chromatography using deuterated mobile phases.

The effect of deuterated solvents in size-exclusion chromatography (SEC) was studied by comparing intrinsic viscosity measurements, SEC calibration curves, and column efficiency using water-soluble polymers. For aqueous SEC, the use of deuterium oxide slightly increases the SEC elution volume. To verify that adsorption onto the packing was absent, data from exclusion experiments were compared at 35 and 50 degrees C. Our results indicate that adsorption is not occurring for pullulan or polyethylene glycol (PEG)/poly(ethylene oxide) (PEO); for the latter, however, the elution volume increased using both D2O and H2O, indicative of slight hydrodynamic volume contraction of PEG/PEO at higher temperatures. A moderate increase in band broadening (moderate decrease in column efficiency) was observed using D2O. Finally, the effects of chloroform versus deuterated chloroform were evaluated, but no hydrodynamic volume changes were observed.