Structure–Activity Relationships of Cbx7 Inhibitors, Including Selectivity Studies against Other Cbx Proteins

Abstract

The five human polycomb (Pc) paralog proteins, chromobox homolog (Cbx) 2/4/6/7/8, are a family of chromodomain containing methyllysine reader proteins that are canonical readers of trimethyllysine 27 on histone 3 (H3K27me3). The aberrant expression of the Cbx7 gene is implicated in several cancers including prostate, gastric, thyroid, pancreas, and colon cancer. Previous reports on antagonizing the molecular recognition of Cbx7–H3K27me3 with chemical inhibitors showed an impact on prostate cancer cell lines. We report here on the design, synthesis, and structure–activity relationships of a series of potent peptidomimetic antagonists that were optimized on a trimethyllysine-containing scaffold to target Cbx7. The ligands were characterized using fluorescence polarization (FP) for their binding efficiency and selectivity against the Pc paralog Cbx proteins. The most selective ligand 9, as indicated by the FP data analysis, was further characterized using the isothermal titration calorimetry (ITC). Compound 9 exhibits a 220 nM potency for Cbx7 and exhibits 3.3, 1.8, 7.3 times selective for Cbx7 over Cbx2/4/8 and 28-fold selective over the HP1 family member Cbx1. Our research provides several potent and partially selective inhibitors for Cbx2/4/7 that do not contain trimethyllysine. Our models and binding data suggest that the aromatic cages of Cbx7/Cbx4 can accommodate larger alkyl groups such as diisobutyl substitution on the lysine nitrogen.

Rational Adaptation of L3MBTL1 Inhibitors to Create Small‐Molecule Cbx7 Antagonists

“…Expression of chromodomain of Cbx7 and its purification, and conducting FP experiments and data analysis of the compounds were performed in similar manner as described in the published reports . Addgene plasmid 25241 for CBX7, deposited by C. Arrowsmith, Structural Genomics Consortium, Toronto (Canada), and encoding for Cbx7 chromodomain residues 7–62, was used for all biochemical binding studies.…”

“…Successful approaches to Cbx7 inhibition have been driven by peptide‐based approaches– and high‐throughput screening for small‐molecule inhibitor approaches . Small‐molecule Cbx7 antagonists MS35472 and MS351 have been shown to de‐repress the p16 gene in PC3 prostate cancer cells …”

Pan-Specific and Partially Selective Dye-Labeled Peptidic Inhibitors of the Polycomb Paralog Proteins

“…chromodomain is the most similar to CBX7 (similarity score of 90%), 3,14 both bind the native histone substrate with similar affinity, 3 and almost all CBX7 ligands reported to date have had similar affinities for CBX4. 9,10,12,13 Interestingly, both 10 and 11 show significantly weaker binding to CBX4 compared to CBX7. Future efforts on selective inhibition of CBX7 may benefit from extended engagement of the peptide-binding groove.…”

“…9,11,12 We have previously reported a peptide-driven approach to identify a series of submicromolar inhibitors targeting CBX4/CBX7 and CBX6. [12][13][14] Potent peptidic inhibitors of CBX4/CBX7 have also been identified by the Frye group and have shown activity in cellular based studies. 9,15 The first small molecule inhibitors of any CBX protein also targeted CBX7.…”

Aza‐amino acid scanning of chromobox homolog 7 (CBX7) ligands

“…The 12 new azapeptide analogues of ligand 1 were evaluated for their affinity for CBX7 in a competitive fluorescence polarization assay that measured the displacement of a dye‐labeled peptide ligand, as previously reported . Most of the new analogues were not measurably active in the assay up to the limits of their most concentrated solutions (0.8–1.8 mM).…”