if a few literature reports by the same research group suggested that the role in the
regulation of cell growth and tumorigenesis might be tissue- and context-dependent
[79, 80], CBX7 constitutes an interesting potential drug target.
A first successful approach to find inhibitors of CBX7 resulted from the
truncation of a 25 amino acid long peptide sequence from protein SETDB1, which
had demonstrated a twofold increase in binding affinity for CBX7 when compared
to H3K27me3-containing sequences [77]. Initial truncation led to a 5-mer compound, Ac-FALKme3S-NH2 (Fig. 6), with promising activity when evaluated in
a fluorescence polarization (FP) assay measuring CBX7-H3K27me3 disruption
(IC 50 ¼ 12 μM). From this starting point and with the aid of molecular modeling
studies utilizing the crystal structure of CBX7 in complex with H3K27me3
(PDB: 2L1B), a series of small trimethyllysine-containing analogs of the initial
peptide were synthesized by the iterative, systematic replacement of one residue
on the scaffold at a time (Leu, Ala, Phe, Ser) while keeping the trimethylated lysine
residue. Modifications at Leu (À1) revealed a high tolerance for hydrophobic and/or
aromatic residues such as Val, Ile, Tyr (analog 11, Fig. 6; IC 50 ¼ 6.2 μM), or a
pentane ring [77]. The co-crystal structures of CBX7 in complex with these analogs
confirmed the ligand conformation predicted by modeling studies [77]. It was found
that substituting the C-terminal serine by simple H-bond-donating substituents
like aminobenzimidazole, propanediols, or a pendent ammonium group increases
the binding potency. Finally, a selected set of compounds that comprise all the
favorable modifications to the FALKme3S-scaffold previously gained by SAR
studies was synthesized, which revealed ligands (e.g., analog 64, Fig. 6) with
affinities around 200 nM for CBX7 and tenfold selectivity over CBX8 and even
400-fold selectivity over CBX1, representing the first reported inhibitors of any
chromodomain [77]. Starting from Ac-FALKme3S-NH2 as a lead peptide, a series
of constrained aza-lysine analogs was also prepared with the aim to study the
influence of side chain geometry and ε-amine methylation on affinity [81]. An
aza-amino acid scan was performed in which each amino acid residue of the lead
peptide was replaced by its corresponding semicarbazide counterpart in order to gain
insight into the conformational preferences of the parent peptide. Although the
azapeptides maintain all side chain structural components present in parent ligand,
a loss of affinity was generally seen across the series. Considering the backbone
conformational preferences of azapeptides, as well as the flatter nature of the
semicarbazide residue, such constraints on the lead peptide were not tolerated
in the protein-binding site, likely because they disturb the preferred β-strand
conformation.
Another peptide-based approach led to the identification of the most potent
CBX7 inhibitor reported to date, UNC3866 (Fig. 6). The study was based on
the amino acid sequence RGFALKme3STHG (Fig. 6), which binds CBX7 with
significantly increased affinity compared to H3K27me3 (K D ¼ 5 μM and 110 μM,
respectively) in isothermal calorimetry (ITC) experiments [82]. Several modifications to RGFALKme3STHG designed to increase the hydrophobicity, cell permeability, and stability against lysine demethylases provided the initial lead peptide,
UNC3567 (Fig. 6), which bound with comparable affinity to the parent peptide
(K D ¼ 6.7 μM). A series of molecular dynamics simulations investigating the
Methyl-Readers and Inhibitors
351
regulation of cell growth and tumorigenesis might be tissue- and context-dependent
[79, 80], CBX7 constitutes an interesting potential drug target.
A first successful approach to find inhibitors of CBX7 resulted from the
truncation of a 25 amino acid long peptide sequence from protein SETDB1, which
had demonstrated a twofold increase in binding affinity for CBX7 when compared
to H3K27me3-containing sequences [77]. Initial truncation led to a 5-mer compound, Ac-FALKme3S-NH2 (Fig. 6), with promising activity when evaluated in
a fluorescence polarization (FP) assay measuring CBX7-H3K27me3 disruption
(IC 50 ¼ 12 μM). From this starting point and with the aid of molecular modeling
studies utilizing the crystal structure of CBX7 in complex with H3K27me3
(PDB: 2L1B), a series of small trimethyllysine-containing analogs of the initial
peptide were synthesized by the iterative, systematic replacement of one residue
on the scaffold at a time (Leu, Ala, Phe, Ser) while keeping the trimethylated lysine
residue. Modifications at Leu (À1) revealed a high tolerance for hydrophobic and/or
aromatic residues such as Val, Ile, Tyr (analog 11, Fig. 6; IC 50 ¼ 6.2 μM), or a
pentane ring [77]. The co-crystal structures of CBX7 in complex with these analogs
confirmed the ligand conformation predicted by modeling studies [77]. It was found
that substituting the C-terminal serine by simple H-bond-donating substituents
like aminobenzimidazole, propanediols, or a pendent ammonium group increases
the binding potency. Finally, a selected set of compounds that comprise all the
favorable modifications to the FALKme3S-scaffold previously gained by SAR
studies was synthesized, which revealed ligands (e.g., analog 64, Fig. 6) with
affinities around 200 nM for CBX7 and tenfold selectivity over CBX8 and even
400-fold selectivity over CBX1, representing the first reported inhibitors of any
chromodomain [77]. Starting from Ac-FALKme3S-NH2 as a lead peptide, a series
of constrained aza-lysine analogs was also prepared with the aim to study the
influence of side chain geometry and ε-amine methylation on affinity [81]. An
aza-amino acid scan was performed in which each amino acid residue of the lead
peptide was replaced by its corresponding semicarbazide counterpart in order to gain
insight into the conformational preferences of the parent peptide. Although the
azapeptides maintain all side chain structural components present in parent ligand,
a loss of affinity was generally seen across the series. Considering the backbone
conformational preferences of azapeptides, as well as the flatter nature of the
semicarbazide residue, such constraints on the lead peptide were not tolerated
in the protein-binding site, likely because they disturb the preferred β-strand
conformation.
Another peptide-based approach led to the identification of the most potent
CBX7 inhibitor reported to date, UNC3866 (Fig. 6). The study was based on
the amino acid sequence RGFALKme3STHG (Fig. 6), which binds CBX7 with
significantly increased affinity compared to H3K27me3 (K D ¼ 5 μM and 110 μM,
respectively) in isothermal calorimetry (ITC) experiments [82]. Several modifications to RGFALKme3STHG designed to increase the hydrophobicity, cell permeability, and stability against lysine demethylases provided the initial lead peptide,
UNC3567 (Fig. 6), which bound with comparable affinity to the parent peptide
(K D ¼ 6.7 μM). A series of molecular dynamics simulations investigating the
Methyl-Readers and Inhibitors
351