equal affinity [82]. UNC3866 bound with modest affinity to chromodomains
of the PcG and the lesser explored chromodomain Y chromosome (CDY) [83]
protein families but was inactive against HP1 chromodomains and other distinct
methyllysine-binding domains. Key insights into UNC3866 selectivity for CBX7
over other CBX proteins were provided by crystallography studies [82, 84].
Consistent with the inhibition of the H3K27me3 recognition by CBX7, UNC3866
demonstrated modest antiproliferative effects in PC3 cells (GI 50 ¼ 7.6 μM) [82],
an advanced stage metastatic prostate cancer model previously shown to have its
growth regulated by CBX7 [85].
Due to the off-target affinity of UNC3866 for CDY proteins, specifically CDY1
(K D ¼ 6.3 μM), CDYL1b (0.91 μM), and CDYL2 (0.85 μM), a series of CDYL
inhibitors based on its structure were developed [86]. Roughly 14,000 diverse
peptide compounds were synthesized by means of a split-and-pool combinatorial
method and then screened through an on-bead strategy utilizing magnetic enrichment [87, 88]. This approach led to the isolation of ligands with the desired
selectivity profile and improved affinity for CDYL2. The method successfully
identified UNC4991 (Fig. 6) with high affinity for CDYL2. Further evaluation
using ITC found UNC4991 to effectively bind both CDYL2 (K D ¼ 0.64 μM) and
CDYL (K D ¼ 0.49 μM) with $fivefold to sixfold selectivity over CBX7. Notably,
this study further supported the importance of the nature of the N-terminal capping
on chromodomain selectivity. In particular, the tert-butyl phenyl moiety of
UNC3866 facilitates CBX7 binding, while isobutyl, cyclohexyl, and pyridyl caps
favor CDYL2 binding [86].
The first small-molecule inhibitors of the CBX7-H3K27me3 interaction
were identified in 2015 by high-throughput screening of a library of over 100 K
compounds, including 2,560 FDA-approved drugs and other compounds selected
from commercial sources [89]. About 60 compounds were identified as modest
inhibitors of CBX7, 6 of which (sennoside A, suramin, aurin tricarboxylic acid,
trypan blue, and Evans blue from the FDA database and MS37452 from the
commercial compounds library) were confirmed by using 2D
1 H15 N HSQC spectra
and the fluorescence anisotropy-binding assay. Suramin (IC 50 ¼ 8.1 μM) and
MS37452 (also termed MS452, K D ¼ 29 μM, IC 50 ¼ 67 μM; Fig. 7) were chosen
for further structural and biological evaluation. The co-crystal structure of suramin
in complex with CBX7 demonstrated that two suramin molecules bound to
two CBX7 molecules in two distinct orientations to create an interwoven, compact
2:2 complex [89]. Yet, although the trypanocidal suramin has also recently demonstrated anticancer activity [90], it is well-characterized as a promiscuous screening
compound in a variety of different screening contexts [91, 92]. Therefore, the
study focused on MS37452. The co-crystal structure of the complex between
CBX7 and MS37452 showed that the ligand adapts two rotamer conformations
in which the methylbenzene moiety is positioned either cis or trans with
respect to the 2,3-dimethoxybenzene [89]. Structural studies demonstrated that the
dimethoxybenzene and piperazine interact with the CBX7 aromatic cage residues
(Phe11, Trp32, and Trp35) in both orientations, but the methylbenzene moiety in
the trans conformation more closely mimics the intermolecular interactions between
Methyl-Readers and Inhibitors
353
of the PcG and the lesser explored chromodomain Y chromosome (CDY) [83]
protein families but was inactive against HP1 chromodomains and other distinct
methyllysine-binding domains. Key insights into UNC3866 selectivity for CBX7
over other CBX proteins were provided by crystallography studies [82, 84].
Consistent with the inhibition of the H3K27me3 recognition by CBX7, UNC3866
demonstrated modest antiproliferative effects in PC3 cells (GI 50 ¼ 7.6 μM) [82],
an advanced stage metastatic prostate cancer model previously shown to have its
growth regulated by CBX7 [85].
Due to the off-target affinity of UNC3866 for CDY proteins, specifically CDY1
(K D ¼ 6.3 μM), CDYL1b (0.91 μM), and CDYL2 (0.85 μM), a series of CDYL
inhibitors based on its structure were developed [86]. Roughly 14,000 diverse
peptide compounds were synthesized by means of a split-and-pool combinatorial
method and then screened through an on-bead strategy utilizing magnetic enrichment [87, 88]. This approach led to the isolation of ligands with the desired
selectivity profile and improved affinity for CDYL2. The method successfully
identified UNC4991 (Fig. 6) with high affinity for CDYL2. Further evaluation
using ITC found UNC4991 to effectively bind both CDYL2 (K D ¼ 0.64 μM) and
CDYL (K D ¼ 0.49 μM) with $fivefold to sixfold selectivity over CBX7. Notably,
this study further supported the importance of the nature of the N-terminal capping
on chromodomain selectivity. In particular, the tert-butyl phenyl moiety of
UNC3866 facilitates CBX7 binding, while isobutyl, cyclohexyl, and pyridyl caps
favor CDYL2 binding [86].
The first small-molecule inhibitors of the CBX7-H3K27me3 interaction
were identified in 2015 by high-throughput screening of a library of over 100 K
compounds, including 2,560 FDA-approved drugs and other compounds selected
from commercial sources [89]. About 60 compounds were identified as modest
inhibitors of CBX7, 6 of which (sennoside A, suramin, aurin tricarboxylic acid,
trypan blue, and Evans blue from the FDA database and MS37452 from the
commercial compounds library) were confirmed by using 2D
1 H15 N HSQC spectra
and the fluorescence anisotropy-binding assay. Suramin (IC 50 ¼ 8.1 μM) and
MS37452 (also termed MS452, K D ¼ 29 μM, IC 50 ¼ 67 μM; Fig. 7) were chosen
for further structural and biological evaluation. The co-crystal structure of suramin
in complex with CBX7 demonstrated that two suramin molecules bound to
two CBX7 molecules in two distinct orientations to create an interwoven, compact
2:2 complex [89]. Yet, although the trypanocidal suramin has also recently demonstrated anticancer activity [90], it is well-characterized as a promiscuous screening
compound in a variety of different screening contexts [91, 92]. Therefore, the
study focused on MS37452. The co-crystal structure of the complex between
CBX7 and MS37452 showed that the ligand adapts two rotamer conformations
in which the methylbenzene moiety is positioned either cis or trans with
respect to the 2,3-dimethoxybenzene [89]. Structural studies demonstrated that the
dimethoxybenzene and piperazine interact with the CBX7 aromatic cage residues
(Phe11, Trp32, and Trp35) in both orientations, but the methylbenzene moiety in
the trans conformation more closely mimics the intermolecular interactions between
Methyl-Readers and Inhibitors
353