was observed against the first bromodomain of BRD4. When tested in the U2OS
cells, target engagement and small-molecule displacement of the CBP bromodomain
from chromatin were verified using a fluorescence recovery after photobleaching
(FRAP) assay.
An improved inhibitor from the Structural Genomics Consortium in Oxford,
I-CBP112 (Figs. 4 and 5), has now been disclosed, with a K d of 151 and 167 nM
for the CBP and p300 bromodomains, respectively, and only low to mid-micromolar
affinity for the BRD4 bromodomains [55]. Although active in cells, and competent
for displacing an isolated bromodomain from chromatin, I-CBP112 was unable to
displace full-length CBP from chromatin in the same FRAP assay, consistent with
additional interactions being necessary to stabilize the protein complex at genomic
loci. However, this molecule did show anticancer activity in bone marrow cells
driven by the MLL-CBP fusion protein impairing clonogenetic growth. Reduced
clonogenetic growth from I-CBP112 treatment was also observed in 12 additional
human cell lines tested.
3.3 BETs
A seminal breakthrough for bromodomain inhibitor discovery occurred in 2010 with
the dual reports of the first submicromolar inhibitors of the BET bromodomains
(BRD2, 3, 4, and T) by GSK [10] and a collaborative study between the Structural
Genomics Consortium in Oxford and the Bradner laboratory at the Dana-Farber
Cancer Institute [9]. These landmark reports which led to invaluable chemical probes
for the BET bromodomains have since set the stage for validating the therapeutic
potential for inhibiting bromodomain function in human clinical trials.
In the GSK study by Nicodeme et al., the researchers developed the 1,2,4-triazolobenzodiazepine inhibitor termed I-BET based on their investigation of inflammation
modulatory compounds for the immune system (Fig. 6) [10]. The lead compound was
developed from an initial reporter assay screen for small-molecule activation of
ApoA1 followed by target identification of the BET bromodomains using an affinity
matrix selection of tethered small molecules. I-BET maintained a K d of 50.5–61.3 nM
for tandem BET bromodomains by ITC and similar affinity in a histone-competitive
FRET assay. Selectivity was further verified against 5 other off-target bromodomains
in a thermal stability study and 38 additional proteins in biochemical assays. In a
cellular context, I-BET-treated bone-derived macrophages stimulated with lipopolysaccharide (LPS) downregulated up to 151 inflammatory genes including expression
of cytokines and chemokines, Il6, Ifnb1, Il1b, and cxcl9. Their results with I-BET
treatment were similar when compared with siRNA genetic knockdown of individual
BETs. However, only the siRNA bromodomain knockdowns affecting TNF levels
supported additional mechanisms for BETs outside of bromodomain interactions. In
2012, 20 years after the initial characterization of a bromodomain structural motif,
this molecule (also called IBET762 and GSK525762) would become the first
Applied Biophysics for Bromodomain Drug Discovery
301
cells, target engagement and small-molecule displacement of the CBP bromodomain
from chromatin were verified using a fluorescence recovery after photobleaching
(FRAP) assay.
An improved inhibitor from the Structural Genomics Consortium in Oxford,
I-CBP112 (Figs. 4 and 5), has now been disclosed, with a K d of 151 and 167 nM
for the CBP and p300 bromodomains, respectively, and only low to mid-micromolar
affinity for the BRD4 bromodomains [55]. Although active in cells, and competent
for displacing an isolated bromodomain from chromatin, I-CBP112 was unable to
displace full-length CBP from chromatin in the same FRAP assay, consistent with
additional interactions being necessary to stabilize the protein complex at genomic
loci. However, this molecule did show anticancer activity in bone marrow cells
driven by the MLL-CBP fusion protein impairing clonogenetic growth. Reduced
clonogenetic growth from I-CBP112 treatment was also observed in 12 additional
human cell lines tested.
3.3 BETs
A seminal breakthrough for bromodomain inhibitor discovery occurred in 2010 with
the dual reports of the first submicromolar inhibitors of the BET bromodomains
(BRD2, 3, 4, and T) by GSK [10] and a collaborative study between the Structural
Genomics Consortium in Oxford and the Bradner laboratory at the Dana-Farber
Cancer Institute [9]. These landmark reports which led to invaluable chemical probes
for the BET bromodomains have since set the stage for validating the therapeutic
potential for inhibiting bromodomain function in human clinical trials.
In the GSK study by Nicodeme et al., the researchers developed the 1,2,4-triazolobenzodiazepine inhibitor termed I-BET based on their investigation of inflammation
modulatory compounds for the immune system (Fig. 6) [10]. The lead compound was
developed from an initial reporter assay screen for small-molecule activation of
ApoA1 followed by target identification of the BET bromodomains using an affinity
matrix selection of tethered small molecules. I-BET maintained a K d of 50.5–61.3 nM
for tandem BET bromodomains by ITC and similar affinity in a histone-competitive
FRET assay. Selectivity was further verified against 5 other off-target bromodomains
in a thermal stability study and 38 additional proteins in biochemical assays. In a
cellular context, I-BET-treated bone-derived macrophages stimulated with lipopolysaccharide (LPS) downregulated up to 151 inflammatory genes including expression
of cytokines and chemokines, Il6, Ifnb1, Il1b, and cxcl9. Their results with I-BET
treatment were similar when compared with siRNA genetic knockdown of individual
BETs. However, only the siRNA bromodomain knockdowns affecting TNF levels
supported additional mechanisms for BETs outside of bromodomain interactions. In
2012, 20 years after the initial characterization of a bromodomain structural motif,
this molecule (also called IBET762 and GSK525762) would become the first
Applied Biophysics for Bromodomain Drug Discovery
301