selective [42]. This study used a promiscuous inhibitor scaffold based on the
pan-bromodomain inhibitor, bromosporine [43]. Virtual screening efforts of over
12,000 compounds were then applied and a thermal stability assay (differential
scanning fluorimetry, DSF) implemented to verify hits. The optimized inhibitor
with a K d of 126 nM for PCAF only had off-target binding to one human
bromodomain, the highly homologous bromodomain of GCN5 (K d ¼ 600 nM).
Target engagement was carried out by a NanoBRET assay in HEK-293 cells and
possessed a 40 min half-life against human liver microsomes demonstrating good
metabolic stability for in vivo studies. This compound is available from the Structural Genomics Consortium along with a negative control enantiomer D-Moses.
During the same time period, Constellation and Genentech patented several PCAF
inhibitors, including a pyridazinone scaffold (WO/2016/112298A1), that GSK
researchers also reported on [44]. The lead compound GSK4027 (Fig. 3) and
negative control GSK4028 are also available from the SGC and have improved
binding affinity for PCAF and GCN5 with a K i of 1.4 nM for both proteins and
>70-fold selectivity against other human bromodomains.
3.2 CBP/p300
The CREB-binding protein (CBP) and homologous protein p300 are large
coactivator proteins possessing histone acetyltransferase activity, histone binding
ability via its bromodomain, and additional protein-protein interaction modules
for binding transcriptional activators, including the KIX, CH1, and CH2 domains
[45]. This protein has been shown to be essential in the development with genetic
knockouts resulting in embryonic lethality [46]. Aberrant function of the protein and
associated transcription factors are linked to Rubinstein-Taybi syndrome [47],
cardiac ischemia [48], leukemia [49], and ovarian, breast, and lung cancers [50].
The bromodomain of CBP was the second target for small-molecule development
in 2006 also by the Zhou lab for disrupting an acetylated p53 interaction [48]. The
tumor suppressor activity of p53 is often attempted to be preserved as an anticancer
therapy; however, in this report Sachchidanand et al. sought to reduce hyperactivity
of p53 during myocardial ischemia which leads to cardiomyocyte death. The
mechanism for such a response is via acetylation of p53 at K382 leading to
p53-mediated recruitment of CBP to chromatin and initiation of gene transcription
in response to DNA damage.
Sachchidanand et al. designed a focused library of acetylated lysine mimics,
consisting of an N-acetyl amine connected to diverse aromatic rings [48]. These
200 compounds were screened by 2D
1 H15 N HSQC NMR in mixtures of 8 compounds. Following deconvolution of hits based on chemical shift perturbation,
computational docking and an NMR solution structure led to MS7972 (PDBID
2D82). A K d of 19 μM was subsequently determined via a tryptophan intrinsic
fluorescence assay in addition to blocking acetylated p53 interactions in vitro.
Preliminary data showed effects at decreasing p21 levels in U2OS cells at high
Applied Biophysics for Bromodomain Drug Discovery
299
pan-bromodomain inhibitor, bromosporine [43]. Virtual screening efforts of over
12,000 compounds were then applied and a thermal stability assay (differential
scanning fluorimetry, DSF) implemented to verify hits. The optimized inhibitor
with a K d of 126 nM for PCAF only had off-target binding to one human
bromodomain, the highly homologous bromodomain of GCN5 (K d ¼ 600 nM).
Target engagement was carried out by a NanoBRET assay in HEK-293 cells and
possessed a 40 min half-life against human liver microsomes demonstrating good
metabolic stability for in vivo studies. This compound is available from the Structural Genomics Consortium along with a negative control enantiomer D-Moses.
During the same time period, Constellation and Genentech patented several PCAF
inhibitors, including a pyridazinone scaffold (WO/2016/112298A1), that GSK
researchers also reported on [44]. The lead compound GSK4027 (Fig. 3) and
negative control GSK4028 are also available from the SGC and have improved
binding affinity for PCAF and GCN5 with a K i of 1.4 nM for both proteins and
>70-fold selectivity against other human bromodomains.
3.2 CBP/p300
The CREB-binding protein (CBP) and homologous protein p300 are large
coactivator proteins possessing histone acetyltransferase activity, histone binding
ability via its bromodomain, and additional protein-protein interaction modules
for binding transcriptional activators, including the KIX, CH1, and CH2 domains
[45]. This protein has been shown to be essential in the development with genetic
knockouts resulting in embryonic lethality [46]. Aberrant function of the protein and
associated transcription factors are linked to Rubinstein-Taybi syndrome [47],
cardiac ischemia [48], leukemia [49], and ovarian, breast, and lung cancers [50].
The bromodomain of CBP was the second target for small-molecule development
in 2006 also by the Zhou lab for disrupting an acetylated p53 interaction [48]. The
tumor suppressor activity of p53 is often attempted to be preserved as an anticancer
therapy; however, in this report Sachchidanand et al. sought to reduce hyperactivity
of p53 during myocardial ischemia which leads to cardiomyocyte death. The
mechanism for such a response is via acetylation of p53 at K382 leading to
p53-mediated recruitment of CBP to chromatin and initiation of gene transcription
in response to DNA damage.
Sachchidanand et al. designed a focused library of acetylated lysine mimics,
consisting of an N-acetyl amine connected to diverse aromatic rings [48]. These
200 compounds were screened by 2D
1 H15 N HSQC NMR in mixtures of 8 compounds. Following deconvolution of hits based on chemical shift perturbation,
computational docking and an NMR solution structure led to MS7972 (PDBID
2D82). A K d of 19 μM was subsequently determined via a tryptophan intrinsic
fluorescence assay in addition to blocking acetylated p53 interactions in vitro.
Preliminary data showed effects at decreasing p21 levels in U2OS cells at high
Applied Biophysics for Bromodomain Drug Discovery
299