within the first bromodomain of BRD4 and was identified as one of the origins
of high selectivity for this compound [30] (Fig. 3). A similar binding mode and
displacement of structured waters has also been reported for a selective BRG1/PB1
bromodomain inhibitor, PFI-3 [28].
Additional chromatin-binding domains are typically found in close proximity to
bromodomains as a means to further increase affinity and specificity in both chromatin and transcriptional complexes. These include proximal PHD domains, such as
those found in BPTF [32] and BRPF [33]; KIX domains, as seen in p300/CBP [34];
as well as tandem bromodomains [20]. TAFII250/TAF1 was the earliest example
where high-resolution structural information was obtained for a multidomain
bromodomain-containing protein, as well as being the first x-ray crystal structure
of a bromodomain (PDBID: 1EQF) [23]. In this case, Tijan and co-workers crystallized the tandem bromodomains. Isothermal titration calorimetry (ITC) analysis,
with both di- and tetraacetylated H4 histones, yielded stoichiometries of either 1:2
or 1:1, supporting engagement of both domains through a single peptide in the
hyperacetylated state. In later studies of the bromodomain and extraterminal (BET)
family of bromodomains, certain diacetylated histones were shown to engage both
the canonical histone binding site, with secondary interactions with the ZA and BC
bromodomain loops, whereas in other cases, engagement of two bromodomains was
observed such as for histone H4 acetylated at positions 8 and 12 [20]. In addition to
histone interactions, Tijan and co-workers speculated on the possibility of weak
DNA interactions with TAFII250. These interactions were thought to occur on the
basic face of the bromodomain and core histone interactions on its acidic face.
Such identifications of weak DNA interactions have since been experimentally
supported [35].
By 2012, driven by the growing structural biology data and disease-associated
behavior of aberrant bromodomain function, there was considerable interest in the
development of small-molecule inhibitors of histone-bromodomain interactions for
drug discovery. As a resource for such initiatives, Filippakopolous and co-workers
performed a large-scale structural analysis of bromodomains and biophysical determination of histone-bromodomain interactions [20]. These researchers contributed
an additional 29 high-resolution bromodomain crystal structures including 25 new
protein structures. Due to improved structural algorithms and BLAST searches, they
were able to reclassify bromodomains into a cluster of 8 protein families consisting
of 61 distinct bromodomains from 46 human proteins. In the class VIII family of
bromodomains, a new β-hairpin insert was identified as a new structural motif. These
families of bromodomains represent the current state of the field today. As of 2018,
there now exist over 1,050 structures of human and nonhuman bromodomains
(Table 1) serving as an excellent starting point for characterizing native interactions
and design of new inhibitors. A figure of the current bromodomain phylogenetic tree
including information on available crystal structures and chemical probes is compiled in Fig. 4.
To address the role of acetylation-dependent bromodomain interactions which
underlie the histone code, these researchers further went on to characterize 485 histone-bromodomain interactions using peptide arrays which included both histone
Applied Biophysics for Bromodomain Drug Discovery
295
of high selectivity for this compound [30] (Fig. 3). A similar binding mode and
displacement of structured waters has also been reported for a selective BRG1/PB1
bromodomain inhibitor, PFI-3 [28].
Additional chromatin-binding domains are typically found in close proximity to
bromodomains as a means to further increase affinity and specificity in both chromatin and transcriptional complexes. These include proximal PHD domains, such as
those found in BPTF [32] and BRPF [33]; KIX domains, as seen in p300/CBP [34];
as well as tandem bromodomains [20]. TAFII250/TAF1 was the earliest example
where high-resolution structural information was obtained for a multidomain
bromodomain-containing protein, as well as being the first x-ray crystal structure
of a bromodomain (PDBID: 1EQF) [23]. In this case, Tijan and co-workers crystallized the tandem bromodomains. Isothermal titration calorimetry (ITC) analysis,
with both di- and tetraacetylated H4 histones, yielded stoichiometries of either 1:2
or 1:1, supporting engagement of both domains through a single peptide in the
hyperacetylated state. In later studies of the bromodomain and extraterminal (BET)
family of bromodomains, certain diacetylated histones were shown to engage both
the canonical histone binding site, with secondary interactions with the ZA and BC
bromodomain loops, whereas in other cases, engagement of two bromodomains was
observed such as for histone H4 acetylated at positions 8 and 12 [20]. In addition to
histone interactions, Tijan and co-workers speculated on the possibility of weak
DNA interactions with TAFII250. These interactions were thought to occur on the
basic face of the bromodomain and core histone interactions on its acidic face.
Such identifications of weak DNA interactions have since been experimentally
supported [35].
By 2012, driven by the growing structural biology data and disease-associated
behavior of aberrant bromodomain function, there was considerable interest in the
development of small-molecule inhibitors of histone-bromodomain interactions for
drug discovery. As a resource for such initiatives, Filippakopolous and co-workers
performed a large-scale structural analysis of bromodomains and biophysical determination of histone-bromodomain interactions [20]. These researchers contributed
an additional 29 high-resolution bromodomain crystal structures including 25 new
protein structures. Due to improved structural algorithms and BLAST searches, they
were able to reclassify bromodomains into a cluster of 8 protein families consisting
of 61 distinct bromodomains from 46 human proteins. In the class VIII family of
bromodomains, a new β-hairpin insert was identified as a new structural motif. These
families of bromodomains represent the current state of the field today. As of 2018,
there now exist over 1,050 structures of human and nonhuman bromodomains
(Table 1) serving as an excellent starting point for characterizing native interactions
and design of new inhibitors. A figure of the current bromodomain phylogenetic tree
including information on available crystal structures and chemical probes is compiled in Fig. 4.
To address the role of acetylation-dependent bromodomain interactions which
underlie the histone code, these researchers further went on to characterize 485 histone-bromodomain interactions using peptide arrays which included both histone
Applied Biophysics for Bromodomain Drug Discovery
295