proteins of this target class are 
 classified into readers , writers , and erasers of marks on histones 
 or other nuclear proteins and dna .
the 
 complex combinations of these posttranslational marks regulate gene 
 expression and have been termed the  histone code  .
bromodomains represent one of the readers 
 of these marks , specifically recognizing acetyl - lysine ( kac ) through 
 an architecturally conserved interaction module .
sixty - one unique bromodomains have been identified from 
 the human genome , each containing a conserved 
 tertiary structure as described by mutjaba et al .
, with the hydrophobic kac binding site at 
 one end formed between the z short helix , the za loop , and 
 the bc loop ( figure 1a ) .
this binding site 
 is primarily hydrophobic , with the carbonyl oxygen of the acetyl - group 
 forming two hydrogen bonds , one to a donor from either asparagine 
 or threonine and the other to a conserved water molecule at the base 
 of the pocket ( figure 1b , c ) .
( a ) conserved protein 
 fold of bromodomains comprising the four canonical helices z , 
 a , b , and c .
all illustrated by falz ( pdb 3qzs ) . through discovery of potent small molecule inhibitors 
 ( figure 2 ) , bet 
 family members
, a definition that will 
 be used throughout the paper : proteins able ( or predicted to be able ) 
 to bind drug - like molecules ( not necessarily a drug ) .
a short hairpin rna screen 
 suggested that inhibition of the bet family may be a therapeutic strategy 
 for aml . through discovery of pan - bet 
 family inhibitor gsk1210151a from the isoxazole class ,
it has been 
 suggested that inhibition of the bet family may be a therapeutic strategy 
 for mll - fusion leukemia , and pan - bet family inhibitor gsk525762a , 
 from the benzodiazepine class , has demonstrated anti - inflammatory 
 potential in mouse models of inflammatory disease and sepsis .
inhibitors of other bromodomains ( crebbp and pcaf ) have been found 
 ( figure 2 ) , but 
 none show the submicromolar inhibition reported for bet family inhibitors 
 so far .
bromodomains are currently an underexplored protein family 
 in both basic biology and drug discovery , however , therapeutic potential 
 is becoming increasingly recognized . with many bromodomain structures 
 publicly available ,
this led us to investigate the structure - based 
 druggability across the protein family .
selected published bromodomain 
 inhibitors . from an initial inspection of various bromodomain 
 binding sites
, we hypothesized that not all bromodomains would be 
 as druggable as the bet family and a wide range of druggabilities 
 would be observed .
further , we wanted to identify variations in the 
 amino acids within the binding site that correlated with predicted 
 druggability .
prediction of the druggability of a novel protein 
 target allows realistic expectations of hit rates before any screening 
 effort is undertaken . for a less druggable target ,
the acceptable 
 potencies and associated ligand efficiencies are likely to be lower 
 than for a more druggable one and there is an associated risk of not 
 finding tractable hit matter . in this scenario , alternative strategies 
 may be sought such as higher screening concentrations , the use of 
 larger and more diverse libraries , or the choice of screening technique 
 employed .
one analysis of the druggability across a protein 
 family was performed by campagna - slater et al . on another epigenetic 
 target family , the histone methyltransferases . in this study ,
sitemap was used alongside the degree of 
 buried surface area of the bound cofactor to assess the druggability . 

all of the histone methyltransferases were predicted to be druggable 
 with dscores from sitemap ranging from 0.96 to 1.13 but with the degree 
 of buried surface area of the cofactor showing some variability .
another study has also recently been published , performed by santiago 
 et al . primarily on methyl - lysine
sitemap 
 was also used in this work , and the authors suggest the methyl - lysine 
 binding proteins to be less druggable than bromodomains .
however , 
 they only consider the eight members of the bet family that may not 
 be representative of the family as a whole .
many structure - based druggability 
 prediction methods have been published in recent years , these include 
 dlid , dogsitescorer , the ebi s drugebility , drugpred , fpocket , mappod , screen , and sitemap .
cover a number of these methods and some of 
 the challenges in computational druggability assessment .
to 
 assess the druggability of bromodomain proteins , we required a tool 
 that is readily available but more importantly allows water molecules 
 to be included in the analysis .
this is necessary as we have identified 
 five water molecules that appear to be conserved for most bromodomains 
 and reduce the overall volume of the pocket . to our knowledge ,
the 
 use of sitemap is consistent with the analyses of the histone methyltransferases 
 and methyl lysine binding proteins highlighted above .
a detailed validation 
 of this method has been published , with sitemap accurately identifying 
 86% of the ligand binding sites from a set of 538 complexes of the 
 pdbbind database as the top scoring site .
, demonstrating comparable performance of sitemap 
 to fpocket on their nonredundant data set ( nrdd ) .
sitemap uses the same definition of druggable as we are 
 using here and uses contributions from the volume of the pocket , the 
 enclosure , and the degree of hydrophobicity to assess druggability . 

the main output from sitemap is two druggability assessment scores : 
 sitescore ( eq 1 ) and dscore ( eq 2 ) where n is the number of site points , e is the enclosure score , and p is the 
 hydrophilic score.12 both scores take contributions from 
 the same properties but with different coefficients .
both scores use 
 a cap of 100 for the number of site points ( for our analysis only 
 two structures reach this cap ) , and sitescore uses a cap of 1.0 for 
 the hydrophilic score , whereas dscore is not capped . for our data 

set , the two scores have high correlation , with r equal to 0.92 . because of the high correlation with 
 sitescore and the suggestion that it is more discriminatory of druggable 
 and undruggable sites , dscore was selected 
 to be used alone in our analyses .
sitemap was applied to a filtered 
 set of the published bromodomain structures extracted from the pdb , and a wide range of predicted druggabilities 
 was observed , from difficult to druggable . from this initial druggability 
 assessment ,
the dscores were compared with the clustering generated 
 from whole sequence similarity of structure - based alignments by filippakopoulos 
 et al .
this analysis showed that whole 
 sequence similarity alone did not sufficiently explain the trends 
 in the druggability that were observed , therefore we inspected the 
 binding sites and identified unique amino acid signatures that showed 
 better correlation with observed druggabilities .
we propose that this 
 new classification is more relevant to small molecule binding than 
 whole sequence similarity due to its focus on the binding site residues . 

it allows druggability prediction of bromodomains without structural 
 characterization and will aid the selection of templates for homology 
 models by comparison to members within the same classification .
crucially , 
 it also enables the medicinal chemist to identify family members that 
 are likely to bind the same inhibitors as the targeted bromodomain , 
 which can be explored either for lead hopping or selectivity screening .
many bromodomain - containing proteins possess 
 multiple bromodomains but also exist as different sequence isoforms . 
 when referring to a single bromodomain of one isoform , we have used 
 this format : bromodomain - containing protein name , followed by isoform 
 if present ( a / b / c if isoforms are identical ) , followed by the bromodomain 
 number . for example , the second bromodomain of the b isoform of brd8 
 would be shortened to brd8b(2 ) , and the single bromodomain of baz1a , 
 which is identical between isoforms a and b , would be shortened to 
 baz1a(a / b ) .
when referring to different chains within a pdb 
 file , we have used this format : pdb code followed by a letter corresponding 
 to the number of the protein chain within the file .
for example , the 
 second chain of the protein brd1 in pdb 3rcw would be shortened to 3rcw_b .
protein chains within each pdb 
 file were separated , ligands and nonconserved water molecules removed , 
 and protonation states assigned using protonate3d in moe .
forty - six chains from 14 pdb files with unresolved 
 binding site residues were filtered out ( supporting 
 information , table s1 ) .
bound state , resolution , presence of 
 unresolved side chains , and presence of conserved water molecules 
 were recorded for each chain . for taf1(a
/ b ) , whereby both bromodomains 
 have been crystallized within one peptide chain , these were separated 
 and treated individually .
individual chains were then preprocessed 
 using the protein preparation wizard in 
 maestro with 
, 
  create disulfide bonds  , and  convert selenomethionines 
 to methionines  options selected .
the preprocessed 
 chains were submitted to sitemap using default parameters and with 
  identify top - ranked potential receptor binding sites  
 to avoid any bias from using ligands / peptides to define pockets .
the 
 minimum number of site points per pocket identified needed to be reduced 
 to 14 from 15 for pb1(a / b / c)(1 ) .
kac binding sites were then selected 
 from all identified sites and all outputted values recorded .
module 
 and the blosum62 matrix with default settings . as used in the full sequence alignment by filippakopoulos 
 et al .
, we have also used brd4(a / b)(1 ) 
 as a reference sequence for numbering of the residues .
surfaces are color - coded using the pocket 
 coloring from moe with green indicating enclosed surface of the protein 
 and white indicating exposed .
having selected sitemap to assess 
 druggability , the next step was to collect the available crystal structures 
 from the pdb .
this yielded 105 different pdb entries covering 33 of 
 the 61 unique human bromodomains .
these pdb entries were then separated 
 into the separate protein chains , as each protein chain within a crystal 
 structure can be of a different conformation , and any chains with 
 unresolved residues in the binding site were removed . through inspection 
 of available bromodomain structures , it was apparent that five water 
 molecules are conserved across most bromodomain kac binding sites . 

no publicly available structures demonstrate the displacement of any 
 of these by a ligand ( figures 1a , 3 , and supporting information , figure s1 ) , suggesting that the water 
 molecules are an important feature of the binding pocket .
frequently , 
 water molecules are removed prior to druggability assessment and we 
 decided to determine druggability in the presence and absence of these 
 conserved water molecules to assess their effect on the dscore .
all 
 five water molecules could be identified in structures of 23 of the 
 28 unique bromodomains passing the requirement of a structure without 
 unresolved binding site residues , although not all of the water molecules 
 were always present in the same structure due to limitations of protein 
 crystallography ( most frequently in low resolution structures ) . to 
 maximize our coverage of the observed protein conformations
while 
 ensuring that all assessed structures contained the same number of 
 water molecules , for structures with missing water molecules , structures 
 of the same protein with the missing water molecules were aligned 
 and the missing water molecules were included from the other structure(s ) . 
 for smarca4 , a high resolution ( 1.50  ) structure and
one bound 
 with nmp were available and both of these structures demonstrate only 
 four of the five water molecules present , so the druggability assessment 
 has been assessed as such , raising the number of bromodomains initially 
 considered to 24 .
details of all the water molecules included can 
 be found in supporting information , table s3 . conserved water molecules in the binding site of brd4(a / b)(1 ) ( pdb 3mxf ) .
the absence of these water molecules led to a larger 
 identified pocket and consequently a higher druggability score , with 
 most of the bromodomains classified in the druggable range ( dscore 
 > 0.85 ) .
crucially , without the water molecules , a smaller range 
 of scores was observed , making the assessment less discriminative 
 between sites ( figure 4 ) .
given that these water molecules enclose the pocket and 
 have a significant effect on the druggability , all subsequent analysis 
 was performed with the water molecules present .
( a ) plot 
 of dscores obtained when conserved water molecules were included in 
 analysis against the same structures with all water molecules removed . 
 linear line of best fit added to plot .
( b ) histogram of same data 
 showing distribution of scores with normal distribution fitted to 
 this data .
colors indicate druggability classification : red , druggable ; 
 yellow , intermediate ; white , difficult .
the 178 qualifying 
 protein chains ( 24 of the 61 unique human bromodomains ) were prepared 
 and submitted to sitemap druggability assessment .
a wide range of 
 druggabilities was observed for the bromodomains from difficult ( dscore 
 <
0.75 ) ( e.g. , baz2b pdb 3q2f , dscore = 0.52 ) to druggable ( dscore > 0.85 ) ( e.g. , 
 pcaf pdb 3gg3_b , dscore = 1.08 ) . scores in between these two have been classified 
 as intermediate ( e.g. , crebbp pdb 3p1e_b , dscore = 0.82 ) ( figure 5 ) . details of all outputted 
 scores from this assessment can be found in supporting 
 information , table s3 .
box - plots showing range and distribution of 
 druggability for each bromodomain across available structures passing 
 imposed filters ( including presence of binding site water molecules )
colors indicate druggability classification : 
 red , druggable ; yellow , intermediate ; white , difficult .
( a ) four outliers 
 removed from druggability assessment ( see group 4 text ) .
most bromodomains contain a small and tight binding 
 site to recognize kac of the protein substrate .
this conveys a basic 
 level of druggability as demonstrated by baz2b ( pdb 3q2f , dscore 0.52 ) and 
 the potential to bind a small fragment with acceptable ligand efficiency ( nmp , baz2b le 0.29 ) .
the differences between the sites stem from the environments around 
 that small and tight pocket . for
the bromodomain family as a 
 whole , even the lowest scoring bromodomain ( pb1(a / b / c)(1 ) ) would be 
 classed as more druggable than a protein 
protein interaction 
 with a large ( > 1000  ) and fairly featureless 
 interface .
sitemap would fail to identify binding sites such as this 
 ( e.g. , siah1 pdb 2a25 ) . at the other end of the druggability scale ,
some bromodomains 
 have demonstrated comparable predicted druggability to what are currently 
 considered druggable targets such as protein kinases ( e.g. , aurora 
 a , pdb 1mq4 , 
 dscore = 0.96 ) or hsp90 ( pdb 1am1 , dscore = 0.99 ) .
when a ligand or peptide binds , the observed conformation of the protein 
 may change to potentially induce a more druggable pocket .
this has 
 been seen for the bcl-2 family of proteins whereby small molecule 
 inhibitors bind to pockets not observed in the apo or peptide bound 
 structures and show improved potency .
this is in line with an increase in the sitemap predicted druggabilities 
 of the bcl - xl binding sites from apo ( pdb 1r2d_a , dscore = 0.76 ) to ligand bound ( abt-737 , 
 pdb 2yxj , dscore 
 = 0.95 ) . to assess whether this could be the case for bromodomains , 

those with both apo and ligand or peptide bound ( holo ) structures 
 available were collated and the dscores from sitemap compared ( table 1 ) .
dscores after four outliers were 
 removed ( see group 4 text for details ) .
colors : white , bromodomains for which a more druggable 
 pocket was not observed for the holo structures ; green , bromodomains 
 for which a more druggable pocket was observed for the holo structures 
 but a comparably druggable pocket was observed in the ensemble of 
 apo structures ; red , bromodomains for which a more druggable pocket 
 was observed for the holo structures and a comparably druggable pocket 
 was not observed in the ensemble of apo structures . from table 1 , it can be seen 
 that only crebbp of the 12 bromodomains with apo and holo structures 
 available show evidence of a more druggable pocket ( 0.050.1 
 increase in median dscore ) in the presence of a ligand or peptide 
 that is not observed in the ensemble of apo structures .
the median 
 dscore for the entire ensemble of structures was classed as intermediate 
 at 0.75 , but the highest scoring structure was classed as druggable 
 at 0.89 ( pdb 3p1c_b ) when bound with kac .
three bromodomains ( brd2(2 ) , baz2b , 
 and pb1(a / b / c)(5 ) ) unexpectedly show reduced median druggability for 
 the ligand or peptide bound structures when compared to the apo .
comparably 
 druggable conformations of the holo structures for brd2(2 ) and pb1(a / b / c)(5 ) 
 within the range of the apo are observed in the full ensemble , but 
 this is not the case for baz2b .
these effects will be discussed later , 
 in the context of the similarity of each bromodomain with other members 
 of the family .
other than for crebbp , in general it does not appear 
 that ligand binding is able to induce a significantly more druggable 
 structure than is observed in the apo ensemble for the bromodomain 
 family . having completed the initial computational druggability assessment , 
 the next step was to identify trends within the data set . when compared 
 with the clustering generated from whole sequence similarity performed 
 by filippakopoulos et al .
, a lack of correlation 
 was observed such as the first and second bromodomains of taf1 being 
 placed in the same cluster despite dscores at either end of the scale . 

it is not surprising to see a lack of correlation between druggability 
 and whole sequence similarity , as when dealing with a druggability 
 assessment it is the nature of the binding site that affects the score , 
 not the rest of the domain .
for this reason , we decided to inspect 
 the structures for binding site for features that vary across the 
 bromodomain family and can be used to order the members of the family 
 into groups .
this led to the identification of eight groups 
 characterizing 49 of the 61 unique bromodomains .
each of these groups 
 is defined by the presence of a unique signature of up to three amino 
 acid residues that is shared by all members of that particular group 
 and gives a characteristic shape to the kac binding site ( table 2 , figure 9 , and supporting information , figure s1 ) .
taken together , 
 the amino acid residues of all group signatures span seven residues 
 that enclose the kac binding site . these were position 81 , which is 
 a tryptophan in the bet family and forms
what has been termed the 
 za - channel , the two residues facing the 
 binding pocket on the za - loop , both leucine at position 92 and 94 
 in the bet family , the residue at position 140 , which is most commonly 
 asparagine and forms the key hydrogen bond donor interaction with 
 the kac carbonyl , residues 144 and 146 on the bc - loop and c - helix , 
 which enclose the hydrophobic shelf in the bet family , and residue 
 149 , which although not enclosing the pocket does influence the position 
 of residue 81 , which can have a large effect on both the za - channel 
 and hydrophobic shelf .
residue 145 on the c - helix has also been included 
 in the analysis , which although not part of any of the signatures 
 has been used in further differentiating some of the bromodomains 
 within each of the groups .
thus , in total , eight residues have been 
 used to characterize the bromodomain binding sites ( figure 6 ) .
number of bromodomains with available 
 structures passing the filters out of the number of members in the 
 group .
eight residues around the binding site used in analysis ( brd4(a / b)(1 ) 
 used as reference , pdb 3mxf ) .
( b ) binding site residues shown with transparent surface representation . to determine which residues were present at each 
 position ,
available bromodomain structures were overlaid with the 
 reference structure , brd4(a / b)(1 ) ( pdb 3mxf ) , and the eight binding site residues 
 were recorded that best aligned with the brd4(a / b)(1 ) residues ( supporting information , table s5 and figure s1 ) . for five of the eight identified residues ( 140 , 144 , 145 , 146 , 
 and 149 ) , the spacial alignment corresponded with the sequence alignment , 
 making the identification of the residues for the bromodomains without 
 a structure straightforward .
for the other three residues , due to 
 the variation in length and position of the za - loop , spatial alignment 
 did not always correspond with the sequence alignment .
here we have 
 used the residue that aligned best in space with the brd4(a / b)(1 ) 
 structure , as this should be more relevant to the nature of the binding 
 pocket . for bromodomains without a structure , the matching residue 
 from a protein within the same grouping from the alignment of filippakopoulos 
 et al . was used ( e.g. , between brpf1b 
 and brpf3 ) . in a few cases ,
this was not always possible due to the 
 lack of a sufficiently homologous bromodomain with a structure being 
 available ( e.g. , mll1 or trim28 ) .
these bromodomains have been excluded 
 from groups that are characterized by the za - loop residues ( 81 , 92 , 
 and 94 ) . using the binding site groupings obtained , a qualitative classification 
 tree was generated ( figure 7 ) .
this allowed plotting of the predicted druggabilities to 
 visualize where the most druggable groups are as well relationships 
 between the groups .
these included the relationship between crebbp 
 and ep300 with the bet family as they share the extended length of 
 the za - loop , the relationship between groups 2 and 3 ( y or f at position 
 146 ) , and between groups 5 and 6 ( y or f at position 94 ) ( see group 
 texts for more details ) .
bromodomain classification tree generated on 
 the basis of eight binding site amino acid signatures showing bromodomain 
 druggability .
druggabilities of ash1l , atad2b , brpf1b , pb1(a / b / c)(3 ) , 
 pb1(a / b / c)(4 ) , and smarca2b assessed using structures failing imposed 
 filters included ( see group text ) .
furthermore , we further divided several groups 
 into subclassifications such as the separation of the baz family within 
 group 4 by exploring changes in whole sequence similarity that may 
 have an effect on the overall fold of the bromodomains such as the 
 za loop position or more subtle changes in the binding site that have 
 smaller effects on the pockets than the signature residues .
we believe that this grouping better explains the trend in druggability 
 assessment than whole sequence similarity , but also that this grouping 
 will predict small molecule selectivity patterns more accurately due 
 to the focus on the binding site .
this should prove useful when determining 
 selectivity of inhibitors and the potential to identify possibilities 
 to transfer hit matter from one bromodomain to another .
another potential 
 use of this grouping is for the building of a homology model .
if the 
 use of the model is to predict the binding mode of a small molecule 
 inhibitor or to select compounds in a virtual screening approach , 
 then the choice of template is very important .
the bromodomains grouped 
 together here share binding site features , and thus members of the 
 same group should represent the best templates for building homology 
 models for binding mode prediction . when comparing the clustering based on whole sequence similarity 
 to the grouping performed here , differences can be observed ( figure 8) .
the two classifications 
 are not dramatically different ( 42/61 placed in the same group ) , which 
 is not surprising , but there is sufficient difference to suggest that 
 when dealing with small molecule inhibitors the binding site classification 
 may be more informative .
groups from left 
 to right in same number order as table 2 with 
 the same coloring .
whole sequence classification colors generated 
 from binding site classification group which shares highest percentage 
 similarity .
an example of the differences is baz1a(a / b ) that 
 shares whole sequence similarity with the bet family but does not 
 share binding site similarity with this family . it is therefore unlikely 
 to bind similar ligands and likely possesses druggability similar 
 to that of the group it has been placed in by binding site classification 
 ( group 4 ) .
another example is that atad2a and atad2b are placed in 
 the same cluster as group 3 by whole sequence similarity but do not 
 share binding site similarity with this group or any other bromodomain .
each of the binding site classification groups will now be discussed 
 individually , commenting on their druggability , but also any trends 
 or inconsistencies within the groups .
the bet family of bromodomains was classified as druggable , which 
 correlates with the fact that a number of potent small molecule inhibitors 
 have been found .
the comparatively high 
 druggability for this family can be explained by a more enclosed upper 
 part of the pocket and thus additional surface area for interaction 
 with small molecules not present in other less druggable bromodomains . 

this is predominantly due to the presence of a tryptophan residue 
 at position 81 and a methionine residue at position 149 that influences 
 the position of the tryptophan residue , forming the za channel .
on 
 the other side of the pocket , the za loop is longer than most other 
 bromodomains , providing additional surface that can be utilized for 
 interaction with small molecules .
these features result in above - average 
 druggability of the sites ( figure 9a ) .
bromodomains aligned with 
 brd4(a / b)(1 ) pdb 3mxf and colors generated using moe pocket coloring : green = enclosed 
 and white = exposed .
pocket colors are used to highlight binding sites 
 and does not represent pockets identified by sitemap used for druggability 
 assessment .
images captured from the same viewpoint except image g 
 at the same orientation as figure 6 . ( a )
( g ) pb1(a / b / c)(1 ) pdb 3iu5 structure representative of group 8 . 

( h ) crebbp pdb 3p1c_b structure . given the high similarity of the pockets , it is 
 not surprising that nonselective bet family inhibitors have been found , 
 although there are subtle differences between the first and second 
 bromodomains of the bet family that could be exploited for selectivity . 

the entire bet family has the same za loop residues facing the pocket , 
 but the first bromodomains possess an aspartate at position 144 whereas 
 the second bromodomains possess a histidine . at position 145
, the 
 entire bet family has an acidic residue but this changes between aspartate 
 and glutamate .
we have separated this group in the classification 
 tree into the first and second bromodomains to reflect these small 
 changes .
an outlier in the druggability assessment was seen 
 for a peptide bound structure of brd2(2 ) ( pdb 2e3k_b , dscore = 0.64 ) . 

the reason for this low score was the position of the tryptophan at 
 position 81 .
for the rest of the bet family ( and other brd2(2 ) structures ) , 
 this residue is directed toward the binding site creating the za channel 
 ( figure 10a ) . in 
 this outlier ,
the tryptophan residue is directed away from the pocket , 
 opening the pocket significantly ( figure 10b ) and inducing a pocket more like crebbp or ep300 ( leucine at position 
 81 ) ( figure 10c ) , greatly reducing the druggability . 
 for the 56 bet family structures passing the initial filters ,
this 
 is the only one for which the tryptophan is oriented away from the 
 binding site , suggesting that this is an unusual conformation and 
 does not appear relevant to small molecule inhibitor binding .
( c ) crebbp 
 pdb 3dwy_a structure 
 showing similarity to brd2(2 ) atypical conformation .
four of these ( cecr2 , falz(a / b ) , gcn5l2 , and pcaf ) were classified 
 at the high end of the druggability scale by sitemap and are within 
 the same cluster by whole sequence similarity .
the key features of 
 these pockets are the aromatic residue at position 146 compared with 
 a small hydrophobic residue in many other bromodomains and a tryptophan 
 at position 81 . together , these signature residues provide a significant 
 amount of hydrophobic surface on this side of the pocket .
the za loop 
 is two amino acids shorter than the bet family , but this part of the 
 pocket is still sufficiently enclosed to provide high druggability 
 ( figure 9b ) .
these bromodomains represent a 
 family that demonstrate high predicted druggability but to date have 
 not been exploited with high affinity compounds .
two outliers 
 were seen for the bromodomain falz(a / b ) ( pdb 2f6n_a and 2fsa_c ) , which both scored 
 significantly less than the other nine structures passing the imposed 
 filters of this bromodomain .
when inspecting the structures and comparing 
 them with more druggable conformations of falz(a / b ) , no obvious changes 
 could be seen , as was the case with brd2(2 ) . however , when examined 
 more closely , it could be seen that both the za loop and the bc loop 
 are moved slightly away from the pocket , inducing a more open conformation 
 and thus reducing the druggability .
the four structures that are peptide 
 bound do not demonstrate this more open conformation and may be stabilized 
 in the closed conformation by the presence of the peptide .
interestingly , 
 the remaining two members of group 2 ( taf1(a / b)(2 ) and taf1l(2 ) ) possess 
 the same signature residues but are not present in the same whole 
 sequence classification as the other members of group 2 .
taf1(a / b)(2 ) 
 also scored in the druggable range ( dscore = 0.89 ) , however , taf1l(2 ) 
 scored in the difficult range ( dscore = 0.73 ) , possibly due to the 
 za loop and tryptophan 81 positions opening the binding site . with 
 only one structure available , this could be an example of a false 
 negative , with an effect similar to the outliers of falz(a / b ) ( pdb 2f6n_a and 2fsa_c ) , and with further 
 conformational sampling of the za loop and tryptophan 81 , a more druggable 
 conformation could be observed .
taf1(a / b)(2 ) and taf1l(2 ) differ to 
 the other members of group 2 by the residues at position 94 , 145 , 
 and 149 within the binding site as well as having reduced sequence 
 similarity ; for these reasons they have been given their own subclassification 
 in the classification tree .
group 3 contains six bromodomains , 
 which all fall into the same classification from the whole sequence 
 similarity and are related to the group 2 by the presence of the aromatic 
 residue at position 146 , enclosing this part of the pocket .
they differ 
 by the lack of the tryptophan at position 81 opening the za channel , 
 so the druggability scores are somewhat lower , placing them in the 
 intermediate category ( figure 9c ) . within the 
 group , brd7 and brd9
have been given their own subclassification due 
 to the changes in za loop residues and having tyrosine rather than 
 phenylalanine at position 146 .
although a crystal structure 
 for brpf1b was not available , an nmr structure ( pdb 2d9e ) was and passed 
 the filters other than the presence of the conserved water molecules . 
 when sitemap druggability assessment was applied to the ensemble of 
 structures , a median dscore of 1.04 was obtained ( supporting information , table s4 ) and is slightly higher but 
 in a similar range to the dscore values obtained from the other members 
 of the group without water present . using the lines of best fit from 
 figure 4a and a subset of the values from this 
 group ,
estimates of the dscore with water molecules were achieved 
 of 0.91 and 0.97 respectively .
these values are higher than other 
 members of the group and places this bromodomain in the druggable 
 category .
the four members of the baz family 
 cluster together by binding site similarity within group 4 , unlike 
 the whole sequence similarity classification .
the group is characterized 
 by a shorter za loop than the bet family , with no residue overlapping 
 with leucine 92 from brd4(a / b)(1 ) in space , making the pocket fairly 
 open and reducing druggability .
the baz family share the tryptophan 
 at position 81 with the bet family , but this does not form the same 
 za channel due to the change in residue at position 149 ( figure 9d ) . for the bet family , this is a methionine , which 
 restricts the movement of the tryptophan forming the za channel , but 
 in the baz family , this residue is small ( alanine or cysteine ) , which 
 results in movement of the tryptophan toward residue 149 , removing 
 the za channel and hydrophobic shelf present in the bet family and 
 heavily reducing the druggability into the difficult category .
the baz family is joined by trim24 , trim33a , and trim66 in this group , 
 and although these do not possess a tryptophan at position 81 , they 
 share a very similar za loop , with no residue overlapping with the 
 leu92 from the bet family .
this open part of the pocket , and the lack 
 of a za channel , give these bromodomains similar pockets to the baz 
 family .
they have been given their own subclassification due to this 
 change in position 81 from tryptophan to a leucine or valine .
four structures of trim24 score highly in the druggability assessment 
 and appear to be outliers ( supporting information , 
 table s4 ) .
when inspecting the sites identified by sitemap , 
 it was apparent that the favorable score is not solely due to the 
 kac binding site but also an extended site ranging from the kac binding 
 site to the interface between the bromodomain and the adjacent phd . 
 for this reason , the analysis performed here has excluded these data 
 points .
the kac binding site is better assessed by the other generated 
 scores , placing it in the difficult range , but there may be small 
 pockets close to the kac binding site which could be exploited by 
 using fragments followed by a linking effort .
baz2b surprisingly 
 indicated reduced druggability of the ligand bound structure when 
 compared to the apo ( 0.18 reduction in dscore ) . from the initial definition 
 of druggability
, it would be expected that in general , holo structures 
 should be as druggable if not more so than their apo counterparts . 

when comparing the two baz2b structures ( pdb 3g0l and 3q2f ) , there are only 
 subtle differences between the two conformations of the binding site , 
 namely that for the ligand bound structure the pocket is slightly 
 narrower due to movements of the za loop and bc loop , increasing the 
 enclosure score ( 0.610.69 ) .
this narrowing most likely occurs 
 to maximize contact with the flat heteroaromatic part of the ligand , 
 but in doing this reduces the volume of the most enclosed part of 
 the pocket ( 105  to 92  ) . for 
 sitemap druggability assessment ( particularly for low druggability 
 sites )
, the reduction in volume counts more toward the dscore than 
 the increase in enclosure , so the overall effect is to reduce the 
 predicted druggability of the ligand bound structure relative to the 
 apo .
group 5 is characterized 
 by the presence of an aromatic residue at position 94 , the effect 
 of this is to provide a  lid  to the pocket , thus increasing 
 the enclosure and therefore the druggability ( figure 9e ) .
structures of pb1(a / b / c)(3 ) and pb1(a / b / c)(4 ) were 
 available ( pdb 3k2j and 3tlp ) , 
 but these structures were excluded from the initial analysis due to 
 them missing some of the conserved water molecules . to include them 
 in the analysis and to allow direct comparison of the dscores , water 
 molecules from the highly similar pb1(a / b / c)(2 ) were included through 
 alignment of the structures .
this yielded median druggabilities for 
 the two bromodomains of 0.57 and 0.70 , respectively , placing them 
 in the difficult category ( supporting information , 
 table s4 ) .
phip(2 ) scored highest and was placed in the 
 druggable range . the second , third , fourth , and sixth bromodomains 
 of pb1 also fall into this grouping but none show as high a druggability 
 as phip(2 ) and also show less whole - sequence similarity , and this 
 has been indicated with a different subclassification with pb1a(6 ) 
 given its own subclassification due to a four - residue shorter za loop . 

the phip(2 ) structure does have a ligand bound , so this reduced druggability 
 of the pb1 members could either be due to lack of protein conformational 
 sampling ( za loop position ) and be a false negative or could be due 
 to more subtle effects influencing the overall conformation of the 
 protein and therefore the druggability .
the members of group 
 6 also share this aromatic residue at position 94 , but without any 
 available structures it is difficult to say whether these would be 
 druggable like phip(2 ) or more challenging like many of the pb1 bromodomains . 

unlike group 5 , group 6 members cluster together by whole sequence 
 similarity , however , there are six other bromodomains that share whole 
 sequence similarity with group 6 but do not appear to share binding 
 site similarity .
the four proteins in this group 
 all fall into the same classification by whole sequence similarity . 
 by whole sequence similarity , group 7 is joined by four other bromodomains 
 ( pb1(a / b / c)(24 ) and pb1a(6 ) ) but do not share the signature 
 residues and do not fall into this group .
pb1(a / b / c)(5 ) was classified 
 in the intermediate druggability range but smarca4 as difficult .
all 
 of these proteins possess a shorter za loop than the bet family , reducing 
 the surface available for interaction with small molecules .
the shape 
 of the kac binding pockets are also different to those of the bet 
 family in the available structures , with the location of the aromatic 
 residue at position 139 moved toward the pocket and leucine at position 
 87 rather than the valine present in most other bromodomains .
this 
 induces a wider entrance , opening the tight binding pocket at the 
 base of the binding site ( figures 9f and 11a ) . for this reason
, 
 it is expected that this group could bind ligands differently to the 
 other groups , as the tightest , most conserved part of the binding 
 site is significantly different .
( a ) pb1(a / b / c)(5 ) structure representative 
 of group 7 .
( c ) overlaid backbones of usual conformation ( pdb 3g0j_a ) in green and 
 unusual ( pdb 3g0j_b ) in blue . orientation and coloring consistent with figure 9 .
one structure of pb1(a / b / c)(5 ) that failed the 
 original filters on the presence of the conserved water molecules 
 ( pdb 3g0j_b ) 
 showed a particularly unusual conformation that is unlike any conformation 
 of this bromodomain or any other bromodomain ( figure 11b ) .
the za loop is moved toward the z helix , which 
 is not possible in many other bromodomains due to the presence of 
 alanine at position 81 ( figure 11c ) but may 
 also be allowed due to the change in shape of the binding site discussed 
 previously .
the effect of this is to close this part of the pocket , 
 which for many of the other bromodomains is the location of the za 
 channel .
this results in an increase in hydrophobicity of the remaining 
 pocket and reduced preference for the conserved water molecules . when 
 sitemap druggability assessment was applied to this structure ( with 
 a single water molecule at the base of the pocket ) a dscore of 0.87 
 was achieved , which is significantly higher than any other conformation 
 of this protein and places it in the druggable range .
this unusual 
 conformation of the protein may represent an opportunity for inhibiting 
 this bromodomain selectively over any other , as this unusual conformation 
 is not expected to be common and may be unique to pb1(a / b / c)(5 ) .
this 
 conformation may also allow for substitution of the water molecules , 
 which appear to be highly conserved for most other bromodomains . as was the case for brpf1b ,
no crystal structure was available for 
 smarca2b , but an nmr structure was available that passed the filters 
 imposed apart from the presence of the conserved water molecules ( pdb 2dat ) . when sitemap druggability 
 assessment was applied to the ensemble of structures , a median dscore 
 of 0.81 was achieved ( supporting information , 
 table s4 ) , which is higher than the dscore for smarca4 without 
 water present but lower than the dscore for pb1(a / b / c)(5 ) .
when converted , 
 as with brpf1b , using the dscore values with and without water molecules 
 of the other bromodomains and the other members of the group from 
 figure 4a , estimates of the dscore with water 
 molecules of 0.53 and 0.64 were obtained .
this places smarca2b in 
 the difficult range with comparable predicted druggability to smarca4 , 
 which shares high whole sequence similarity . for the binding of kac to bromodomains
, 
 a key hydrogen bond is formed between the carbonyl of the acetyl group 
 and a donor from either an asparagine or threonine residue at position 
 140 .
for group 8 , this key residue is 
 replaced with tyrosine ( eight bromodomains ) or aspartic acid ( mll1 : 
 although protein construct has been engineered and may not be true 
 for full length protein ) .
this changes the nature of the pocket significantly , 
 and it has been suggested that these domains may not bind kac , or 
 if they do , the manner in which they do would be unlike most other 
 bromodomains .
mll1 has been given its 
 own subclassification due to it possessing an aspartate at position 
 140 as have the sp family due to their high sequence similarity to 
 each other over the other members .
a structure for pb1(a / b / c)(1 ) 
 is available which shows how tyrosine 140 reduces the size of the 
 pocket ( figure 9 g ) , and sitemap assessed this 
 site as the least druggable of the bromodomains with only a very small 
 pocket being identified .
with such a low assessed druggability , the 
 only opportunity to target this site with a small molecule inhibitor 
 would be to displace the conserved water molecules .
but , even with 
 the water molecules removed , the site only achieves a dscore in the 
 low end of the intermediate range suggesting that pb1(1 ) would be 
 very challenging to bind small molecules to the equivalent of the 
 kac binding site of other bromodomains .
a structure for ash1l 
 ( pdb 3mqm ) , 
 another member of this group , is also available , but the conserved 
 water molecules are not present as is the case for pb1(a / b / c)(1 ) , 
 so it was removed by the initial filter .
the site scored comparably 
 to pb1(a / b / c)(1 ) without water molecules with a median dscore of 0.74 , 
 suggesting that this bromodomain would be similarly difficult to target 
 with a small molecule inhibitor .
the remaining bromodomains 
 failed to be placed into any groups larger than two , with little similarity 
 to any of the other groupings described here .
of those with available 
 structures , none showed any particular druggability as assessed by 
 sitemap , except crebbp , which was classified in the intermediate range . 

crebbp is interesting as it possesses the same longer za loop as the 
 bet family , with similar residues facing the binding site that provide 
 similar interaction potential .
however , the tryptophan at position 
 81 in the bet family , which forms the za channel and hydrophobic shelf , 
 is a considerably smaller leucine , resulting in a loss of these features 
 and a decrease in predicted druggability ( figure 9h ) .
another unusual feature is the presence of an arginine 
 at position 145 , which provides the potential to form charged interactions 
 with this strongly basic center .
flexibility of both the za loop and 
 the unusual arginine could explain the large changes in predicted 
 druggability of crebbp , with the highest scoring protein conformation 
 being placed in the druggable category and the lowest in the difficult . 
 with high sequence similarity and binding site similarity
, the bromodomain 
 of ep300 would be expected to bind similar ligands to crebbp and have 
 similar potential for a more druggable pocket to be induced . similarly to pb1(a / b / c)(3 ) and pb1(a / b / c)(4 ) , a structure of atad2b 
 ( pdb 3lxj_d ) 
 was available that was filtered out due to missing two of the conserved 
 water molecules .
all five water molecules were present in a structure 
 of the similar atad2a ( pdb 3dai ) , and through aligning the two structures , the missing 
 water molecules of atad2b were included .
sitemap druggability assessment 
 yielded a score of 0.64 , placing this bromodomain in the difficult 
 category . when targeting any protein with small molecule inhibitors , selectivity 

for the bromodomains , the highly conserved small 
 and tight binding site ( binding acetyl part of kac ) at the base of 
 the pocket makes prediction of selectivity for a low molecular weight 
 ( < 200 da ) fragment challenging as it is the environment around 
 this site which will determine the selectivity for larger molecules . 
 from this analysis ,
the first proteins that should be tested for selectivity 
 issues would be those within the same group ( figure 7 ) .
there are , however , some similarities between groups that 
 may give rise to comparable binding of small molecules .
the groups 
 that are related to each other that have been previously discussed 
 ( groups 2 and 3 and groups 5 and 6 ) may bind similar ligands , but 
 there are differences that may be exploited for selectivity . also 
 as discussed , crebbp and ep300 show some similarity through the za 
 loop to the bet family , but also phip(2 ) and wdr9(a / b)(2 ) show some 
 similarity in the shape of the binding site with the same hydrophobic 
 shelf and above average druggability .
other than these , selectivity 
 would be expected between groups for molecules larger than small fragments . adequately assessing the full ensemble of protein conformations 
 is an issue that affects any prediction that uses crystal structures , 
 as by their nature a static image is observed . to address this issue , 

we have used as many experimentally observed conformations of the 
 bromodomains as possible , and scores generated by the druggability 
 assessment do vary between conformations of the same protein , including 
 different protein chains within the same crystal structure . for this 
 reason , we can not rule out that some proteins that were classified 
 as difficult or intermediate may be false negatives and may have the 
 potential to be druggable with additional conformational sampling . 
 however , the range of scores observed for different conformations 
 of the same bromodomain appear to be less than those between different 
 bromodomains due to changes in the eight selected residues identified 
 here ( figure 5 ) . for this reason ,
it is possible 
 that new bromodomain conformers may show slightly increased druggability 
 than predicted from the currently available structures ( e.g. , intermediate 
 instead of difficult or druggable instead of intermediate ) .
however , 
 it is unlikely that large leaps will occur ( e.g. , for a protein classified 
 as difficult to move into the druggable category ) , and other than 
 a few special cases discussed in the text ( brd2(2 ) , falz(a / b ) and 
 crebbp ) , these have not so far been observed .
predicted druggabilities of available bromodomain 
 structures were assessed and a range of scores observed .
the bet family 
 members were predicted to be druggable , consistent with literature 
 evidence .
one group ( group 2 ) showed comparable or increased predicted 
 druggability relative to the bet family and represents a currently 
 unexplored group of proteins that may have relevance in drug discovery 
 as their biology is revealed .
many of the other bromodomains showed 
 lower predicted druggability and some of these were classed as difficult 
 based on their dscore .
the comparatively low score suggests that these 
 will show lower hit rates in screening efforts and that it will be 
 more challenging to identify and optimize hit matter .
however , it 
 should be noted that even these bromodomains are far more druggable 
 than featureless protein  protein interactions .
trends 
 within the data set were then sought and rationalized by unique signatures 
 characterizing the binding pockets , leading to a new classification 
 of the bromodomains into groups with similar amino acids in key positions 
 and similar predicted druggabilities .
this classification showed significant 
 differences to the whole sequence classification , suggesting that 
 it may prove more useful to drug discovery directed toward the acetyl - lysine 
 binding site .
our proposed classification also allows medicinal 
 chemists who work on a particular bromodomain to identify other family 
 members that are likely to bind similar inhibitors .
this information 
 can be explored to select proteins for counterscreening or to identify 
 bromodomain inhibitors that can be explored in a target hopping approach .
furthermore , our results highlight the significance of water molecules 
 in the computational analysis of bromodomain binding sites .
a number 
 of conserved water molecules occupy the base of the pocket and so 
 far no example has been reported in which these have been replaced 
 by small molecules . for this reason , all bromodomains have been treated 
 equally with all of these water molecules kept as part of the binding 
 site and the druggability assessment performed as such . the corresponding 
 assessment without the water molecules present
has also been performed , 
 which places more of the bromodomains in the druggable category and , 
 crucially , appears to increase the observed score more for the less 
 druggable sites , making it less discriminatory between druggable and 
 difficult sites .
this work represents the first analysis of 
 this type for the bromodomain family and will prove useful for drug 
 discovery projects aiming to identify inhibitors of the acetyl - lysine 
 binding site of bromodomains .
 bromodomains are readers of the epigenetic code that 
 specifically bind acetyl - lysine containing recognition sites on proteins . 
 
 recently the bet family of bromodomains has been demonstrated to be 
 druggable through the discovery of potent inhibitors , sparking an 
 interest in protein  
 protein interaction inhibitors that directly 
 target gene transcription . here 
 , we assess the druggability of diverse 
 members of the bromodomain family using sitemap and show that there 
 are significant differences in predicted druggability . 
 furthermore , 
 we trace these differences in druggability back to unique amino acid 
 signatures in the bromodomain acetyl - lysine binding sites . these signatures 
 were then used to generate a new classification of the bromodomain 
 family , visualized as a classification tree . 
 this represents the first 
 analysis of this type for the bromodomain family and can prove useful 
 in the discovery of inhibitors , particularly for anticipating screening 
 hit rates , identifying inhibitors that can be explored for lead hopping 
 approaches , and selecting proteins for selectivity screening . 
Introduction
Materials and Methods
Results and Discussion
Conclusion