the bromodomain ( brd ) 
 family of proteins recognize acetylated - lysine 
 ( kac ) in proteins and represent a set of protein  protein interaction 
 modules that are becoming increasingly explored in the field of drug 
 discovery .
the bet family of brds is a 
 subset of this larger bromodomain family and is made up of four members : 
 brd2 , brd3 , brd4 , and brdt in humans , with each containing two brd 
 modules that share high sequence similarity and highly similar binding sites .
the bet family shares the same conserved tertiary structure of 
 bromodomain proteins , with the kac binding site being formed as a central 
 cavity by an atypical left - handed four - helix bundle flanked by the 
 za loop and the bc loop ( figure 1a ) .
this binding 
 site is primarily hydrophobic , with key polar interactions being formed 
 between the acetyl carbonyl of kac and a family - conserved asparagine 
 residue as well as a structurally conserved water molecule . mimicking 
 the interaction of this acetyl group
has been the basis for generating 
 small - molecule inhibitors of the readout function of the bromodomain 
 proteins ( figure 1b ) , which was exemplified 
 by the discovery of ( + ) -jq1 ( figure 1c ) . outside 
 of the highly enclosed base of the pocket
, inhibitors of the bet family 
 have shown that occupying the adjacent regions , known as the hydrophobic 
 shelf ( occupied by phenyl in figure 1c ) and 
 za channel ( occupied by thiophene in figure 1c ) , leads to nanomolar potency and a high degree of selectivity toward 
 other bromodomains ( figures 1c and 2 ) .
( a ) 
 structure of first bromodomain of brd4 bound to an acetylated 
 peptide ( pdb i d : 3uvw ) .
( b ) interaction of kac with brd4 ( pdb i d : 3uvw ) . ( c ) ( + ) -jq1 bound 
 to brd4 with a chlorophenyl ring occupying the hydrophobic shelf and 
 thiophene occupying the za channel ( pdb i d : 3mxf ) .
the inhibition of the bet family of bromodomains has been 
 proposed 
 as a therapeutic strategy in multiple disease areas including cancer , 
 inflammation , and obesity . here , we have focused 
 on brd4 , which was identified as a therapeutic target in aml , other cancers , and inflammatory disease , as a representative member of the bet subfamily .
a number of small - molecule 
 inhibitors of bet family members have now been published .
a key feature of these inhibitors is a kac mimetic 
 that anchors the molecule into the brd binding site via hydrogen bonds 
 and hydrophobic interactions , which is critical for potent binding . 

a limited number of chemotypes that possess these features had been 
 published when this work started and has been increasing ( figure 2 ) .
these include triazolodiazepine 
 ( e.g. , ( + ) -jq1 ) , isoxazole ( e.g. , gsk1210151a ) , dihydroquinazolinone ( e.g. , pfi-1 ) , tetrahydroquinoline , benzimidazole ( e.g. , bic1 ) , indolizine , thiazolidinone , triazolopyridine , and tetrahydrothienopyridine scaffolds .
structure , 
 activity , and le of published bet - family inhibitors 
 classified by kac mimetic . upon starting this work , only the triazolodiazepine , dihydroquinazolinone , 
 and benzimidazole kac mimetics had been disclosed . to offer more possibilities 
 for drug - design efforts against brd4 and other bromodomains
different 
 chemotypes binding to the same protein often show different physicochemical 
 properties , distinct biological profiles , and offer additional opportunities 
 for intellectual property generation . here
, we describe a virtual 
 screening approach that focuses on 
 kac mimetics and identifies novel scaffolds that fit this profile . 

furthermore , we designed the virtual screen in a fashion largely independent 
 of the bromodomain targeted , and we present the validation of the 
 approach against brd4 .
the enclosed kac binding site of bromodomains 
 imposes strict geometric 
 constraints on inhibitors , requiring excellent shape complementarity 
 in this part of the pocket . to meet these constraints
, we wanted to 
 take advantage of the availability of brd4 and other bromodomain crystal 
 structures by utilizing molecular docking .
however , the docking of 
 several million commercially available compounds and in particular 
 the processing of the results is still a time - consuming and labor - intense 
 task .
frequently , methods of higher throughput , such as similarity- 
 or pharmacophore - based searching , are used to select a smaller set 
 of compounds that is then subjected to docking .
our approach to preselect 
 a set of compounds for which docking can be managed recognizes the 
 key role of the kac mimetic in all known bromodomain inhibitors .
we 
 initially selected commercially available compounds that feature an 
 kac mimetic chemotype , that is , compounds that feature a moiety that 
 has the potential to match the hydrogen - bond pattern and the steric 
 constraints of the kac binding site .
specifically , we created an extensive 
 set of chemotpyes by exploring published data , intuitive design , and 
 similarity searches .
this set consisted of known chemotypes and , critically , 
 also many chemical structures not yet described as bromodomain inhibitors . 

next , we converted these chemotypes into substructures searches and 
 retrieved commercially available compounds that feature these kac 
 mimetic substructures .
this set of compounds was then subjected to 
 docking against the first bromodomain of brd4 ( brd4(1 ) ) .
after an 
 extensive filtering of the results and the purchasing and testing 
 of selected compounds , we identified six inhibitors , including four 
 unprecedented kac mimetics . to validate our results further , we generated 
 crystal structures of selected compounds bound to brd4(1 ) and established 
 early sar around the novel chemotypes through synthesis and the purchase 
 of derivatives .
our work is relevant to scientists working on 
 bromodomain inhibitors 
 because it addresses three goals : first , through the focus on kac 
 mimetics , it efficiently preselects a subset of commercially available 
 compounds and ensures that a large number of kac mimetics are included 
 in the docking step ; second , through the docking step , it meets the 
 tight geometric requirements of the kac binding site ; and third , this 
 approach is applicable to other bromodomains for which crystal structures 
 are available .
furthermore , we report an extensive compilation of 
 novel chemotypes that can act as kac mimetics and will thus be useful 
 for other design approaches and scaffold hopping , and we report novel 
 inhibitors of brd4 that have the potential to be developed into significantly 
 more potent compounds .
our approach centered on 
 identifying a series of chemotypes that had the potential to mimic 
 kac and fit the geometric constraints of the brd4(1 ) binding pocket . 

these were then converted into substructure searches that were used 
 to mine commercially available compounds for inhibitors of brd4 using 
 the emolecules database .
one branch we will 
 refer to as the  literature substructures  branch , which 
 was based on the kac mimetic substructures extracted from published 
 bromodomain inhibitors .
these were also modified to generate structurally 
 related substructures that maintained the key pharmacophore ( e.g. , 
 1,2,4-triazole to isoxazole ) .
the other branch we will refer to as 
 the  similarity searching  branch , which was based on 
 pharmacophore , shape , and 2d fingerprint similarity searches derived 
 from the published ( + ) -jq1 and the crystal structure of it bound to 
 brd4(1 ) ( pdb i d : 3mxf ) .
the results of each of these branches was submitted to docking , 
 and through a series of filters designed to reduce docking false positives , 
 compounds were selected for purchase ( figure 3 ) .
we generated a library 
 of bromodomain - focused substructures through the identification of 
 the kac mimetic substructure from published bromodomain inhibitors . 
 to expand the number of substructures considered
an example of this approach starting from ( + ) -jq1 is highlighted in 
 figure 4 .
the substructures carried forward 
 to the rest of the approach are shown in the supporting 
 information , figure s1 .
example of 
 the generation of substructures from ( + ) -jq1 for the 
 literature substructures approach .
with this branch , we were 
 looking for novel kac mimetics featuring chemotypes distinct from 
 already known inhibitors . to identify such chemotypes , we used similarity 
 searches ( e.g. , shape or pharmacophore ) that allowed the identification 
 of distinct chemotypes that nevertheless share the pharmacophoric 
 features critical for binding of the probe compound ( + ) -jq1 .
these 
 similarity searches gave us a rich set of putative kac mimetics from 
 2.4 million commercially available compounds .
however , we reasoned 
 that as a result of the tight geometric constraints of the brd4 binding 
 site many of these would not be able to bind , despite a high similarity , 
 because even a simple change ( e.g. , from a methyl to an ethyl group ) 
 may lead to clashes within the kac pocket . to enrich for compounds 
 that fit the tight geometric constraint of the binding site
, we thus 
 performed a docking step and extracted kac mimetics with sufficient 
 shape complementarity .
it is important to note that this docking step 
 serves the purpose of creating a virtual library of kac mimetics in 
 conjunction with the similarity search and is distinct from the final 
 docking step that will be described later and served the purpose of 
 selecting compounds for purchase .
specifically , pharmacophore , shape , 
 and 2d fingerprint searches 
 were performed on 2.4 million commercially available compounds for 
 the initial similarity search .
three pharmacophore searches were used , 
 probing the base of the acetyl - lysine binding site , the hydrophobic 
 shelf , and the za channel , using the structure of brd4(1 ) as an excluded 
 volume ( pdb i d : 3mxf ) .
the first search contained all of the features known to contribute 
 to the potent binding of inhibitors : two acceptors within the acetyl - binding 
 site at the base of the pocket as well as hydrophobic substituents 
 in the za channel and the hydrophobic shelf positions .
this placed 
 fairly tight constraints on the molecules being screened against the 
 probe , and as a result only a few molecules were able to match these 
 criteria . to allow for more molecules to match and to focus on the 
 kac mimetic parts of the molecules , these constraints were relaxed 
 by removing the requirement for the molecule to occupy the hydrophobic 
 shelf .
one further search was performed that required only one of 
 the two acceptors within the acetyl binding site at the base of the 
 pocket because examples of single - acceptor - atom kac mimetics are known 
 ( e.g. , dihydroquinazolinone derivatives ) .
one shape - based and one 
 2d fingerprint similarity search were also applied to find molecules 
 with a similar shape and similar functional groups to the entire ( + ) -jq1 
 molecule .
one additional shape - based search was also applied to ( + ) -jq1 , 
 with the chlorophenyl and tertiary - butyl ester removed to focus on 
 the kac part of the binding site .
each of these similarity searches 
 was expected to identify different compounds and therefore maximize 
 the chances of finding hits .
a key step of our virtual screening 
 approach was the use of an extensive set of substructures that we 
 obtained from published data , intuitive design , and similarity searches , 
 as already described .
these substructures were consequently used to 
 identify all commercially available examples of each of the substructures , 
 employing substructure searches on the emolecules database .
the results were then submitted to a second 
 docking step and subsequent selection for purchase , as detailed below . 

this docking step was performed in the same manner as the one from 
 the similarity searching branch , but it was necessary because of the 
 different ligands present .
it is important to note that this substructure 
 search also ensured that all examples of a particular substructure 
 of interest were identified and fed into the final docking step .
this 
 is in contrast to our experience with similarity searches where a 
 number of representatives of a particular chemotype are frequently 
 missed , for example , because of different tautomers , protomers , or 
 conformers of the starting library compounds .
the results of each of the 
 substructure searches were subsequently docked against brd4(1 ) to 
 predict the binding mode and to calculate a score , from which examples 
 of each substructure can be selected for purchase .
one of the limitations 
 of using docking as a part of a virtual screen is that it generates 
 false positives , reducing the enrichment of actives .
many of the false positives , however , involve 
 the scoring of the molecules , with the pose generation considered 
 to be sufficiently accurate to be useful . a recent report by ferreira et al .
these 
 include failure to penalize high - energy conformations of the molecules 
 as well as the presence of heteroatoms that are not engaged in favorable 
 interactions with the receptor . using this knowledge
, we defined 
 the following criteria that are necessary to be fulfilled by a molecule 
 and its docking pose to be considered for purchase:(1)no obvious high - energy 
 conformations 
 ( using the csd as validation ) ( see the experimental 
 section for details).(2)the majority of the heteroatoms form 
 interactions with the receptor or are satisfied by internal hydrogen 
 bonds , and key interactions defined by the hypothesis are formed.(3)no clashes with the receptor 
 or vacant 
 sites unlikely to be filled by water molecules or protein movement.(4)no reactive groups or 
 groups known 
 to interfere in biochemical assays .
no obvious high - energy 
 conformations 
 ( using the csd as validation ) ( see the experimental 
 section for details ) .
the majority of the heteroatoms form 
 interactions with the receptor or are satisfied by internal hydrogen 
 bonds , and key interactions defined by the hypothesis are formed .
no clashes with the receptor 
 or vacant 
 sites unlikely to be filled by water molecules or protein movement . no reactive groups or 
 groups known 
 to interfere in biochemical assays . to implement these criteria , we manually 
 inspected the docking 
 poses as a pragmatic approach .
this allowed for the defined criteria 
 to be implemented quickly and each individual molecule to be considered 
 on its own merit .
at least 500 of the top ranked ligands from each 
 docking run were inspected , and representative compounds from each 
 scaffold that fulfilled these criteria were selected , resulting in 
 a set of 150 compounds .
our approach 
 is heavily based on 
 docking and extensively takes advantage of human input for the final 
 selection of compounds .
to benchmark our approach , we sought a valid 
 control experiment to compare our results to .
many retrospective analyses 
 suggest that ligand - based methods frequently outperform the equivalent 
 structure - based approaches when it comes to discriminating between 
 active and inactive compounds ; therefore , we chose 
 an experiment based on these approaches as a control .
we based our 
 selection on the same 2d fingerprint and shape - based similarity searches 
 as the similarity searching part of our virtual screening approach 
 starting from the entire ( + ) -jq1 ligand and its bound conformation 
 to brd4(1 ) , but in this experiment docking and subsequent visual inspection 
 were not utilized . to select compounds and to ensure that a 
 minimum level of diversity was achieved ,
the top 50 compounds from 
 each method , ranked by similarity to ( + ) -jq1 with unique murcko scaffolds , were selected for purchase .
one hundred compounds 
 in total were selected using this approach in a fully automated way , 
 and there was no overlap between the compounds selected by the control 
 experiment and those from the virtual screen .
using our virtual screening 
 protocol detailed above and including 
 the compounds selected from the control experiment , 250 compounds 
 were purchased and prepared as dmso stock solutions .
ten of these 
 compounds were insoluble in dmso , and as such they were not carried 
 forward to biochemical screening .
the remaining 240 compounds were 
 initially screened using the alphascreen assay format at a single 
 concentration up to 250 m , depending 
 on compound solubility .
six compounds exhibited an inhibition of brd4(1 ) , 
 but only two of these compounds yielded an ic50 : 1 had an ic50 of 4.7 m , and 2 had an ic50 of 80.9 m .
the ic50 of 
 the remaining compounds was higher than the solubility and as such 
 could not be determined . despite the modest activity of some of the 
 hits , we next sought to cocrystallize all novel kac mimetics that 
 showed significant binding , and we obtained crystal structures of 
 four compounds bound to brd4(1 ) . finally , we investigated derivatives 
 of five of these compounds through purchase and synthesis , yielding 
 compounds that showed significantly improved activity .
2 was not followed up further because of publications appearing after 
 the virtual screen was performed detailing derivatives of this isoxazole 
 scaffold .
the structures of these hits and the data gathered for each of the 
 compounds are summarized in table 1 .
the docking 
 poses used to initially select these six compounds can be found in 
 the supporting information , figure s2 . see the experimental 
 section for details .
following hplc purification of a 
 commercial sample . following the identification of six 
 compounds as inhibitors of brd4(1 ) ,
crystal structures of four of 
 the compounds in the kac binding site were obtained ( figure 7 ) .
this allows the binding mode to be used to design 
 more potent analogues and to compare the docking poses to the experimentally 
 observed binding mode that the docking was trying to predict .
extremely 
 high accuracy was attained for the docking of 3 and 4 , for which the docking poses were within experimental error 
 of the crystal structure poses ( rmsd  0.30  ) . in the 
 case of these two compounds , the protein showed very limited movement 
 from its conformation bound to ( + ) -jq1 with an rmsd of 0.37  
 between the model used ( pdb i d : 3mxf ) and each of the triazolopyrimidine bound 
 structures , which may have contributed to the success of the docking 
 experiment . in the case of 1 and 5 , the 
 docking predicted the experimentally observed pose of the ligand ( rmsd 
 < 2.0  ) but with a few subtle differences . some of these 
 differences were the result of water molecules bridging interactions 
 that the docking was unable to consider .
comparisons between the predicted 
 and experimentally determined poses can be seen in figure 8 .
details of all of the crystallographic binding 
 modes will be discussed in the context of each of the series below .
crystal - structure 
 binding modes of four of the identified inhibitors 
 bound to brd4(1 ) .
the right panel gives a through - protein view highlighting the 
 binding mode of each kac mimetic .
( a ) compound 1 , ( b ) compound 3 , ( c ) compound 4 , and ( d ) compound 5 . overlay of the observed crystallographic pose 
 ( cyan carbons ) with 
 the docking pose in the model from pdb i d : 3mxf ( yellow carbons ) following alignment 
 of the two proteins using moe .
( a ) compound 1 , ( b ) compound 3 , ( c ) compound 4 , and ( d ) compound 5 . compounds for which more information 
 has been gathered will now 
 be described .
the chloropyridone 
 scaffold was selected 
 using the literature substructures branch derived from the known dihydroquinazolinone 
 inhibitors .
the evolution of the initial dihydroquinazolinone substructure 
 involved the removal of the phenyl ring , and we hypothesized that 
 the chloropyridone motif would be able to mimic the remaining dihydropyrimidone 
 substructure ( figure 9 ) .
the carbonyl and nh 
 donor were maintained through this modification , with the chlorine 
 atom occupying the same location as the methyl group it replaced . 

a substructure search performed on the emolecules database yielded 
 82 commercially available examples of this substructure . following 
 the docking and selection process detailed above , 1 and 7a were selected for purchase .
1 was the most potent of the 240 compounds initially 
 tested , with an ic50 of 4.7 m and associated le 
 of 0.28 .
structurally related 7a , also tested in the 
 original batch of 240 compounds , was found to be inactive .
the only 
 other commercially available compounds containing the chloropyridone 
 and triazole functionalities were 7b and 7c . these were subsequently purchased and tested ( table 2 ) and found to be significantly less active . a crystal structure 
 of 1 bound to brd4(1 ) was obtained with the carbonyl 
 of the pyridone occupying a similar position to the carbonyl oxygen 
 of the acetyl - group of kac , forming the same interactions with the 
 nh donor of the asn140 side chain and conserved water molecule .
the 
 chlorine atom occupies the base of the pocket that the methyl group 
 of kac would usually occupy , and the nh donor of the pyridone forms 
 a water - mediated hydrogen bond to the carbonyl of the conserved asn140 
 side chain .
this is the first example of a chlorine substituent occupying 
 the base of the kac binding pocket , which can potentially be incorporated 
 into other templates , replacing the methyl group commonly used at 
 this position .
the remaining interactions of 1 involve 
 the triazole that forms an interaction with one of the conserved water 
 molecules and occupies the za - channel region of the pocket as well 
 as hydrophobic interactions of the phenyl and pyridine rings ( figure 7a ) .
3 and 4 both 
 contain the same triazolopyrimidine scaffold and were initially 
 selected on the basis of the hypothesis that they mimic kac .
the observation 
 that 3 and 4 show significant inhibition 
 as well as , importantly , the cocrystal structures of these compounds 
 with brd4(1 ) validated this hypothesis and confirmed the triazolopyrimidine 
 as a novel kac mimetic .
the triazolopyrimidine engages in the same 
 interactions in both structures as the carbonyl of the acetyl group 
 of kac , albeit through two nitrogen atoms compared to each lone pair 
 of the carbonyl ( figure 7 ) .
this strong complementarity 
 is likely the reason for the accurate prediction of the docking experiment 
 as well as for the minimal movement of the protein from the reference 
 model ( pdb i d : 3mxf ) .
a number of commercial derivatives of this scaffold were 
 subsequently purchased ( supporting information , figure s3 ) .
gratifyingly , 8b demonstrated a substantially improved activity toward brd4(1 ) , with 
 an ic50 of 24 m and associated le of 0.33 , demonstrating 
 that this class of compounds can be further improved .
the comparison 
 of the matched pair compounds 4 and 8a , 
 which only differ by the presence of an amino substitution 
 at the triazolopyrimidine , suggested that introduction of the amino 
 group into compounds 3 and 8b may lead to 
 an improvement of activity .
the improvement in potency can be rationalized 
 by the observation that the amino group forms an interaction with 
 the conserved asparagine , as seen in the crystal structure of 4 with brd4(1 ) ( figure 7c ) , and completes 
 the acceptor  acceptor 
donor motif that is observed for 
 published inhibitors ( e.g. , dihydroquinazolinones ) as well as some 
 of the other inhibitors identified here ( 2 , but also 1 and 6 if the carbonyl is considered as 2 acceptors ) .
, the improved activity of 8c over 4 is likely due to an interaction of 
 the benzylamino group of 8c with the hydrophobic shelf , 
 as observed from the crystal structures of 3 bound to 
 brd4(1 ) .
this interaction is known to provide improvements in potency 
 in other series of bet - family inhibitors , including ( + ) -jq1 , and is 
 not found in the complex of 4 with brd4(1 ) .
compound 8d shared comparable activity to 8c , showing 
 that the methylation of the benzylic amine is not required for activity . 

finally , introduction of an amino group into 8b to yield 8e maintained the activity but in this case did not lead to 
 an improvement in potency .
however , compounds 8b  e demonstrate that there is scope for further improvement 
 of the initial hits 3 and 4 and that a reasonable 
 le ( 0.3 ) can be achieved .
reagents and conditions : ( a ) pocl3 , reflux , 2 h ; ( b ) r1 amine , etoh , reflux , 2 h. 5 demonstrated 
 inhibition of 
 brd4(1 ) , but its ic50 was lower than its solubility and 
 could not be determined ; however , the crystal structure bound to brd4(1 ) 
 revealed two interesting features .
first , the acetyl group was not 
 acting as the kac mimetic but formed an interaction with the backbone 
 nh donor of asp88 , an interaction that has not been observed in any 
 published bromodomain crystal structure to date ( figure 10 ) .
although the crystallographic orientation of 
 the molecule was predicted by the docking pose , this particular interaction 
 with the backbone nh donor of asp88 was not , suggesting another way 
 in which potency and potentially selectivity can be achieved from 
 the brd4(1 ) binding site .
the second interesting feature of 
 this binding mode is that the quinoline nitrogen does not appear to 
 be forming a direct interaction with the protein . from the crystal 
 structure , electron density
could be observed in close proximity to 
 the quinoline nitrogen ( figure 11 ) , suggesting 
 an interaction through a bridging water molecule .
interestingly , the 
 distance between the oxygen of the water molecule and the ligand appeared 
 to be very short ( 2.14  ) , and we speculated that the active 
 ingredient of this compound was in fact the quinoline n - oxide ; however , 
 mass spectrometry did not detect the n - oxide .
we nevertheless decided 
 to synthesize the n - oxide 14a , and 5 was 
 resynthesized along with it ( scheme 2 ) , and 
 both compounds were tested for brd4(1 ) inhibition .
the resynthesized 
 batch of 5 demonstrated activity 
 similar to the original sample , however , the pure n - oxide generated 
 a significantly improved ic50 of 42 m ( table 4 ) .
this is a good example of a single - atom change 
 in a ligand resulting in the displacement of a single water molecule 
 and achieving an increase in activity .
interaction of the acetyl 
 group of 5 with backbone 
 donor of asp88 in brd4 .
alternate views of the binding mode of 5 can be seen 
 in figure 7d .
electron density of 5 including a water molecule in 
 close proximity to the quinoline nitrogen ( 2fo  fc map ) .
reagents 
 and conditions : ( a ) 
 tf2o , et3n , chcl3 , rt , 2 h ; ( b ) acetamidophenylboronic 
 acid , pd(pph3)4 , csco3 , dioxane , 
 120 c , 30 min ; ( c ) mcpba , dcm , reflux , 3 h. see the experimental 
 section for details . although pyrrolopyridone 6 demonstrated the lowest activity of the compounds initially 
 identified as hits , it also contained the fewest heavy atoms , suggesting 
 decent ligand efficiency despite its modest activity .
in addition , 
 it represented another example of a novel kac mimetic , and we sought 
 to demonstrate that somewhat larger derivatives show improved inhibition . 
 although few pyrrolopyridones were commercially available , a number 
 of related pyrrolopyrimidones could be sourced .
gratifyingly , the 
 phenyl substituted compounds 15a and 15b showed substantially improved activities , with an le above 0.3 ( table 5 ) .
this improvement 
 in potency is consistent with the phenyl ring occupying the za channel , 
 as would be expected from the docking pose of 6 ( supporting information , figure 
 s2 ) .
see the experimental 
 section for details . within 
 our virtual screening approach , two branches were used : the 
 literature substructures and similarity searching branches . as hypothesized , 

overall , 
 a hit rate of 2.5% was achieved for all of the compounds tested , and 
 a hit rate of 4.2% was achieved for the 143 compounds selected by 
 our virtual screening approach ( table 6 ) .
strikingly , 
 all of the active compounds had been identified from our virtual screen 
 rather than from the control experiment , highlighting the success 
 of the approach .
we believe that the superiority of our virtual screening 
 approach compared to the control branch is due to several factors . 

first of all , our kac mimetic approach reduced the number of commercially 
 available compounds sufficiently to enable docking , yet it ensured 
 that a large number of kac mimetics were taken forward into the docking 
 step .
second , the application of docking rather than just a similarity 
 search ensured that the purchased compound have a high likelihood 
 of meeting the tight geometric constraint of the kac binding site . 
 finally , our extensive manual filtering and human input after docking 
 removed several potential false positives . within the compounds 
 selected by the virtual screen , 76 came from the literature substructures 
 approach and two hits were identified .
sixty seven compounds came 
 from the similarity searching approach , from which four of the six 
 actives were identified .
four kac mimetics that were unprecedented 
 for bromodomain inhibition were discovered , fulfilling the main goal 
 of this work .
we initially expected that the literature substructures 
 branch 
 would yield the higher hit rate because of the presence of validated 
 and structurally related warheads .
interestingly , this turned out 
 not to be the case , with a higher hit rate being achieved by the similarity 
 searching branch .
a possible reason for this observation is that the 
 published inhibitors are optimized for the brd4(1 ) binding site and 
 possess good shape - complementarity as well as a kac mimetic warhead . 

compounds selected for the literature substructures branch possessed 
 this warhead , but without optimization suboptimal binding in the upper 
 part of the pocket may have abrogated any activity .
we 
 describe a structure - based virtual screening approach to identify 
 inhibitors of bromdomains together with the validation of this approach , 
 resulting in the discovery of novel inhibitors of brd4(1 ) .
the key 
 elements of this approach are the extensive design and use of substructure 
 queries to compile a set of commercially available compounds featuring 
 novel putative kac mimetics followed by subjection of this set to 
 docking for final compound selection . using this approach , we selected 
 and tested 143 compounds and identified novel hits , including four 
 unprecedented acetyl - lysine mimetics . a control experiment that failed 
 to identify hits
however , through use of cocrystal structures and structure - based design , 
 we were able to improve the potency considerably into a range frequently 
 found for screening hits .
this work is therefore a good example of 
 how novel but initially modestly potent compounds are identified with 
 a fast and very limited screening effort and then quickly optimized 
 through the use of cocrystal structures . because of the similarity 
 between bromodomain binding sites and 
 the focus on the kac mimetic , this approach is applicable to the large 
 fraction of bromodomains for which structural information of sufficient 
 resolution is available .
furthermore , we believe that the novel kac 
 mimetics disclosed here will serve as valuable starting points for 
 the development of potent inhibitors of brd4(1 ) and other related 
 bromodomains .
we used the icr vendor 
 collection , which is a library of 2.4 million commercially available 
 compounds from 11 vendors for which details have been described by 
 langdon et al .
single protonation 
 states were generated using filter . at the time of 
 performing the virtual screen against brd4(1 ) , the only inhibitors 
 published were the triazolodiazepines , bic1 , and simple dihydroquinazolinones . 

a number of published structures were available , but the only suitable 
 ligand - bound structures were with triazolodiazepines .
for this reason , 
 the structure used for the virtual screen was that of ( + ) -jq1 bound 
 to the brd4(1 ) ( pdb i d : 3mxf ) , which was used in the structure - based pharmacophore 
 searches and the bound pose of ( + ) -jq1 for shape - based similarity 
 searches , and it was also the structure used for the docking experiments . 

water molecules with the following uids were treated as part of the 
 pocket for docking experiments and pharmacophore excluded volumes : 
 9 , 12 , 15 , 23 , 33 , and 209 .
we performed three pharmacophore 
 searches on the basis of the ( + ) -jq1 molecule bound to brd4(1 ) ( pdb 
 i d : 3mxf ) using 
 the receptor as an excluded volume .
the first pharmacophore was the 
 most complex containing five points , including two acceptors from 
 the triazole ring of ( + ) -jq1 , a hydrophobic substituent for the methyl 
 group at the base of the pocket , and two further hydrophobic substituents 
 at the za - channel and hydrophobic shelf positions of the binding site . 

this pharmacophore characterized each feature known to significantly 
 contribute to the potency of ( + ) -jq1 and found just 6540 matching 
 ligands .
the next pharmacophore was a derivative of the first , relaxing 
 the requirement for both acceptors on the triazole ring to just one 
 of the two .
the third pharmacophore was also a derivative of 
 the first one , but this time with the pharmacophoric point corresponding 
 to the hydrophobic shelf removed . again
this increased the number 
 of matching ligands but not as significantly as for the second pharmacophore 
 to 41  890 .
( a ) pharmacophore searches 1 and 2 ( with 2 requiring only 
 one acceptor ) . 

we performed two shape - based 
 similarity searches : one on the full ( + ) -jq1 ligand in its bound conformation 
 from pdb i d 3mxf and one on a reduced version of the ligand with the ester and chlorophenyl 
 groups removed to focus on the kac mimetic part of the binding site 
 ( figure 13 ) .
both searches were performed with 
 two acceptor pharmacophore points from the triazole part of the ( + ) -jq1 
 ligand .
the top 100  000 compounds ranked by the tanimotocombo 
 scoring function from each search were taken through to subsequent 
 docking steps .
shape - based similarity searches used with acceptor 
 pharmacophoric 
 points included ( red spheres ) .
a single 2d similarity 
 search was performed using the ecfp4 fingerprint and the ( + ) -jq1 ligand 
 as a template in pipeline pilot .
the top 100  000 
 ligands ranked by tanimoto similarity were taken through to subsequent 
 docking steps .
the structure was 
 preprocessed using the protein preparation wizard in maestro , with the assign 
 bond orders , create disulfide bonds , and convert selenomethionines 
 to methionines options selected .
grids for glide docking were then 
 generated using the receptor grid generation tool in maestro using 
 an enclosing box of size 20  around the center of ( + ) -jq1 and 
 other settings left at the default .
docking 
 was performed using glide with default 
 settings . for more than 5000 ligands ,
the compounds 
 were initially docked using glide in htvs mode and the 5000 top ranked 
 compounds by glidescore were docked using glide in sp mode . for fewer 
 than 5000 ligands ,
water molecules included were treated as rigid , with hydrogen 
 positions used as defined by the protonate3d step . to analyze the observed dihedral angle of certain 
 substructures in the csd
, initial substructure 
 searches and the dihedral in question were defined using conquest 
 and the data output and analyzed using vista .
a high - energy conformation 
 was defined as one that possessed a dihedral angle not observed in 
 the csd for sufficiently populated substructures .
all reagents were diluted in 50 mm hepes , 100 
 mm nacl , 0.1% bsa , ph 7.4 supplemented with 0.05% chaps and allowed 
 to equilibrate to room temperature prior to addition to plates .
a 
 24-point 1:2 serial dilution of the ligands was prepared over the 
 range of 1500 m , and 4 l was transferred to 
 low - volume 384-well plates ( proxiplatetm-384 plus , perkinelmer ) followed 
 by 4 l of his - tagged protein ( brd4(1 ) , 250 nm ) .
the plates 
 were sealed and incubated at room temperature for 30 min before the 
 addition of 4 l of biotinylated peptide at an equimolar concentration 
 to the protein ( the peptide used for brd4(1 ) : h - sgrgk(ac)ggk(ac)glgk(ac)ggak(ac)rhrk(biotin)-oh ; 
 cambridge research biochemicals , uk ) .
the plates were sealed and incubated 
 for a further 30 min before the addition of 4 l of streptavidin - coated 
 donor beads ( 25 g / ml ) and 4 l of nickel chelate acceptor 
 beads ( 25 g / ml ) under low - light conditions .
the plates were 
 foil sealed to protect from light , incubated at room temperature for 
 60 min , and read on a pherastar fs plate reader ( bmg labtech ) using 
 an alphascreen 680 excitation/570 emission filter set .
ic50 was calculated in prism 5 ( graphpad software ) after normalization 
 against corresponding dmso controls , and they are given as the final 
 concentration of compound in the 20 l reaction volume .
aliquots of the 
 purified proteins were 
 set up for crystallization using a mosquito crystallization robot 
 ( ttp labtech ) .
coarse screens were typically set up onto greiner 3-well 
 plates using three different drop ratios of precipitant to protein 
 per condition ( 100 + 50 , 75 + 75 , and 50 + 100 nl ) .
all crystallizations 
 were carried out using the sitting - drop vapor - diffusion method at 
 4 c .
brd4(1 ) crystals with 1 were grown by mixing 
 150 nl of protein ( 9.9 mg / ml and 5 mm final ligand concentration ) 
 with an equal volume of reservoir solution containing 0.2 m sodium 
 sulfate , 0.20 m nabr , 0.1 m btprop ph 8.5 , 20.0% peg6k , 10.0% ethylene 
 glycol .
brd4(1 ) crystals with 3 were grown by mixing 
 100 nl of protein ( 9.0 mg / ml and 5 mm final ligand concentration ) 
 with 200 nl of reservoir solution containing 0.20 m na(malonate ) , 
 0.1 m btprop ph 8.5 , 20.0% peg3350 , 10.0% ethylene glycol .
brd4(1 ) 
 crystals with 4 were grown by mixing 150 nl of the protein 
 ( 9.0 mg / ml and 10 mm final ligand concentration ) with an equal volume 
 of reservoir solution containing 0.20 m na2so4 , 20.0% peg3350 and 10.0% ethylene glycol .
brd4(1 ) crystals with 5 were grown by mixing 150 nl of protein ( 9.9 mg / ml and 5 
 mm final ligand concentration ) with an equal volume of reservoir solution 
 containing 0.2 m sodium sulfate , 0.20 m licl , 0.1 m tris ph 8.0 , 20.0% 
 peg6k , 10.0% ethylene glycol . in all cases , diffraction - quality crystals 
 grew within a few days .
all crystals 
 were cryo - protected using the well solution supplemented with additional 
 ethylene glycol and were flash frozen in liquid nitrogen .
data were 
 collected in - house on a rigaku fre rotating anode system equipped 
 with a raxis - iv detector at 1.52  .
indexing and integration 
 was carried out using mosflm , and scaling 
 was performed with scala .
initial phases 
 were calculated by molecular replacement with phaser using the known model of brd4(1 ) ( pdb i d : 2oss ) .
initial models 
 were built by arp / warp followed by manual 
 building in coot .
thermal 
 motions were analyzed using tlsmd , and 
 hydrogen atoms were included in late refinement cycles .
data collection 
 and refinement statistics can be found in the supporting information , table s1 .
the models and structure factors have been deposited with pdb accession 
 codes 4mep ( brd4(1)/ 
 compound 1 ) , 4men ( brd4(1)/ compound 3 ) , 4meq ( brd4(1)/ compound 4 ) , and 4meo ( brd4(1)/ compound 5 ) . commercially available
column 
 chromatography was performed on a biotage sp1 purification system 
 using biotage flash silica cartridges .
semiprep hplc purification 
 was achieved using 1000 l standard injections ( with needle 
 rinse ) of the sample at a 20 mg / ml concentration in dmso onto a phenomenex 
 gemini column ( 10 m , 250  21.2 mm , c18 , phenomenex ) . 

chromatographic separation at room temperature was carried out using 
 a gilson gx-281 liquid handler system combined with a gilson 322 hplc 
 pump ( gilson ) over a 15 min gradient elution ( grad15 min20mlslipo.m ) 
 from 40:60 to 100:0 methanol / water ( both modified with 0.1% formic 
 acid ) at a flow rate of 20 ml / min .
vis spectra were acquired 
 at 254 nm on a gilson 156 uv  vis detector ( gilson ) .
collection 
 was triggered by uv signal and collected using a gilson gx-281 liquid 
 handler system ( gilson ) .
lc  ms and hrms analysis was performed on an agilent 
 1200 series hplc and diode array detector coupled to a 6210 time - of - flight 
 mass spectrometer with dual multimode apci / esi source .
analytical 
 separation was carried out at 30 c on a merck purospher star 
 column ( rp-18e , 30  4 mm ) using a flow rate of 1.5 ml / min in 
 a 4 min gradient elution with detection at 254 nm .
the mobile phase 
 was a mixture of methanol ( solvent a ) and water containing formic 
 acid at 0.1% ( solvent b ) .
gradient elution was as follows : 1:9 ( a / b ) 
 to 9:1 ( a / b ) over 2.5 min , 9:1 ( a / b ) for 1 min , and then reversion 
 back to 1:9 ( a / b ) over 0.3 min , and finally 1:9 ( a / b ) for 0.2 min .
samples 
 were prepared as solutions in a deuterated solvent and referenced 
 to the appropriate internal nondeuterated solvent peak or tetramethylsilane . 

compound 1 was purchased as a 5 mg 
 solid sample from chembridge ( supplier i d 25722975 ) . h 
 nmr ( 500 mhz , dmso )  2.21 ( s , 3h ) , 7.45 ( s , 1h ) , 7.477.55 
 ( m , 4h ) , 7.66 ( t , 1h ) , 7.72 ( s , 1h ) , 8.07 ( d , 1h ) , 8.11 ( d , 2h ) , 8.51 
 ( d , 1h ) .
hrms m / z calcd for c19h15cln5o [ m + h ] , 364.0960 ; 
 found , 364.0961 .
compound 2 was purchased as a 5 mg 
 solid sample from key organics ltd .
h nmr ( 500 mhz , dmso )  2.13 ( s , 3h ) , 6.93 ( s , 2h ) , 7.547.65 
 ( m , 4h ) . hrms m / z calcd for c11h10f3n2o [ m + h ] , 243.0740 ; found , 243.0741 .
h nmr ( 500 mhz , cdcl3 )  2.37 ( s , 3h ) , 2.60 ( s , 3h ) , 3.20 ( s , 3h ) , 5.31 ( s , 
 2h ) 6.07 ( s , 1h ) , 7.137.20 ( m , 4h ) , 8.34 ( s , 1h ) . hrms m / z calcd for c15h18n5 [ m + h ] , 268.1557 ; found , 268.1558 .
compound 4 was 
 purchased as a 5 mg solid sample from princeton biomolecular 
 research inc .
h nmr ( 500 mhz , 
 dmso )  6.34 ( s , 2h ) , 7.18 ( s , 1h ) , 7.577.63 ( m , 3h ) , 
 8.14 ( d , 2h ) .
the methyl peak was not observed and was expected to 
 be below the dmso peak .
hrms m / z calcd for c12h12n5 [ m + h ] , 226.1087 ; found , 226.1091 .
compound 5 was initially purchased 
 as a 5 mg solid sample from chembridge ( supplier i d 50866471 ) .
h nmr ( 500 mhz , dmso )  2.07 ( s , 3h ) , 7.21 ( d , 1h ) , 
 7.36 ( s , 1h ) , 7.487.56 ( m , 2h ) , 7.70 ( d,1h ) , 7.75 ( t , 1h ) , 
 7.81 ( s , 1h ) , 7.84 ( d , 1h ) , 8.01 ( d , 1h ) , 10.14 ( s , 1h ) .
hrms m / z calcd for c18h16n2o [ m + h ] , 277.1335 ; found , 277.1333 .
triflic anyhydride ( 0.95 ml , 5.65 mmol ) was added 
 to a solution of 2-methylquinolin-4-ol 12 ( 750 mg , 4.71 
 mmol ) and triethylamine ( 0.66 ml , 4.71 mmol ) in anhydrous dcm ( 5 ml ) 
 under nitrogen and stirred for 2 h. the reaction mixture was concentrated 
 in vacuo .
the resulting purple oil was purified by column chromatography 
 ( etoac to 5% meoh in etoac ) to afford title compound ( 863 mg , 63% ) . 

lc  ms ( esi , m / z ) tr 2.82 min , 292.18 [ m + h ] .
h nmr ( 500 mhz , cdcl3 )  2.87 ( s , 3h ) , 7.35 
 ( s , 1h ) , 7.68 ( t , 1h ) , 7.86 ( t , 1h ) , 8.05 ( d , 1h ) , 8.17 ( d , 1h ) .
n-(3-(2-methylquinolin-4-yl)phenyl)acetamide ( 5 ) . to a solution of 2-methylquinolin-4-yl trifluoromethanesulfonate 13 ( 280 mg , 0.79 mmol ) in dioxane / water ( 2:1 , 4.5 ml ) were 
 added acetamidophenylboronic acid ( 283 mg , 1.58 mmol ) , tetrakis triphenylphosphine 
 palladium ( 45.6 mg , 0.04 mmol ) , and cesium carbonate ( 386 mg , 1.185 
 mmol ) .
the reaction mixture was heated at 120 c for 30 min under 
 microwave conditions .
the reaction mixture was diluted with etoac 
 ( 20 ml ) and washed with water ( 20 ml ) .
the aqueous layer was re - extracted 
 with etoac ( 20 ml ) , and the combined organic layers were dried ( mgso4 ) and concentrated in vacuo .
the residue was purified by column 
 chromatography ( 20% etoac in hexane to 100% etoac ) to afford the title 
 compound ( 141 mg , 65% ) .
lc  ms ( esi , m / z ) tr 1.97 min , 277.13 [ m + 
 h ] .
h nmr ( 500 mhz , cdcl3 )  
 2.23 ( s , 3h ) , 2.81 ( s , 3h ) , 7.237.29 ( m , 2h ) , 7.447.57 
 ( m , 3h ) , 7.647.74 ( m , 3h ) , 7.89 ( d,1h ) , 8.15 ( d , 1h ) .
hrms m / z calcd for c18h17n2o [ m + h ] , 277.1335 ; found , 277.1336 .
h nmr ( 500 mhz , dmso )  4.05 ( s , 3h ) , 6.24 ( d , 1h ) , 
 6.39 ( d , 1h ) , 6.81 ( t , 1h ) , 7.26 ( d , 1h ) , 10.81 ( s , 1h ) . hrms m / z calcd for c8h9n2o [ m + h ] , 149.0709 ; found , 149.0709 . 3-chloro-5-(3-isopropyl-1-(3-methylpyridin-4-yl)-1h-1,2,4-triazol-5-yl)pyridin-2(1h)-one 
 ( 7a ) .
compound 7a was purchased as a 5 mg 
 solid sample from chembridge ( supplier i d 90638099 ) . h 
 nmr ( 500 mhz , dmso )  1.24 ( m , 1h ) , 1.32 ( d , 6h ) , 2.08 ( s , 3h ) , 
 7.34 ( s , 1h ) , 7.47 ( d , 1h ) , 7.68 ( s , 1h ) , 8.59 ( d , 1h ) , 8.71 ( s , 1h ) . 

hrms m / z calcd for c16h17cln5o [ m + h ] , 330.1116 ; found , 
 330.1114 .
compound 7b was purchased 
 as a 5 mg solid sample from chembridge ( supplier i d 95921162 ) .
h nmr ( 500 mhz , dmso )  0.930.99 ( m , 7h ) , 2.07 
 ( s , 2h ) , 4.29 ( s , 4h ) , 6.90 ( d , 1h ) , 6.977.03 ( m , 2h ) , 7.41 
 ( s , 1h ) , 7.69 ( s , 1h ) .
hrms m / z calcd 
 for c19h20cln4o3 [ m + 
 h ] , 387.1218 ; found , 387.1207 .
compound 7c was purchased as a 5 mg 
 solid sample from chembridge ( supplier i d 26575894 ) .
h 
 nmr ( 500 mhz , dmso )  2.34 ( s , 3h ) , 7.367.42 ( m , 3h ) , 
 7.517.57 ( m , 2h ) , 7.66 ( s , 1h ) .
hrms m / z calcd for c14h11clfn4o [ m + h ] , 305.0600 ; found , 305.0592 .
h nmr ( 500 mhz , dmso )  7.56 ( s , 
 1h ) , 7.627.69 ( m , 3h ) , 8.158.20 ( m , 2h ) , 8.63 ( s , 
 1h ) .
the methyl peak was not observed and was expected to be below 
 the dmso peak .
hrms m / z calcd for 
 c12h11n4 [ m + h ] , 211.0978 ; 
 found , 211.0976 .
h nmr ( 500 mhz , dmso )  3.13 ( s , 3h ) , 5.39 ( s , 2h ) , 6.44 ( s , 
 1h ) , 7.00 ( s , 2h ) , 8.49 ( s , 1h ) .
the methyl peak was not observed 
 and was expected to be below the dmso peak .
hrms m / z calcd for c12h13cln5s [ m + h ] , 294.0575 ; found , 294.0571 .
2-amino-5-methyl - triazolo[1,5-a]pyrimidin-7(3h)-one 9 ( 500 
 mg , 3.028 mmol ) was added to cooled ( 0 c ) , stirring pocl3 ( 3 ml ) .
the reaction mixture was allowed to warm to rt and 
 was heated to reflux for 2 h. the reaction mixture was concentrated 
 in vacuo , and the residue was dissolved in dcm ( 20 ml ) .
water ( 20 
 ml ) was added , and the resultant precipitate was isolated by filtration . 

the residue was purified by triturating with dcm and used in subsequent 
 reactions without further purification ( 345 mg , 62% ) .
lc  ms 
 ( esi , m / z ) tr 1.15 min , did not ionize [ m + h ] .
h nmr ( 500 mhz , tfa )  2.91 ( s , 3h ) , 7.72 ( s , 1h ) .
n - methyl-1-(p - tolyl)methanamine . 4-methylbenzyl chloride 
 ( 0.933 ml , 7.112 mmol ) was added to a solution of 40% aqueous methylamine 
 ( 10 ml , 18.031 mmol ) in etoh ( 10 ml ) and stirred at rt for 12 h. the 
 reaction mixture was concentrated in vacuo , the residue was dissolved 
 in water ( 20 ml ) , and the ph adjusted to 12 with 2 m naoh .
the 
 resulting yellow oil was purified by reverse - phase c18 flash column 
 chromatography ( water ) to afford the title compound ( 112 mg , 12% ) . 

lc  ms ( esi , m / z ) tr 0.85 min , 136.11 [ m + h ] .
h nmr ( 500 mhz , cdcl3 )  2.36 ( s , 3h ) , 2.46 
 ( s , 3h ) , 3.73 ( s , 2h ) , 7.15 ( d , 2h ) , 7.22 ( d , 2h ) .
7-chloro-5-methyl - triazolo[1,5-a]pyrimidin-2-amine 10 ( 50 mg , 0.275 mmol ) was added 
 to a stirred solution of n - methyl-1-(p - tolyl)methanamine ( 74 mg , 0.55 mmol ) and k2co3 ( 38 mg , 0.275 mmol ) in anhydrous ethanol ( 0.5 ml ) .
the reaction 
 mixture was heated to reflux for 2 h. the reaction mixture was concentrated 
 in vacuo , and the residue was dissolved in etoac ( 10 ml ) and water 
 ( 10 ml ) .
the aqueous layer was re - extracted with etoac ( 10 ml ) , and 
 the combined organic layers were dried ( mgso4 ) and concentrated 
 in vacuo .
the residue was purified using an scx-2 column ( meoh ) to 
 afford the title compound ( 5.63 mg , 42% ) .
lc  ms ( esi , m / z ) tr 2.63 
 min , 283.17 [ m + h ] .
h nmr ( 500 mhz , cdcl3 )  2.36 ( s , 3h ) , 2.49 ( s , 3h ) , 3.08 ( s , 3h ) , 4.66 ( brs , 
 2h ) , 5.19 ( s , 2h ) , 5.94 ( s , 1h ) , 7.12 ( d , 2h ) , 7.15 ( d , 2h ) .
hrms m / z calcd for c15h19n6 [ m + h ] , 283.1666 ; found , 283.1665 .
7-chloro-5-methyl - triazolo[1,5-a]pyrimidin-2-amine 10 ( 50 mg , 0.275 mmol ) was added to 
 a stirred solution of p - tolylmethanamine ( 1.36 mmol ) 
 and k2co3 ( 38 mg , 0.275 mmol ) in anhydrous ethanol 
 ( 0.5 ml ) .
the reaction mixture was heated to reflux for 2 h , with 
 reaction progress monitored by lc  ms .
the residue was dissolved in ethyl acetate 
 ( 10 ml ) and water ( 10 ml ) .
the aqueous layer was re - extracted with 
 ethyl acetate , and the combined organic layers were dried ( mgso4 ) and concentrated in vacuo .
the residue was purified by column 
 chromatography ( 0% meoh in etoac to 20% meoh in etoac ) to afford the 
 title compound ( 31 mg , 42% ) .
lc  ms ( esi , m / z ) tr 2.47 min , 269.15 
 [ m + h ] .
h nmr ( 500 mhz , cdcl3 ) 
  2.38 ( s , 3h ) , 2.48 ( s , 3h ) , 4.51 ( d , 2h ) , 4.61 ( brs , 2h ) 5.94 
 ( s , 1h ) , 6.14 ( s , 1h ) , 7.21 ( d , 2h ) , 7.24 ( d , 2h ) .
hrms m / z calcd for c14h17n6 [ m + h ] , 269.1509 ; found , 269.1510 .
7-chloro-5-methyl - triazolo[1,5-a]pyrimidin-2-amine 10 ( 50 mg , 0.275 mmol ) 
 was added to a stirred solution of 1-(5-chlorothiphen-2-yl)-n - methylmethanamine ( 111 mg , 0.688 mmol ) and k2co3 ( 38 mg , 0.275 mmol ) in anhydrous ethanol ( 0.5 ml ) . 

the reaction mixture was heated to reflux for 2 h , with reaction progress 
 monitored by lc 
the residue was dissolved in ethyl acetate ( 10 ml ) and water 
 ( 10 ml ) .
the aqueous layer was re - extracted with ethyl acetate , and 
 the combined organic layers were dried ( mgso4 ) and concentrated 
 in vacuo the residue was purified by column chromatography ( 0% meoh 
 in dcm to 10% meoh in dcm ) to afford the title compound ( 4.32 mg , 
 42% ) .
lc  ms ( esi , m / z ) tr 2.71 min , 309.07 [ m + h ] .
h nmr ( 500 mhz , cdcl3 )  2.50 ( s , 3h ) , 3.07 
 ( s , 3h ) , 4.75 ( brs , 2h ) , 5.29 ( s , 2h ) , 5.95 ( s , 1h ) , 6.77 ( m , 2h ) . 
 hrms m / z calcd for c12h14cln6s [ m + h ] , 309.0684 ; found , 
 309.0684 .
mcpba ( 37.5 mg , 0.317 mmol ) was added 
 slowly to a stirring solution of n-(3-(2-methylquinolin-4-yl)phenyl)acetamide 5 ( 50 mg , 0.181 mmol ) in anhydrous dcm ( 1.3 ml ) .
the reaction 
 mixture was heated at 45 c for 3 h. the reaction mixture was 
 cooled to room temperature and quenched with aq sat .
the reaction mixture was extracted with dcm ( 2  10 ml ) , dried 
 ( mgso4 ) , and concentrated in vacuo .
the residue was purified 
 by column chromatography ( 0% meoh in dcm to 10% meoh in dcm ) to afford 
 the title compound ( 7.2 mg , 14% ) .
lc  ms ( esi , m / z ) tr 2.61 min , 293.13 
 [ m + h ] .
h nmr ( 500 mhz , cdcl3 ) 
  2.17 ( s , 3h ) , 3.04 ( s , 3h ) , 7.19 ( d , 1h ) , 7.28 ( s , 1h ) , 7.46 
 ( t , 1h ) , 7.607.66 ( m , 2h ) , 7.69 ( d , 1h ) , 7.86 ( t , 1h ) , 7.97 
 ( d , 1h ) , 8.10 ( brs , 1h ) , 8.82 ( d , 1h ) . hrms m / z calcd for c18h17n2o2 [ m + h ] , 293.1285 ; found , 293.1287 .
compound 15a was 
 purchased as a 5 mg solid sample from sigma - aldrich ( supplier cnc 
 i d 310754082 ) .
h nmr ( 500 mhz , dmso )  2.30 ( s , 
 3h ) , 4.03 ( s , 3h ) , 7.18 ( d , 2h ) , 7.80 ( d , 1h ) , 7.85 ( s , 1h ) , 7.89 
 ( d , 2h ) , 12.15 ( s , 1h ) . hrms m / z calcd for c14h13n3o [ m + h ] , 240.1131 ; found , 240.1133 .
compound 15b was purchased 
 as a 5 mg solid sample from sigma - aldrich ( supplier cnc i d 310754657 ) .
h nmr ( 500 mhz , dmso )  4.08 ( s , 3h ) , 7.22 ( t , 2h ) , 
 7.87 ( 1h , s ) , 7.88 ( 1h , s ) , 8.06 ( 2h , dd ) , 12.03 ( 1h , s ) .
hrms m / z calcd for c13h11fn3o [ m + h ] , 244.0881 ; found , 244.0877 .
 bromodomains 
 ( brds ) are epigenetic readers that recognize acetylated - lysine 
 ( kac ) on proteins and are implicated in a number of diseases . 
 we describe 
 a virtual screening approach to identify brd inhibitors . 
 key elements 
 of this approach are the extensive design and use of substructure 
 queries to compile a set of commercially available compounds featuring 
 novel putative kac mimetics and docking this set for final compound 
 selection . 
 we describe the validation of this approach by applying 
 it to the first brd of brd4 . 
 the selection and testing of 143 compounds 
 lead to the discovery of six novel hits , including four unprecedented 
 kac mimetics . 
 we solved the crystal structure of four hits , determined 
 their binding mode , and improved their potency through synthesis and 
 the purchase of derivatives . 
 this work provides a validated virtual 
 screening approach that is applicable to other brds and describes 
 novel kac mimetics that can be further explored to design more potent 
 inhibitors . 
Introduction
Methods
Results and Discussion
Conclusions
Experimental Section