bromodomains are acetyl - lysine specific 
 epigenetic effector domains 
 that have emerged as new targets for the design of protein interaction 
 inhibitors modulating gene transcription and chromatin structure . targeting bromodomains received a lot of attention recently after 
 selective inhibitors of bet ( bromo and extra - terminal ) bromodomains 
 showed excellent efficacy in a number of cancer models as well as in inflammation and first 
 inhibitors entered now in clinical testing . since then , targeting 
 bromodomains has been mainly focused on bet family members with only 
 few tool compounds available for non - bet bromodomains . however , favorable druggability has been predicted for many bromodomains and the family is well covered in terms of available 
 crystal structures enabling rational design of selective tool compounds . the excellent druggability of bromodomains has recently been demonstrated 
 by high hit rates as well as the frequent 
 potent activity of inhibitors developed for other targets such as 
 kinases . tripartite 
 motif 24 protein ( trim24 ) , also known as tif1 , 
 contains a plant homeodomain / bromodomain dual epigenetic reader domain 
 and a ring type e3 ubiquitin ligase domain . trim24 overexpression 
 has been recently associated with poor overall survival and tumor 
 progression of breast - cancer patients , and high expression levels have been reported in glioblastoma , hepatocellular carcinoma , gastric cancer , nonsmall cell 
 lung cancer , and head and neck squamous 
 cell carcinoma . in addition , in prostate 
 cancer , trim24 is upregulated by mutations in spop , an e3 ubiquitin 
 ligase substrate - binding protein . trim24 
 acts as an e3-ubiquitin ligase of p53 , promoting degradation of this 
 key dna damage regulator . furthermore , 
 it functions as a coactivator of the estrogen receptor and it interacts with the ligand bound retinoic 
 acid ( ra ) receptor repressing its transcriptional activity . knockdown of trim24 in lung cancer cells decreased 
 the protein levels of a number of cyclins and p - rb and increased p27 
 expression , whereas a recent knockdown 
 study of trim24 in colon cancer cell lines showed that deletion of 
 this protein impairs cell growth and leads to induction of apoptosis . thus , trim24 may regulate levels of cancer - relevant 
 proteins on the transcriptional as well as the protein level . a recent study showed that ectopic expression of trim24 in isogenic 
 human mammary epithelial cells ( hmec ) increased cellular proliferation 
 and induced malignant transformation . transcription analysis of these 
 cells revealed a glycolytic and tricarboxylic acid cycle gene signature , 
 resulting in increased glucose uptake and activated aerobic glycolysis . deletion of trim24 in mice results in cell 
 cycle defects , leading to the development of liver cancer as a result 
 of retinoic acid receptor repression . the three - dimensional structure of the trim24 phd / bromodomain 
 revealed 
 that this dual reader domain functions as a cooperative unit recognizing 
 unmodified h3k4 with its phd zinc finger domain and h3k23ac with its 
 bromodomain . the context dependent and 
 complex roles of trim24 in regulating protein turnover and transcription 
 and its role in cancer raise the question of the consequences of inhibition 
 of druggable domains in trim24 such as the bromodomain . to enable 
 functional studies addressing this issue and the potential of targeting 
 the trim24 bromodomain for cancer therapy , we developed a potent chemical 
 tool molecule based on the 1,3 benzimidazolone scaffold , which has 
 been recently identified as an acetyl - lysine competitive inhibitor 
 of bromodomains of the brpf family . to develop a trim24 specific inhibitor , we 
 used an alpha screen 
 assay to evaluate commercial 1,3 benzimidazolones that were all decorated 
 with a sulfonamide substitution in position 5 and had a number of 
 different substituents in r1 . these inhibitors had nm ic50 values 
 for brpf1b and about 10-fold less potency for the closely related 
 brpf2 ( brd1 ) . however , the most interesting compounds of this series 
 ( 35 ) also had 34 m 
 activity against trim24 , which prompted us to explore this scaffold 
 for the development of trim24 inhibitors ( table 1 ) the methoxy 
 analogue 8 lost activity against trim24 but was still 
 a potent brpf 
 bromodomain inhibitor ( ic50 values of 0.27 and 1.2 m , respectively . this trend was observed for all compounds that harbored 
 a methoxy 
 group in r2 and variations in r1 ( compounds 922supporting information table 1 ) . however , phenoxy substitutions in position 6 considerably improved 
 trim24 activity ( table 2 ) . in particular , compound 24 showed ic50 
 values of 480 
 nm for the bromodomain of trim24 . however , inhibitors of this series 
 were still more potent for brpf1b and brpf2 bromodomains . we 
 explored therefore different substitutions in position 6 in 
 combination with r1 ( supporting information table 
 1 ) . interestingly , methoxy - phenoxy substituted inhibitors showed 
 good activity for trim24 but selectivity over brpf bromodomains was 
 not achieved ( table 3 ) . the best inhibitor 
 of this series has an ic50 value of 0.43 m for trim24 and 0.34 
 m for brpf1 , respectively . isothermal titration calorimetry data showed that 34 bound 
 the trim24 bromodomain with a kd of 222 
 nm ( figure 1 ) and had a kd for the brpf1 bromodomain of 137 nm and for brd1 of 1130 
 nm . binding to brpf3 was considerably weaker , resulting in an alphascreen 
 ic50 value of about 7 m . binding was strongly driven by enthalpy , 
 with a large favorable enthalpy change of 12.1 , 15.7 , 
 and 12.1 kcal / mol for trim24 , brpf1and brd1 , respectively , 
 and was opposed by entropy ( ts of 3.2 kcal / mol ( trim24 ) and 6.5 kcal / mol ( brpf1 ) ) . a table of all fitted parameters and a figure of titration data has 
 been included in supporting information figure 
 1 . are raw heat effects ( left panel ) of 30 consecutive 
 injections of trim24 injected in a solution of 34 in 
 20 mm hepes buffer , ph 7.5 , 150 mm nacl , and 0.5 mm tcep . normalized 
 injection heats as well as a nonlinear least - squares fit for a single 
 binding site model are shown in the right panel . next we evaluated the selectivity of 34 against 
 a 
 comprehensive panel of bromodomains using a temperature shift assay . by screening a panel of 45 bromodomains , we 
 found excellent selectivity of 34 for brpf1b/2 and trim24 
 ( figure 2 ) . ( a ) shown 
 are temperature shift 
 data ( tm ) for 45 human bromodomains . the bar 
 diagram shows the mean of three replicates as well as the standard 
 error . tm smaller than 1 degree were not considered 
 significant as indicated by a dotted line . ( b ) temperature shifts 
 mapped to the phylogenetic tree of the human bromodomain family . to get insight into the binding 
 mode of 34 , we determined 
 the cocrystal structure with the trim24 phd / bromodomain . the 
 trim24 cocrystal structure revealed the expected globular domain 
 organization of the phd and bromodomain , showing tight interaction 
 between the two reader domains ( figure 3a ) . the inhibitor was well - defined by electron density , 
 and 34 showed the expected binding mode of the acetyl - lysine 
 mimetic benzimidazolones moiety ( figure 3b ) , forming the canonical hydrogen bond with the 
 conserved asparagine n980 and a water mediated hydrogen bond to y935 
 linking the inhibitor also to the conserved water network at the bottom 
 of the binding pocket . interestingly , the two aromatic rings stack 
 against the za loop , effectively occupying the space at the rim of 
 the acetyl - lysine binding site , a binding mode that has recently been 
 reported also for a baz2b bromodomain inhibitor . similar to the stacking conformation observed in baz2b , 
 it is likely that this inhibitor conformation is not the prevalent 
 conformation in solution , providing potentially a rationale for the 
 observed unfavorable binding entropy measured in the itc experiments . the r2 methoxy phenyl ring fits perfectly into a hydrophobic cavity 
 lined by a923 and l922 , explaining the loss of binding activity for 
 r2 methoxy substitutions . the benzimidazolone ring forms mainly hydrophobic 
 interactions with residues on both sites of the acetyl - lysine binding 
 cavity ( v932 , v928 , v986 , p929 ) . sar revealed that the sulfonamide 
 substitutions ( r1 ) can tolerate many different ring systems . this 
 observation is compatible with the crystal structure , which shows 
 that this substituent is in a solvent exposed position . however , polar 
 interactions of the r1 aromatic decoration with residues in the bc 
 loop ( e985 ) could potentially increase potency and specificity for 
 trim24 as brpf1b has an isoleucine at this position . crystallographic 
 data collection statistics are summarized in supporting 
 information table 2 , and additional figures including a comparison 
 with acetyl - lysine containing peptide complexes have been included 
 in supporting information figure 2 . comparison 
 of the brpf1b and brd1 ( brpf2 ) acetyl - lysine binding site are shown 
 in figure 3c as well as in supporting information figure 2 . as expected , residues contacting 34 are conserved but differences exist in the rim region of 
 the binding sites that may be used for the design of selective trim24 
 inhibitors . ( a ) 2fo  fc density 
 map contoured at 2 around 34 and ribbon diagram 
 of the phd and bromodomain structure . ( b ) details of the interaction 
 of 34 with the trim24 acetyl - lysine binding site . ( c ) 
 comparison of the acetyl - lysine binding site of the bromodomains of 
 brpf1b and brd1 ( brpf2 ) . carbon atoms of residues present in each 
 structure are colored as indicated in the figure . further comparisons 
 of structural features of brpf and trim24 bromodomains as well as 
 a sequence alignment have been included in supporting 
 information figure 2 . cellular activity of 34 was demonstrated using 
 frap 
 ( fluorescent recovery after photobleaching ) assays ( figure 4 ) . ( b ) 
 time dependence of the fluorescent recovery of the bleached area . ( d ) time dependence of the fluorescent recovery of the bleached area 
 for brpf1b . at least 10 nuclei were bleached for each experiment , 
 and the mean recovery time as well as the sem are shown in ( a ) and 
 ( c ) . frap experiments showed that 34 effectively reduced 
 fluorescent recovery half - time at 1 m concentration for full 
 length trim24 fused to gfp to levels similar to the bromodomain inactivating 
 mutant n980f , suggesting that the inhibitor effectively displaces 
 trim24 from chromatin . full length brpf1b linked to gfp showed , however , 
 predominantly cytoplasmic expression , prohibiting development of frap 
 assays . to demonstrate that the bromodomain of brpf1b is inhibited 
 in the nucleus of cells , we used a construct containing a triplicated 
 bromodomain and a nuclear localization signal . to increase affinity 
 of this construct further , we also increased global acetylation levels 
 using the pan - hdac inhibitor saha . brpf bromodomains have been shown 
 to interact with h4k12ac and h2ak5ac with modest affinity ( 5080 
 m ) , explaining the small assay window and the small increase 
 of recovery times after exposure to saha . however , the strategy adding saha provided a sufficiently large 
 assay window for the triplicated brpf1b construct as reported for 
 the bromodomain in cbp . the 
 inhibitor 34 also reduced recovery times , supporting 
 target engagement of the compound . because overexpression of 
 trim24 has been linked to tumorigenesis , 
 we tested the effect of compound 34 on a small panel 
 of diverse cancer cell lines . however , 34 showed no significant 
 cytotoxicity in the cell lines tested ( figure 5 ) . the breast cancer cell line mcf-7 was resistant to trim24 bromodomain 
 inhibition in the concentration range tested , whereas other cell lines 
 such as the myeloma mm1s model showed modest sensitivity ( gi50 > 
 10 
 m ) . a more comprehensive evaluation of the role of trim24 in 
 cancer cell proliferation and chromatin biology would require the 
 development of a tool compound with improved selectivity . cell viability 
 affected by 34 for a selection of cancer 
 cell lines . in this study we identified a potent trim24/brpf1/2 
 dual inhibitor 
 ( 34 ) that was selective for these bromodomains when screened 
 against a comprehensive panel of bromodomains . however , selectivity 
 of this inhibitor series between trim24 and brpf bromodomains was 
 not achieved with this series . the cocrystal structure in complex 
 with the bromo / phd domain of trim24 revealed an acetyl - lysine mimetic 
 binding mode of the benzimidazolone moiety and a stacked conformation 
 of the two aromatic decorations , creating a good shape complementarity 
 with the trim24 binding site . the identified inhibitor was cell active 
 and displaced ectopically expressed full - length trim24 from chromatin . the identified trim24 inhibitor had modest cytotoxicity on a panel 
 of cancer cell lines . the presented sar and the identified inhibitor 
 represents an excellent starting point for further optimization of 
 trim24 inhibitors as well as a valuable tool for better understanding 
 of the biology of the targeted bromodomain proteins .
trim24 is a transcriptional regulator 
 as well as an e3 ubiquitin 
 ligase . 
 it is overexpressed in diverse tumors , and high expression 
 levels have been linked to poor prognosis in breast cancer patients . 
 trim24 contains a phd / bromodomain offering the opportunity to develop 
 protein interaction inhibitors that target this protein interaction 
 module . 
 here we identified potent acetyl - lysine mimetic benzimidazolones 
 trim24 bromodomain inhibitors . 
 the best compound of this series is 
 a selective brpf1b / trim24 dual inhibitor that bound with a kd of 137 and 222 nm , respectively , but exerted 
 good selectivity over other bromodomains . 
 cellular activity of the 
 inhibitor was demonstrated using frap assays as well as cell viability 
 data .