bromodomains are epigenetic 
 readers that specifically recognize 
 acetyl - lysine ( kac ) marks on proteins . targeting
this protein interaction 
 module with small molecules has recently emerged as a potential therapeutic 
 strategy for the treatment of several diseases including cancer and 
 inflammation . to date , most inhibitor development 
 efforts have been focused on the bet family of bromodomain proteins 
 for which several inhibitors have now entered clinical testing .
bromodomains 
 have good predicted druggability , and 
 selective chemical tool compounds have been developed even for less 
 attractive binding sites that possess open or highly charged acetyl - lysine 
 binding pockets such as baz2 and atad2 .
in addition , potent inhibitors have been developed 
 for highly druggable bromodomains present in brpf , cbp , and brd9 .
interestingly , 
 several recent inhibitor development projects have highlighted the 
 success of fragment - based approaches identifying inhibitors , in particular 
 for poorly druggable bromodomains .
p300/cbp - associated factor ( pcaf , also known as histone acetyltransferase 
 kat2b ) is a multidomain protein that harbors an acetyltransferase 
 ( hat ) and e3 ubiquitin ligase domains as well as a c - terminal bromodomain 
 that may associate with the hats p300 and cbp .
while the roles 
 of the acetyltransferase and the e3 ubiquitin ligase activities have 
 been shown to be required for cell proliferation and apoptosis , little is known about the regulatory function of the pcaf bromodomain 
 in cellular processes .
selective inhibitors , so - called chemical probes , 
 would therefore be interesting reagents to unravel the functions of 
 the pcaf bromodomain and to assess its therapeutic potential as a 
 targeting site for drug development .
an important role of the 
 pcaf acetyl - lysine recognition module 
 has already been demonstrated for the replication of aids viruses . 

the pcaf bromodomain targets the hiv tat protein acetylated at k50 , 
 an essential association that activates hiv-1 transcription and promotes 
 the integrated proviral replication .
development 
 of pcaf bromo - domain inhibitors has therefore been proposed as a potential 
 strategy for the treatment of aids , and this strategy 
 has been confirmed by early n1-aryl - propane-1,3-diamine - based 
 compounds that have been shown to prevent pcaf / tat association .
this compound class has recently been 
 followed up by the development 
 of a series of 2-(3-aminopropylamino ) pyridine 1-oxide derivatives 
 that however did not lead to improvement of the micromolar potency 
 of current pcaf bromodomain inhibitors .
this prompted us to identify more diverse chemical starting points 
 using fragment screening by thermal shifts assays and nmr as well 
 as structure - based approaches .
to extend structural knowledge from our previous 
 apo crystal structure , we initially determined 
 the crystal structure 
 of the pcaf - acetyl - lysine complex to provide insights into acetyl - lysine 
 recognition .
the binding mode of the acetyl - lysine in pcaf strongly 
 resembled that observed in other bromodomains with the kac carbonyl 
 forming two canonical hydrogen bonds to a conserved n803 and a conserved 
 water molecule .
this water is part of a conserved intricate network 
 of structural waters located at the bottom of the pocket that bridge 
 interaction with the za loop y760 and the backbone carbonyl of e750 
 ( figure 1a ) . despite 
 sharing the conserved interactions , the kac binding groove in pcaf 

was fenced on one side by the bulky residue y809 , resulting in a tight 
 and restricted pocket ( figure 1b ) .
large aromatic residues in this position are not common 
 for human bromodomains , present in less than a third of proteins in 
 the family .
( a ) detailed interactions 
 between the bound acetyl - lysine ( kac , shown in yellow stick ) and the 
 pcaf bromodomain .
( b ) electrostatic 
 surface representation reveals a tight central kac pocket that is 
 extended by wide cavities lined by the za and bc loop regions .
another interesting structural 
 feature was the extension of the 
 binding pocket by a shelf created by the za loop harboring a mixture 
 of hydrophobic and polar surface residues ( figure 1b ) .
however , recently the development of baz2 
 inhibitors demonstrated that such large binding pockets can be efficiently 
 occupied for instance by aromatic ring systems of the baz2-icr probe 
 that are aligned by intramolecular aromatic -stacking .
the low affinities of the previously developed 
 compounds prompted 
 us to search for other kac mimetics that would be compatible with 
 the constraint nature of the central pcaf pocket .
we employed different 
 techniques to screen two fragment libraries . in one set of experiments
, 
 we used a thermal stability shift assay to screen a small fragment 
 set at 1 mm and identified several fragments that resulted in positive 
 melting temperature ( tm ) shifts in the 
 range of 12 c ( figure 2 ) .
the highest tm shifts were recorded for 1-methylquinolin-2(1h)-one ) ( 1 , br004 ) , 1h - indole-5-carboxamide 
 ( 2 , br005 ) , 2-methylisoindolin-1-one ( 3 , 
 br013 ) , 4-methoxybenzo[d]isoxazol-3-amine ( 4 , mb360 ) , 
 2,3-dihydrobenzo[b]dioxine-5-carboxamide ( 5 , mb364 ) , and ( 4-(1,2,3-thiadiazol-4-yl)phenyl)methanamine 
 ( 7 , mb093 ) , most of which comprised known kac mimetic 
 scaffolds . in parallel , we used target immobilized nmr screening ( tins ) 
 to screen a diverse collection of approximately 950 commercially available 
 fragments .
we first confirmed that binding 
 of kac to immobilized pcaf could be observed in 2% dmso ( supporting 
 information , figure s1 ) .
subsequently mixtures 
 of fragments were screened with a maximum of four compounds per mix 
 such that the dmso concentration was never more than 2% .
consistent 
 with earlier predictions , we observed a tins profile indicating that 
 pcaf is highly ligandable ( figure 2c ) . on the basis of the profile , 63 hits were selected , 
 a 6.6% hit rate .
in contrast to tins , binding of kac to immobilized 
 pcaf in the presence of dmso could not be detected by spr .
consequently , 
 only those hits that were readily soluble in an aqueous buffer were 
 orthogonally validated by spr .
the binding of 17 hits could be fully 
 characterized by spr , and three were selected for structural studies 
 based on novel features ( supporting information , figure s2 and table s1 ) . these 
 fragments (
9 , 10 , and 11 ) also 
 resulted in substantial tm shifts ( figure 2 ) .
fragments containing 
 kac mimetics identified as potential binders 
 for pcaf bromodomain . chemical structures of the identified fragments 
 are shown in ( a ) together with average tm shifts ( sem ) using triplicate experimental data listed in 
 ( b ) .
the t / r ratio 
 of each compound screened was calculated as a height weighted average 
 of the ratio of the peak amplitude of each nmr resonance in the presence 
 of pcaf over that in the presence of the reference protein .
the t / r ratios were then binned , and the frequency 
 is plotted above .
the asymmetry and tailing to the left ( i.e. , a large 
 number of compounds displaying preferential binding to pcaf ) are indicative 
 of high ligandability of pcaf .
we next attempted to verify the 
 binding modes of the identified 
 fragments and successfully determined the complex crystal structures 
 for seven kac mimetic fragments .
as expected from their chemical structure , 
 all of these fragments occupied the kac binding site through groups 
 that mimicked the hydrogen bond interaction of acetyl - lysine .
because 
 the co - crystallized compounds were small , the contacts with the bromodomain 
 were limited only to the canonical hydrogen bond with n803 and the 
 typical water - mediated contact with y760 ( figure 3a ) . however , additional interactions were 
 also observed for fr11 , of which the 1-ethanol decoration was oriented 
 toward the open za cavity and formed both direct and water - mediated 
 hydrogen bonds to the backbones of the za loop v752 and p751 .
superimposition 
 of all structures revealed that the co - crystallized fragments fit 
 tightly into the narrow kac pocket , and most fragments formed aromatic 
 interactions with y809 that lines the central acetyl - lysine binding 
 groove of the pcaf bromomdomain ( figure 3b ) .
no significant structural alterations 
 were observed when comparing all complexes , suggesting that the pcaf 
 bromodomain contains a rigid acetyl - lysine binding pocket .
some kac 
 mimetic groups of the identified fragments were not specific for pcaf 
 and have been previously shown to bind to other bromodomains , for 
 example , 1 also interacts with atad2 and the isoxazole 8 with brd4 and crebbp with highly conserved binding modes .
( a ) detailed interactions between the bound fragments ( yellow 
 stick ) within the pcaf kac binding site .
the conserved water molecules 
 at the bottom of the pocket are shown in pink spheres , and an additional 
 water molecule involving in additional water - mediated interactions 
 observed in the complex with 11 is highlighted by magenta 
 sphere .
( b ) superimposition of the bound fragments and kac revealed 
 canonical acetyl - lysine mimetic binding modes and no significant conformational 
 changes upon inhibitor binding within the pocket .
coordinates have 
 been deposited under accession codes : 5fe1 , 5fe2 , 5fe3 , 5fe4 , 5fe6 , 5fe7 , and 5fe5 . with the success in identifying the kac mimetic fragments 
 and obtaining 
 the complex structures , we next expanded the ligand series to more 
 decorated compounds by choosing two diverse scaffolds ( figure 4a ) .
two compounds , cpd1 ( 12 ) ( n-(1,4-dimethyl-2-oxo-1,2-dihydroquinolin-7-yl)acetamide ) 
 and cpd2 ( 13 ) ( n-(1,4-dimethyl-2-oxo-1,2-dihydroquinolin-7-yl)methanesulfonam ) , 
 were derivatives based on fragment hit 1 containing an 
 additional 4-methyl group and two different decorations at the seventh 
 position , either with n - linked acetamide or sulfonamide .
a third inhibitor , 
 cpd3 ( 14 ) ( n - methyl-5-(2-oxo-2-(phenylamino)ethoxy)-2-((tetrahydro-2h - pyran-4-yl)oxy)benzamide ) , contained instead a benzene 
 core harboring an acetamide extension , a kac mimetic group resembling 
 fragment 1 .
in addition , this latter compound was also 
 further decorated by tetrahydropyran and n - phenylacetamide 
 linked to the benzene ring via an ethoxy linkage at the ortho and 
 meta positions relative to the acetamide group , respectively .
( a ) chemical structures of 
 three in - house available compounds that contain the fragment - related 
 kac mimetic scaffolds .
( b ) average tm shift 
 values from triplicate experiments for the selected compounds .
( c ) 
 itc binding data for the interactions between 13 and 14 with pcaf with the isotherms of raw titration heat shown 
 on the top and the normalized binding heat with the single - site fitting 
 ( red line ) shown at the bottom .
the binding of these three compounds 
 was initially confirmed by 
 dsf with 12 and 13 , showing a similar degree 
 of stabilization with tm shifts 
 of 2.62.9 c at 1 mm concentration ( figure 4b ) .
because of signal 
 interference , 14 was used at half the concentration of 
 the others but nonetheless produced a similar tm shift of 3.3 c .
as expected , the increase 
 of the tm shift values of these 
 three ligands suggested stronger binding to pcaf bromodomain .
we used 
 isothermal titration calorimetry ( itc ) to determine binding affinities 
 in solution . in agreement with the dsf results ,
the measured kd of 13 was observed to be 21 
 m , 
 which was 3-fold higher than that of the more decorated 14 ( kd of 7 m ) ( figure 4c ) .
the increase 
 in the affinity of the latter was due to significantly larger negative 
 binding enthalpy , suggesting more favorable polar interaction of this 
 inhibitor .
however , the ligand efficiency ( le ) decreased to 0.24 for 14 compared to le of 0.34 for 13 . to gain insight 
 into the selectivity of the most potent fragment hits
, we performed 
 a temperature shift screen using a panel of 48 recombinant bromodomains 
 ( supporting information , figure s3 ) .
this 
 selectivity screen revealed strong interaction of 14 to 
 bromodomains present in cecr2 ( 7.1 c ) , brd7 ( 6.5 c ) , suggesting 
 that this fragment can also be optimized targeting other bromodomains .
, both inhibitors showed strong 
 inhibition for brd9 ( supporting information , table s2 ) .
this is not surprising because 1-methylquinoline-2-ones 
 have been developed into highly specific brd9 inhibitors .
the crystal structures of the ligand  pcaf 
 complex allowed 
 us to evaluate the interactions of the introduced decorations .
all 
 compounds could be unambiguously positioned based on the well - defined 
 electron density map ( figure 5 ) . as expected in complexes with 12 and 13 ,
the 1-methylquinoline-2-one group occupied the kac binding 
 groove and formed similar canonical interactions as observed in the 
 pcaf complex with 1 , while the decoration of both compounds 
 oriented toward the za pockets .
in contrast , the 13 n - linked sulfonamide was observed to replace a water molecule 
 that bridged interactions between e756 and k753 in the kac complex 
 ( figure 1a ) , enabling 
 the formation of two direct hydrogen bonds to these two residues as 
 well as a water - mediated contact to the carbonyl backbone of w746 
 ( figure 5 ) .
interestingly , 
 a sulfonamide at this analogous position also engaged within the kac 
 site of baz2b in the gsk2801 inhibitor complex .
crystal structures of pcaf in complexes with 12 , 13 , and 14 . detailed interactions between the 
 bound compounds with the pcaf bromodomain are shown .
the conserved 
 water at the bottom of the pocket is displayed in pink spheres , while 
 an additional water molecule involving in contact between the 13 and protein is shown in magenta sphere .
coordinates have been 
 deposited under accession codes : 5fe8 , 5fe9 , and 5fdz , respectively .
the binding mode of 14 was slightly different 
 potentially 
 due to its larger decorations and its different acetyl - lysine mimetic 
 moiety ( figure 5 ) . 

the acetamide interacted with pcaf in a typical kac mimetic manner , 
 with the benzene core stacking against the y809 in a similar fashion 
 observed also for the other fragments .
the tetrahydropyran expanded 
 into the open za pocket , while the n - phenylacetamide 
 extension occupied the bc pocket with the phenyl ring located above 
 n803 and oriented perpendicularly to y802 and p804 within a distance 
 for -stacking .
considering its affinity , such 
 limited interactions were rather surprising when compared to the binding 
 mode of the weaker 13 , of which the extended group engaged 
 in more hydrogen bond interactions .
we hypothesize therefore that 14 may gain its increased binding potency through a larger 
 contact surface .
hence , an extension of the kac - mimetic fragments 
 resulted in improved binding affinities despite the lack of direct 
 polar interaction with the protein .
in addition , the large surrounding 
 space adjacent to the za and bc loops provides few spatial constraints , 
 enabling the binding of diverse chemical moieties , albeit with little 
 shape complementarity .
it is likely that elongated , flexible , and 
 uncharged substituents that could span the interface as well as engage 
 a few interactions may be beneficial for increasing ligand potency 
 through targeting the za and bc pockets in the pcaf bromodomain .
the fragment - based approach coupled with structural information 
 presented here offers a platform expanding the knowledge on pcaf kac - mimetic 
 scaffolds and binding modes to aid development of potent and selective 
 pcaf bromodomain inhibitors .
the structural models provide valuable 
 data for the rational design of inhibitors to the rigid and poorly 
 enclosed pcaf kac binding pocket .
comparison of inhibitor binding 
 modes highlights the importance of aromatic interactions with y809 
 and provides several diverse acetyl - lysine mimetic chemotypes .
these 
 chemotypes may serve as attractive starting points for inhibitor development 
 efforts targeting this interesting bromodomain .
all fragments that 
 were cocrystallized formed an acetyl - lysine mimetic hydrogen bond 
 to n803 . thus , for future development of these fragments into more 
 potent hits the fragment need to be grown rather than linked in order 
 to increase potency .
the pcaf bromodomain 
 ( aa 715831 ) containing an n - terminal his6-tag was 
 recombinantly expressed in escherichia coli rosetta .
the protein was initially purified by ni - affinity 
 chromatography and subsequently cleaved the tag with tev protease . 

the cleaved protein was further purified using size exclusion chromatography 
 and stored in 25 mm hepes , ph 7.5 , 150 mm nacl , and 0.5 mm tcep .
fragments were purchased 
 from chemical vendors and were at least 95% pure according vendor 
 specifications .
the purity of the synthesized compounds 12 , 13 , and 14 was assessed by analytical 
 hplc using an agilent 1100 equipped with photodiode array detector 
 ( dad ) , quaternary gradient pump , and micro plate sampler ( agilent 
 220 ) .
separation was performed upon centurysil c18-aq + 5 m , 
 50 mm  4.6 mm ( johnson ) .
the flow rate of the mobile phase was 
 kept at 3.5 ml / min .
mobile phases b and c were acetonitrile with 0.35% 
 cf3co2h and water with 0.35% cf3co2h , respectively .
the gradient conditions were as follows : 
 00.5 min 1% b and 99% c , 3.7 min 90% b and 10% c , 5 min 99% 
 b and 1% c. the injection volume was 10 l . 12 and 13 were 90% pure by hplc at 254 nm and 99% 
 by evaporative light scattering detection ( elsd ) .
the protein at 2 m 
 in 10 mm hepes , ph 7.5 and 250 mm nacl was mixed with the fragments / compounds 
 at 1 mm concentration ( unless stated ) .
the assays , data evaluation , 
 and melting temperature ( tm ) calculation 
 were performed using a real - time pcr mx3005p machine ( stratagene ) 
 with the protocols described previously .
all isothermal calorimetry ( itc ) 
 experiments were carried out on nanoitc ( ta instruments ) at 15 c 
 in the buffer containing 25 mm hepes , ph 7.5 , 150 mm nacl , and 0.5 
 mm tcep .
titration was performed by injecting pcaf ( 250 m ) 
 into the reaction cell containing the compounds ( 1520 m ) . 

the corrected data of the integrated heat of titration were fitted 
 to an independent single binding site model based on the manufactor 
 protocol , from which the thermodynamics parameters ( h and ts ) , equilibrium 
 association and dissociation constants ( ka and kd ) , and stoichiometry ( n ) were calculated .
purified , biotinylated pcaf was captured onto a neutraavidin - coated 
 cm5 sensor chip surface in 20 mm sodium phosphate ph7.5 , 150 mm nacl , 
 2 mm dtt , and 0.05% p20 tween .
an acetyl - lys 16 peptide derived 
 from h4 was used to monitor the ligand binding capacity of the immobilized 
 pcaf bromodomain at regular intervals .
apo crystals 
 of pcaf ( 1418 mg / ml ) were obtained using the vapor 
 diffusion method at 4 c and either single - peg - based ( 2135% 
 peg 3350 , 0.1 m bis - tris ph 5.57.0 ) or peg - smear - based ( 2140% 
 medium - molecular - weight peg smears buffered either with 0.1 m bis - tris 
 ph 6.07.5 or 0.1 m tris ph 7.58.8 ) reservoir solutions .
ligand 
 complex crystals , soaking was performed using the mother liquor supplemented 
 with the fragments / compounds at 1020 mm concentration 
 and 20% ethylene glycol .
diffraction data were collected either in - house 
 or at the diamond light source .
initial structure solution was achieved 
 using molecular replacement with phaser from ccp4 suite and the published coordinates 
 of pcaf .
manual model building alternated 
 with refinement were performed in coot and refmac , respectively .
 the 
 p300/cbp - associated factor plays a central role in retroviral 
 infection and cancer development , and the c - terminal bromodomain provides 
 an opportunity for selective targeting . here 
 , we report several new 
 classes of acetyl - lysine mimetic ligands ranging from mm to low micromolar 
 affinity that were identified using fragment screening approaches . 
 
 the binding modes of the most attractive fragments were determined 
 using high resolution crystal structures providing chemical starting 
 points and structural models for the development of potent and selective 
 pcaf inhibitors . 
Introduction
Results
and Discussion
Conclusion
Experimental Section