prostate cancer ( pca ) is one of the major 
 causes of cancer death in men worldwide .
the molecular basis of the disease involves an irregular behavior 
 of the functions mediated by the androgen receptor ( ar ) .
human ar 
 belongs to the nuclear receptor ( nr ) superfamily of transcription 
 factors , which regulate gene transcription upon ligand binding .
the structure of nrs is extensively documented 
 in the literature , and in general , nrs 
 share the following common organization : a variable amino - terminal 
 activation function domain ( af-1 ) , a highly conserved dna - binding 
 domain ( dbd ) , a hinge region that contains the nuclear localization 
 signal , a conserved c - terminal ligand - binding domain ( lbd ) comprising 
 a 12 helical structure that encloses a central ligand binding pocket 
 ( lbp ) , and a second activation function domain ( af-2 ) that is located 
 at the carboxy - terminal end of the lbd and mediates ligand - dependent 
 transactivation .
ar is activated by the endogenous hormone testosterone 
 ( tes ) and its more potent metabolite dihydrotestosterone ( dht ) , both 
 of which bind in the lbp .
the binding of these endogenous modulators 
 induces a reorganization of helix 12 to the so - called  agonist  
 conformation , generating a structured hydrophobic surface ( af-2 ) suitable 
 for the recruitment of tissue - specific nr coactivators .
such nr coactivators 
 can be thought of as  master switches  , directing and 
 amplifying the subsequent transcriptional activity of the target nr . 
 in a recent work , an additional secondary function site called binding 

function 3 ( bf-3 ) has been reported on the surface of the ar that 
 could also play a relevant role in the allosteric modulation of the 
 af-2 .
nr drug development has traditionally 
 focused on advancing full or partial agonists / antagonists interacting 
 within the lbp of the lbd .
pca has been 
 treated by intervention at the early stages through utility of classical 
 antiandrogens , which act by displacing the natural hormones from the 
 pocket and inducing a conformational change of the helix 12 so that 
 coactivators can not be recruited .
tissue specificity , detrimental 
 side effects , and a loss of the pharmacological effect ( acquired drug 
 resistance ) over time are major and ongoing concerns with such lbp 
 targeting treatment regimes .
it has been demonstrated 
 that it is possible to inhibit the transcriptional activity of the 
 nrs by directly blocking the critical receptor : coactivator interaction .
this alternative approach to traditional nr modulation may furnish 
 greater pharmacological insight and afford opportunities to modulate 
 not only under tissue specific circumstances but without adversely 
 affecting natural ligand binding and so preserving the beneficial / nondisease 
 linked functions of the receptors .
specifically , the steroid receptor 
 coactivator ( src ) family has been postulated as a feasible target 
 for pharmacological intervention .
the 
 viability of targeting ar  coactivator interaction using small 
 molecules has been recently demonstrated .
moreover , it 
 has been postulated that circumventing the lbp will overcome the problem 
 of drug resistance in pca . here
we describe the discovery and characterization of a novel 
 class of selective non - lbp  true 
antiandrogens , characterized 
 by full ar antagonism in inhibiting the recruitment of coactivators 
 and lacking intrinsic partial agonistic properties .
mechanistically , 
 these compounds are totally differentiated from the recent description 
 of true lbp antiandrogens like mdv3100 and rd162 , while their selectivity and druglike nature underpin the potential 
 of a non - lbp intervention strategy in advanced prostate cancer resistant 
 to  classical therapy 
, first described for the true 
 non - lbp targeting antiandrogens pyrvinium pamoate ( pp ) and harmol 
 hydrochloride ( hh ) .
the biological 
 data obtained both on target with time - resolved fluorescence resonance 
 energy transfer ( tr - fret)/fluorescence polarization ( fp ) assays and 
 in cellular pca models demonstrate the non - lbp antagonist activity 
 of the series and an alternative mechanism of inhibition , furnishing 
 a new class of nonpeptidic , small molecule ar : coactivator selective 
 disruptors as leads for the development of novel treatments for prostate 
 cancer .
a virtual ( computational ) screen 
 of six vendor compound databases ( see experimental 
 section ) was performed through a combination of 3d pharmacophore 
 generation and docking .
seven x - ray structures of coactivator peptide 
 bound ar were used to define key ligand - derived pharmacophoric features 
 of the most represented motifs occurring in known ar coactivators . initially ,
common key interaction motifs within 
 the peptide of the form fxxlf , lxxll , or fxxlw were considered to 
 generate a consensus af-2 pharmacophore .
subsequently , a second site - derived 
 pharmacophore model was advanced based on the specific characteristics 
 of the androgen receptor af-2 region , which demonstrates known selectivity 
 toward the fxxlf coactivator motif ( figure 1b ) .
the cocrystallization of the ar lbd bound with 
 dht in the presence of the fxxlf peptide ( pdb i d 1t7r ) provided the structural basis of the af-2 interaction for 
 docking studies . virtual screening and identification of diarylhydrazide 
 scaffolds . (
a ) a series of coactivator peptides cocrystallized in 
 the af-2 groove was employed ; for illustrative purposes we present 
 the fxxlf coactivator motif from pdb entry 1t7r .
the af-2 groove is represented in dark 
 gray . for clarity reasons , only lys720 and glu897
are shown and dht 
 is not illustrated ; ( b ) a 3d pharmacophore 
 model was derived containing the common features between ar coactivators 
 and the two aromatic features of the fxxlf motif .
pharmacophores were 
 used to screen vendor compound databases and to guide the docking 
 of putative  hits  into the af-2 site .
( c , d ) two first round 
 actives 1 ( mdg173 ) and 2 ( mdg15 ) docked 
 poses in the af-2 site , with the surface rendered and only key amino 
 acids shown .
partial mapping of initial hits to the pharmacophore 
 suggested additional virtual screening to identify more potent family 
 members .
images were generated with molecular operating environment 
 ( moe ) and pymol . from the virtual screen ,
a first series of compounds 
 with predicted target affinity was selected from commercially available 
 databases ( see experimental section ) and evaluated 
 for biological activity using tr - fret and fp techniques .
this initial 
 screen ( figures 1c , d and 2 ) identified two small molecules , 1 and 2 , both diarylhydrazides , as possible non - lbp ar antagonists . 

non - lbp modulatory activity was evidenced by demonstration of an ic50 in the range of 50100 m in ar tr - fret coactivator 
 displacement assay and their inability to displace bound fluorescently 
 labeled ligand from the lbp through an fp assay . these first round 
  hit  molecules map only partially to the screening 
 pharmacophore ( figure 1c , d ) .
accordingly , an 
 optimization round of screening was initiated to explore the utility 
 of the scaffold for more effective disruption of ar : coactivator interaction . from these initial data ,
a simple molecular similarity search was 
 performed ( tanimoto coefficient > 70% ) to furnish a new screening 
 series of 37 compounds bearing the desired diarylhydrazide scaffold . 

this second round screen identified four small molecules ( figure 2 ) , 3 ( mdg 483 ) , 4 ( mdg 
 292 ) , 5 ( mdg 506 ) , and 6 ( mdg 508 ) , with 
 improved activity ( ic50 < 50 m in an ar tr - fret 
 assay ) . these ligands were taken forward for additional investigation 
 and characterization .
the series of diaryl - substituted 
 hydrazides identified through the vs process ( figure 2 ) inhibited the recruitment of the fluorescent labeled d11-fxxlf 
 coactivator peptide in the presence of an agonist ( dht ) concentration 
 equal to ec80 using time - resolved fret assays .
d11-fxxlf 
 is a peptide developed from random phage display technology that resembles 
 the src family of coactivator proteins in its flanking sequence but 
 that also has an ar n - terminal interaction domain .
thus , it is a biological mimic of the n - terminal and the 
 src coactivator interactions with the lbd . a 12-point dose 
response 
 curve was determined for those compounds that inhibited coactivator 
 binding in the micromolar range , acting as full ar antagonists , 36 ( figure 3a 
 and table 1 ) .
the background 
 signal , representing diffusion - enhanced fret in the absence of ar , 
 was subtracted from the fret value of each compound and from the maximal 
 signal , representing fxxlf - bound ar in presence of dht .
diarylhydrazides inhibit the ar recruitment 
 of a fluorescent - labeled d11-fxxlf peptide but do not displace a potent 
 fluorescent ligand from the ar - lbp .
( a and b ) compounds were tested 
 in a tr - fret assay across a concentration range from 100 m 
 to 45 nm in the presence of a concentration of dht = ec80 in ar - lbd wt ( a ) and ar - lbd t877a ( b ) .
error 
 bars represent the standard error of the mean ( sem ) for n = 6 values .
data was fitted using log antagonist concentration vs 
 response ( variable slope ) with graphpad prism 5 ( see experimental section for details ) .
( c ) fluorescence polarization 
 data is plotted as percent maximal activity represented by ar - lbd 
 and fluorophore complex ( 0% inhibition ) .
the minimum control value 
 represents free fluorophore ( free f ) in solution ( 100% inhibition ) . 

the tr - fret assay can not differentiate between 
 direct coactivator antagonists acting on the lbd surface and classical 
 ar antagonists , which also functionally disrupt coactivator recruitment 
 by displacing dht from the ligand binding pocket . to characterize 
 the nature of the antagonist effect ,
compounds were tested for their 
 ability to displace a potent fluorescent ligand ( fluorophore ) from 
 the ar lbp through a fluorescence polarization ( fp ) assay at a single 
 point concentration ( 50 m ) , using cyproterone acetate ( cpa ) 
 at the same concentration as a reference , a known ar lbp - mediated 
 antagonist .
all compounds tested showed 0% inhibition of the ar - lbd 
 and fluorophore complex , indicating a non - lbp - mediated mechanism of 
 ar transactivation inhibition ( figure 3c ) .
compound 3 gave an unusually high value of millipolarization 
 units ( mp ) , 20% higher than the maximal control ( figure 3c ) .
this could be indicative of solubility issues in the assay 
 buffer and therefore could generate a false negative result .
it is 
 known that fp assay outcomes can be influenced by intrinsic fluorescence 
 of the test compounds and/or light scattering phenomena due to poor 
 solubility and precipitation . to minimize the possibility of such 
 false negative or positive reporting ,
none 
 of the compounds tested showed competing autofluorescence in the assay 
 conditions or was shown to be a false negative .
results are shown 
 in the supporting information   ( supplementary 
 figure 2 ) . to further validate the utility of these ligands 
 in pca
, on - target binding experiments were also performed using the 
 recombinant t877a ar mutant characteristic of advanced 
 stage androgen - independent pca . in tr - fret
, the compounds demonstrated 
 similar activity to that observed in the wild type assays , indicating 
 their potential in advanced phases of prostate cancer ( figure 3b ) .
activity data are in agreement 
 for 4 and 5 in both ar wt and art877a .
the 
 higher confidence mp values and experimental reproducibility obtained 
 for 4 and 5 in coactivator studies were 
 used as the basis to advance these compounds to cellular characterization 
 and receptor subtype selectivity evaluations .
we undertook to profile the selectivity of these compounds for 
 ar over other members of the same phylogenetic branch of the steroidal 
 nuclear receptor subfamily .
compound binding affinities for progesterone 
 receptor ( pr ) , glucocorticoid receptor ( gr ) , estrogen receptor  
 ( er- ) , and estrogen receptor  ( er- ) were determined 
 using tr - fret ( table 2 ) .
er- 
 and estradiol  er- complex and do not displace fluorescent - labeled 
 coactivator src1 - 4 from dexamethasone  gr complex at concentrations 
 up to 100 m ( supporting information , supplementary figures 35 ) . compound 5 binds 
 pr with comparable affinity to that observed for ar , while 4 demonstrates approximately 2-fold binding selectivity for the ar 
 over pr . in functional evaluation
we determined that the diarylhydrazide 
 compounds are full ar antagonists , with a partial antagonistic profile 
 demonstrated in pr , displacing src14 from progesterone 
the non - lbp nature of 
 this interaction was confirmed by an fp assay ( supporting information , supplementary figure 6 ) .
compounds were tested at a concentration range from 100 to 
 1 m in the presence of a concentration of progesterone = ec80 .
error bars represent the standard error of the mean 
 ( sem ) for n = 6 values .
data was fitted using log 
 antagonist concentration vs response ( variable slope ) with graphpad 
 prism 5 ( see experimental section for details ) . 

ic50 values are shown in 
 table 2 . to ascertain the translational ( clinical ) 
 potential of these ligands ,
compounds were evaluated in cellular models 
 of prostate cancer ( lncap , an androgen - dependent 
 cell line and pc-3 , an androgen - independent 
 cell line ) and in normal prostatic epithelia cell line pwr-1e .
cell viability was assessed after 24 h of incubation 
 with the test compounds at three different concentrations ( figure 5 ) .
the classical antiandrogen cpa was used as a 
 reference , showing a minor effect at 50 m in the androgen independent 
 cell line pc-3 . at 50 m 4
reduces cell viability 
 to a 5060% , whereas 5 acts consistently across 
 the three cell lines , retaining cell viability at around 80% .
compounds were tested at 5  10 , 1  10 , and 5  10 m. error bars represent the sem of two independent experiments done 
 in triplicate ( n = 6 ) .
the diarylhydrazides were evaluated for their effects 
 on the ar signaling pathway and on hormone - dependent cellular growth 
 of lncap cells .
compound 5 was well - tolerated after 5 
 days of treatment at 10 and 20 m concentrations and enabled 
 observation of a specific reduction in dht - treated cell count ( figure 6a ) .
prostate specific antigen ( psa ) 
 is a serine protease normally secreted by the prostate epithelia . 

psa is widely used as a 
 marker for pca , as its serum levels are 
 increased in this condition .
compound 5 was shown to 
 reduce dht - induced psa secretion in a dose  response fashion 
 as quantified by an elisa experiment in lncap cells ( figure 6b ) .
it is well - documented that classical antiandrogens 
 ( i.e. , those binding within the lbp / competing with endogenous ligands ) 
 have partial agonistic properties , which make them less useful in 
 the management of advanced prostate cancer . arising from this inherent agonism , in an androgen - deprived lncap 
 cell line , antiandrogens such as cpa
can actually activate the ar pathway and stimulate cell growth . in direct contrast to the behavior of traditional antagonists ,
5 shows no detectable agonist or partial agonist activity 
 at tested concentrations , consistent with an alternative mechanism 
 to that of the classical antiandrogens . finally , treatment with 5 at 10 m
was found to antagonize cpa partial agonist 
 activity ( measured as secreted psa levels in the cellular media in 
 an elisa experiment ) , suggesting its potential benefit in combination 
 therapy for advanced stages of prostate cancer ( figure 6c ) . to further challenge this hypothesis ,
the compounds also demonstrated similar effects in this alternate 
 system , supporting the hypothesis of their functioning as true antiandrogens 
 ( supporting information , supplementary 
 figure 7 ) .
( a and b ) 5 reduces androgen - stimulated 
 cell growth and dht - dependent ar signaling measured as psa levels 
 secreted in the cellular media in a dose - dependent fashion .
( c ) 5 at 10 m reduces cpa - induced ar signaling ( in absence 
 of androgens ) measured as psa levels secreted in the cellular media 
 in a dose - dependent fashion .
data are presented as the mean of two 
 independent experiments , and bars show sem for n = 
 6 values ( a ) .
secreted psa ( ng / ml ) was measured considering the optical 
 density at 450 nm minus the optical density at 540 nm and interpolating 
 the values from the standard curve .
data are presented as mean of 
 two independent experiments , and bars show sem for n = 4 values ( b and c ) .
classical antiandrogen therapy is known 
 to have limited beneficial effects in hormone - insensitive pca .
alternative 
 ar inhibitors are therefore needed in the treatment of pca . in this 
 study , we demonstrate the successful implementation of a virtual screening 
 approach in the identification of small molecule ar modulators , where 
 the structural motif of ar coactivators was included in a 3d pharmacophore . 

we report the discovery , identification , and characterization of a 
 novel series of diarylhydrazide non - lbp - binding antiandrogen compounds , 
 with demonstrated ability to displace ar coactivators and with established 
 potency in ar - dependent prostate cancer cell lines .
activity was measured 
 with a tr - fret assay and a non - lbp - mediated mechanism of inhibition 
 was confirmed by fp assay .
these compounds are shown to function without 
 any demonstrated intrinsic or partial agonist activity in ar and therefore 
 can be classified as true non - lbp antiandrogens .
the nature of nr coactivators and the high homology of nr coactivator 
 binding sites are such that , to more fully profile the potential utility 
 of these ligands , their selectivity was evaluated across members of 
 the subclass of steroid receptors , including er- and er- , 
 gr , ar , and pr .
the selectivity of the diarylhydrazide scaffold 
 for the ar was demonstrated through tr - fret evaluation in the estrogen 
 and glucocorticoid receptors , where agonist bound receptor recruitment 
 of coactivator was unimpaired at screening concentrations up to 100 
 m .
we additionally investigated the potential cytotoxicity 
 of the diarylhydrazides in three different cell lines , selecting 5 for its favorable cytotoxic profile ( cell viability was 
 retained at around 80% in different prostatic cellular models ) .
unmodified diarylhydrazide screening hits were also shown to have 
 2-fold selectivity for ar over pr , with partial antagonist activity 
 demonstrated for the scaffolds in a pr functional assay , remarkable 
 given the high ( > 60% ) homology of these nr family members .
futhermore , given the established utility of mifepristone ( a pr 
 modulator which also has antiandrogenic activity ) in the treatment 
 of castration resistant prostate cancer , the narrower 
 selectivity window observed for these ar ligands in pr over the other 
 nr s assessed is not a significant concern in the context of 
 the therapeutic area under consideration .
classical antiandrogens 
 can be also distinguished for their different behaviors at a cellular 
 level .
save for two recent examples , all lbp antiandrogens 
 described to date have also intrinsic partial agonist activity , demonstrated by induction of psa in the absence 
 of hormone stimulation in lncap cells . in this study ,
the novel non - lbp 
 diarylhydrazide antiandrogen 5 did not induce psa expression 
 in absence of hormone stimulation when compared to cpa . in androgen - deprived 
 lncap cells ,
5 reduces psa expression in combination 
 with cpa , antagonizing its partial agonist activity in a dose responsive 
 fashion .
this result supports the hypothesis of a nonclassical mechanism 
 of ar inhibition for these diarylhydrazide ligands and it also demonstrates 
 the potential application of these and other non - lbp antiandrogen 
 small molecules targeting alternative ar sites in combination with 
 existing prostate cancer therapy .
through application of virtual screening 
 methodologies , we present and characterize novel diarylhydrazide scaffolds 
 as true antiandrogens  displacing ar  coactivator interaction 
 and having a full antagonistic profile on ar ( both wt and t877a ) , 
 partial antagonistic profile for pr , and selectivity for the other 
 members of the nr-3 family ( gr , er- , and er- ) .
the initial small molecule non - lbp true ar modulators provided by 
 this study will be used to further characterize the ar 
coactivator 
 interface , to understand the basis of selectivity , and to further 
 guide rational drug design in the search of other novel scaffolds 
 directed at this interface .
black , low volume , 384-well assay plates ( corning , ny , cat . no .
3676 ) 
 were used to perform the assay ( total volume 20 l ) , and tr - fret 
 signal was measured with pherastar equipment ( bmg labtech ) using a 
 lanthascreen optic module ( excitation , 335 nm ; emission , 520 nm channel 
 a and 495 nm channel b ) .
tr - fret values were calculated at 10 
 flashes per well , using a delay time of 100 s and integration 
 time 200 s as recommended by the invitrogen assay guidelines . 

a serial dilution of compounds was first prepared in 
 100 dmso ( sigma - aldrich ) starting from the maximum desired concentration 
 to achieve a 12 point range concentration using 96-well polypropylene 
 plates ( nalgene nunc , rochester , ny ) .
each 100 solution was 
 diluted to 2 concentration with tr - fret coregulator buffer a 
 ( invitrogen proprietary buffer ) , yielding a final concentration of 
 1% dmso in each well .
ten microliters of 2 solution was then 
 added to the 384-well plate , following addition of 5 l of 4 
 ar - lbd and 5 l of d11-fxxlf / tb anti - gst antibody in agonist 
 mode and 5 l of d11-fxxlf / tb anti - gst antibody / dht ( included 
 at a concentration equal to ec80 as determined by running 
 the assay in agonist mode first).d11-fxxlf and tb antibody were premixed in 
 light protecting vials prior to use .
a final concentration of 5 mm 
 dtt was used in the assay buffer in order to prevent protein degradation . 

all plates ( agonist and antagonist mode ) were incubated between 2 
 and 4 h at room temperature protected from light prior to tr - fret 
 measurement .
ic50 values were determined by testing each 
 ligand at concentrations ranging from 100 m to 45 nm using 
 2- and 3-fold dilutions to generate a 12 point dose 
data was fitted using the sigmoidal dose response ( variable 
 slope ) available from graphpad prism 5.the z factor for these assays 
 was > 0.5 , as calculated by the equation provided by zhang et al .
in line with the assay protocol , a 
 known agonist , dihydrotestosterone ( dht , cat no .
a8380 , sigma ) , and 
 a known antagonist , cyproterone acetate ( cat no .
a control with no ar - lbd present was 
 included to account for diffusion - enhanced fret or ligand - independent 
 coactivator recruitment .
a negative control with 2 dmso was 
 present to account for any solvent vehicle effects .
the 
 assay was adapted to exclude possible nonspecific aggregation mechanism 
 of inhibition by adding very low concentration of detergent triton 
 x-100 ( 0.01% ) to the assay buffer following the shoichet review guidelines ( supporting information , supplementary figure 1 ) .
p3018 ) was 
 used to investigate the binding of the test compound to the lbp site , 
 occupied by a high - affinity fluorophore ligand ( fluormone ) .
the 100 test compound solutions in dmso were diluted in ar 
 green buffer ( invitrogen ) to achieve 2 concentrations and placed 
 in a 384-well plate ( corning , cat no .
ar - lbd and fluormone ( 2 ) mix were prepared separately 
 and then added to each compound dilution to achieve a final concentration 
 lbd - fluormone of 50 and 2 nm , respectively
. plates were incubated 
 protected from light for at least 4 h. controls included a maximum 
 mp positive control , which consists of the ar - lbd and fluormone mix 
 ( 2 ) , and a minimum mp control , containing only fluormone ( 2 ) . 

a vehicle control was added to account for dmso effect , and a blank 
 control containing buffer only .
fluorescence polarization was measured 
 with pherastar equipment ( bmg labtech ) using an optic module with 
 excitation at 485 nm and emission at 530 nm .
lncap cells ( androgen - dependent ) , pc-3 
 ( androgen - independent ) , and pwr-1e ( normal prostatic epithelia ) were 
 cultured in rpmi-1640 glutamax ( invitrogen ) , f12k ( invitrogen ) , and 
 k - sfm media ( invitrogen ) .
the first two were supplemented with 10% 
 fetal bovine serum ( fbs ) , penicillin ( 100 units / ml ) , and streptomycin 
 ( 100 g / ml ) .
k - sfm was supplemented with 5 ng / ml epidermal growth 
 factor ( egf ) and 0.05 mg / ml bovine pituitary extract ( bpe ) .
cells 
 were propagated at 1:3 or 1:6 dilutions at 37 c in 5% co2 . for cell 
 viability ( end point )
assays lncap , pc-3 , and pwr-1e cells were seeded 
 at 2.5  10/ml density in 200 l volume of 
 a 96-well plate in triplicate and incubated for 24 h prior testing . 

test compounds were included at different concentrations to achieve 
 a final concentration of 0.5% dmso in each well .
cell viability was assessed 
 after 24 h of treatment using 10% alamarblue reagent ( invitrogen ) 
 for each well .
cell viability was monitored by the reduction of resazurin , 
 a blue , cell - permeable , nontoxic compound , to resorufin , a red and 
 highly fluorescent product .
viable cells continuously convert resazurin 
 to resorufin , increasing the overall color and fluorescence of the 
 media surrounding cells .
fluorescence intensity can be quantitatively 
 determined with a fluorescence microplate reader at excitation / emission 
 544 nm/590 nm ( spectramax gemini ) . for hormone - dependent cell 
 proliferation assays in androgen - deprived lncap cells ,
cells were 
 seeded at 2  10 cells / ml in a 24-well plate in triplicate . 

cells were plated in phenol red free rpmi glutamax ( invitrogen ) supplemented 
 with 10% charcoal - stripped fbs to deplete endogenous steroids 48 h 
 prior to the assay , as described in previous reports .
the optimal condition for the treatment was found to be 
 5 days and the concentration of dht included to stimulate the cells 
 was 0.1 nm .
cells were treated with different concentrations of test 
 compounds with or without 0.1 nm dht to achieve a final concentration 
 of 0.1% dmso in each well .
a control for the vehicle was included 
 to ensure that no effect on viability could be detected .
media and 
 treatments were replaced every second day , after washing the cells 
 twice with 1 pbs .
supernatants were collected after 5 days for 
 evaluation of secreted psa levels , and cell proliferation was assessed 
 for the same plate using alamarblue in order to exclude nonspecific 
 effects due to toxicity issues
. secreted levels 
 of prostate specific antigen were evaluated with a commercially available 
 kit ( quantikine human kallikrein 3/psa immunoassay , r&d systems ) . 

, 50 l of standards and cell culture samples were added 
 to precoated wells containing assay diluent rd1w ( r&d systems ) 
 and incubated for 2 h at room temperature .
unbound material was washed 
 several times and 200 l of horseradish peroxidase ( hrp ) labeled 
 psa conjugate antibody was added to each well and further incubated 
 for 2 h at room temperature .
wells were washed and treated with colored 
 substrate ( tetramethylbenzidine ) for an additional 30 min , after which 
 50 l of stop solution ( 2 n sulfuric acid ) was added per well 
 and optical density ( 450 nm with correction at 540 nm ) was read with 
 a plate reader within 30 min ( versamax ) .
a virtual screen was designed to 
 select compounds mapping onto the peptide binding surface ( af2 ) of 
 the ar receptor , based on an ensemble of documented x - ray crystal 
 structures ( pdb i d 1t73 , 1t74 , 1t76 , 1t79 , 1t7f , 1t7 m , 1t7r , and 1t7 t ) .
molecular operating environment ( moe ) software was employed to preprocess the proteins and 
 to remove the coactivator peptides from the complexes .
an initial 
 pharmacophore was generated using the moe pharmacophore elucidator 
 and considering the most significant features , which involved hydrophobic , 
 donor , and acceptor features .
a second pharmacophore was developed 
 including two additional hydrophobic / aromatic features to represent 
 the phe side chains present in the fxxlf coactivator motif ( 1t7r ) , so as to increase 
 the selectivity for ar over other families of nuclear receptor .
these 
 pharmacophore models were then applied for in silico screens of small - molecule 
 commercial libraries to identify compounds that resemble the  active 
 principle  of the starting peptides .
a number of vendor databases 
 were selected for screening of ligands , including amsterdam ( 5389 compounds ) , peakdale ( 8188 ) , asinex platinum collection 
 ( 75  258 ) , specs ( 175  800 ) , 
 maybridge ( 56  870 ) , and zinc ( 4.6 million ) compounds .
a bayesian analysis was performed on the 
 peptide structures to estimate parameters of an underlying distribution 
 based on the observed distribution .
the above databases were then 
 filtered for those compounds with properties similar to the peptides , 
 thus focusing the search on the ar ligand chemical space .
all molecules were standardized for stereochemistry 
 and charges and ionized at a ph of 7.4 and all calculable tautomers 
 were enumerated . at this stage the conformational flexibility of the 
 screening compounds was explored using the omega software ( openeye scientific package ) .
a maximum of 50 
 conformations were generated for each molecule in the data set .
the virtual molecules were overlaid 
 on and compared to the generated pharmacophore of the active ligands , 
 and those molecules that compared favorably were advanced for additional 
 virtual screening and scoring . the fast rigid exhaustive docking ( fred ) software as implemented in openeye scientific s 
 package was used to exhaustively examine all possible poses within 
 the protein site , filtering for shape complementarity and scoring . 

the smaller databases ( amsterdam and 
 peakdale ) were screened on all 13 crystal 
 structures and only ligands scoring well on more than one crystal 
 structure were considered .
the larger databases specs , asinex , maybridge , and zinc were screened on the 1t7r crystal structure .
a structural similarity search was 
 conducted on 1 and 2 using a tanimoto coefficient 
 of > 70% on the specs compound database .
thirty - seven compounds were purchased and four small molecules were 
 selected for optimization and characterization studies based on their 
 improved on - target activity determined by tr - fret .
all screening compounds 
 described in this work were purchased as commercial samples from specs 
 nv .
compound purity in all instances 
 was greater than 95% as determined by lcms and nmr .
 prostate cancer ( pca ) therapy typically involves administration 
 of  classical  antiandrogens , competitive inhibitors 
 of androgen receptor ( ar ) ligands , dihydrotestosterone ( dht ) and testosterone 
 ( tes ) , for the ligand - binding pocket ( lbp ) in the ligand - binding domain 
 ( lbd ) of ar . 
 prolonged lbp - targeting leads to resistance , and alternative 
 therapies are urgently required . 
 we report the identification and 
 characterization of a novel series of diarylhydrazides as selective 
 disruptors of ar interaction with coactivators through application 
 of structure and ligand - based virtual screening . 
 compounds demonstrate 
 full (  true  ) antagonism in ar with low micromolar potency , 
 selectivity over estrogen receptors  and  and glucocorticoid 
 receptor , and partial antagonism of the progesterone receptor . 
 mdg506 
 ( 5 ) demonstrates low cellular toxicity in pca models 
 and dose responsive reduction of classical antiandrogen - induced prostate 
 specific antigen expression . 
 these data provide compelling evidence 
 for such non - lbp intervention as an alternative approach or in combination 
 with classical pca therapy . 
Introduction
Results
Discussion
Conclusion
Experimental Section