there is a critical need to develop potent 
 and selective therapeutic 
 agents capable of targeting malignant tissue without compromising 
 normal cell viability .
while chemotherapeutic agents ( e.g. , doxorubicin 
 and docetaxel ) remain widely used in the clinic , they lack inherent 
 selectivity desired to limit toxicity to normal cells . in addition , administration of chemotherapeutic agents 
 can induce drug resistance , resulting in disease progression .
thus , the development of more targeted therapies 
 could circumvent nonspecific interactions and potentially overcome 
 drug resistance in cancer therapy .
intriguing studies are currently 
 exploring new methods to engage 
 biomolecular targets with high affinity and specificity , including 
 the generation of multivalent and heterobifunctional constructs .
advances 
 in chemical synthesis techniques , such as cross - coupling and conjugation 
 strategies , have enabled chemists to decorate a plethora of molecular 
 species with targeting moieties , providing access to elaborate molecular 
 architectures that can be tailored to occupy distinct binding sites 
 within one or multiple biomacromolecules .
although these types of 
 compounds fall outside the molecular weight range of typical drug 
 compounds ( 5003000 da ) , increasing interest in developing 
 new chemical entities that can modulate biomolecular targets in novel 
 ways and address selectivity requirements are emerging . to date
, 
 there have been only limited examples evaluating the potential 
 for targeting the androgen receptor ( ar ) with steroidal conjugates . 

the ar is an important drug target for treatment of prostate cancer 
 and has been the subject of research for several decades .
a large 
 number of bioactive compounds targeting ar have been identified via 
 screening efforts . in this review ,
we 
 begin by providing a rationale for continued studies in prostate cancer 
 pharmacology targeting the ar .
particular focus is placed on examining 
 current approaches to specifically engage and modulate ar activity 
 with steroid conjugates utilizing rational design principles .
androgens are a class of steroid 
 hormones that consist of 19-carbon 
 derivatives of cholesterol and are synthesized by the testis and adrenal 
 glands .
they are also precursors for estrogens , 
 the female sex hormones , produced by hydroxylation , elimination , and 
 aromatization of androgens through the enzyme aromatase .
functioning 
 primarily through the ar , which is a ligand - dependent transcription 
 factor , androgens play a fundamental role in the development and survival 
 of male reproductive tissues , such as the prostate , by influencing 
 gene expression levels .
the body 
 maintains control of testosterone ( the most abundant androgen 
 in men ) levels within a normal reference range of 240800 
 ng / dl .
low 
 levels of testosterone resulting from zinc deficiency or aging can 
 lead to fatigue and erectile dysfunction .
by contrast , high levels of testosterone have been linked to a variety 
 of diseases , including prostate cancer .
prostate cancer remains the most common cancer among men and 
 is 
 globally estimated to affect 900  000 patients every year . as the second leading cause of cancer - related 
 deaths in men ( 258  000/year ) , approximately one out of every 
 six men will be diagnosed with prostate cancer in the u.s .
if detected 
 early , an arsenal of therapeutic options currently provide a promising 
 chance for long - term survival .
however , 40% of patients will 
 develop castration - resistant prostate cancer ( crpc ) , arising from 
 drug resistance ( vida infra ) , which is associated with poor survival 
 rates .
the ar is a 110 kda protein that shares sequence 
 homology with 
 other nuclear hormone receptors in the superfamily , including the 
 progesterone receptor ( pr ) , glucocorticoid receptor ( gr ) , and estrogen 
 receptor ( er ) .
the ar consists of four 
 basic elements : n - terminal domain , dna binding domain , hinge region , 
 and the ligand binding domain ( lbd ) .
the 
 first domain is the 559 amino acid long intrinsically disordered n - terminal 
 domain , which contains the ligand - independent activation function 
 1 ( af-1 ) .
activation function sites encode signature motifs containing 
 lxxll or fxxlf sequences to recruit co - regulatory proteins that are 
 essential for transcription .
the most highly conserved region within 
 all nuclear hormone receptors , including ar , is the centrally located 
 dna binding domain , consisting of two zinc finger domains that recognize 
 specific dna consensus sequences known as the androgen response elements 
 ( figure 1a ) .
the third domain , dubbed the hinge 
 region , connects the dna binding domain to the ligand - binding domain 
 ( figure 1b ) . the ligand - binding domain ( lbd ) 

contains ligand - dependent activation function 2 ( af-2 ) , forms the 
 ligand - binding pocket , and mediates interactions between the ar and 
 heat shock proteins ( figure 1b ) .
importantly , af-2 can interact with an fxxlf 
 binding motif located within the n - terminal domain , a feature unique 
 to ar .
x - ray crystal structure of ( a ) androgen 
 receptor ( ar ) dna binding 
 domain ( ribbon , red ) in complex with the androgen response elements 
 ( sticks , pdb code 1r4i ) and ( b ) ar ligand binding domain ( ribbon , gray ) and portion of 
 hinge region ( ribbon , blue ) in complex with native ligand ( sticks , 
 green , pdb code 1i37 ) .
( c ) amino acids residues that establish high affinity binding 
 with native ligand dht ( pdb code 2ama ) .
the crystal structure of the ar lbd bound to native ligand 
 ( dht ) 
 reveals the amino acid residues critical for maintaining high binding 
 affinity ( figure 1c ) .
although van der waals forces contribute to binding affinity , hydrogen 
 bonds establish stronger interactions with the native ligand .
arg752 forms a hydrogen bond with the o3 atom 
 ( ketone ) of the steroid ligand .
mutagenesis of arg752 has been shown 
 to compromise binding affinity , suggesting the importance of this 
 interaction for achieving high affinity .
in addition , asn705 and thr877 form hydrogen bonds with the 17- 
 hydroxyl group of the steroid ligand .
mutagenesis of asn705 and thr877 
 have also resulted in reduced binding affinity and specificity , establishing 
 their importance to maintaining high affinity .
it is important to note that modifications to the 17- hydroxyl 
 group can result in diminished binding affinity , while even large 
 substituent modifications at the 17- position often retain 
 strong binding interactions .
the ar is a ligand - dependent 
 transcription factor that is stabilized 
 in the cytoplasm by chaperone proteins ( figure 2 ) .
competitive displacement of the chaperones 
 by dihydrotestosterone ( dht ) , an androgen biosynthesized from testosterone 
 through the enzyme 5-reductase , activates the ar . upon activation
, a conformational change brings 
 the n- and c - termini into proximity and facilitates ar dimerization . upon translocation into the nucleus , ar binds to palindromic 5-tgttct-3 
 consensus sequences ( androgen response elements ) in the promoter regions 
 of target genes .
this event stimulates the recruitment 
 of necessary cofactors , including lxxll or fxxlf motif - containing 
 proteins , and other components of the transcriptional machinery to 
 regulate gene expression .
abbreviations : 
 dht , dihydrotestosterone ; hsp , heat shock protein ; p , phosphorylation 
 site ; fxxlf , coactivator protein .
the 
 ar mediates a variety of androgen - dependent diseases including 
 benign prostatic hypertrophy ( bph ) , prostatic intraepithelial neoplasia 
 ( pin ) , and prostate cancer .
it has been 
 proposed that prostate cancer often originates from high - grade prostatic 
 intraepithelial neoplasia ( hgpin ) , a process in which subtle alterations 
 in the shape and size of prostate cells occur .
more importantly , progression 
 of prostate cancer has been linked to elevated expression of ar in 
 malignant tissue , suggesting that ar plays a central role in prostate 
 cancer cell biology .
although many hypotheses 
 regarding the involvement of ar in prostate cancer progression have 
 been postulated , the precise molecular mechanisms are not fully understood .
patients diagnosed with localized or metastatic prostate cancer 
 usually undergo androgen deprivation therapy ( reduction of circulating 
 androgen levels ) , through chemical castration ( gonadotropin - releasing 
 hormone agonists ) or surgical castration . unfortunately , these methods do not completely eliminate circulating 
 levels of androgens , as the tumor itself is capable of local androgen 
 synthesis , due to the expression of androgen biosynthetic enzymes .
this has led to numerous research efforts focusing 
 on the development of inhibitors that interfere with key enzymes , 
 such as cytochrome p450 17a1 ( cyp17a1 ) , in androgen biosynthesis as 
 exemplified by the recent fda approval of abiraterone ( zytiga ) .
the standard treatment approach for prostate 
 cancer involves androgen 
 deprivation therapy in conjunction with small molecule anti - androgens 
 that block ar signaling ( figure 3a ) .
anti - androgens compete with dht for binding 
 to ar , thus inhibiting ar transactivation through a variety of mechanisms , 
 including disruption of nuclear localization , interruption of dna 
 binding , and interference with coactivator recruitment .
unfortunately , most patients receiving anti - androgen therapy eventually 
 develop drug resistance as indicated by rising levels of serum prostate - specific 
 antigen ( psa ) , a gene regulated by ar , leading to the lethal disease 
 state termed castration - resistant prostate cancer or crpc .
small molecule inhibitors targeting the ar : ( a ) anti - androgens ; 
 ( b ) activation function 2 inhibitors ; ( c ) allosteric ( bf3 site ) regulators ; 
 ( d ) n - terminal domain inhibitors .
purple denotes approved therapies 
 for androgen - dependent prostate cancer , and orange represents approved 
 therapies for castration - resistant prostate cancer .
current mechanisms proposed for advancement to 
 crpc include the 
 following:(1)alterations in ar co - regulatory protein 
 balance;(2)somatic gain 
 of function mutations 
 within ar , with the majority in the lbd , resulting in activation by 
 other steroid hormones and anti - androgens;(3)generation of new fusion gene products;(4)ar  ligand - independent  

activation via cross - talk with other signaling pathways.these mechanisms have garnered significant attention because 
 of their ability to  reactivate  ar and disease progression , 
 and provide a conceptual underpinning to guide development of new 
 therapeutic interventions .
nevertheless , currently crpc is primarily 
 treated with chemotherapeutic agents , immunotherapy , or abiraterone 
 ( vida supra ) .
alterations in ar co - regulatory protein 
 balance ; somatic gain 
 of function mutations 
 within ar , with the majority in the lbd , resulting in activation by 
 other steroid hormones and anti - androgens ; generation of new fusion gene products ; ar  ligand - independent  
 activation via cross - talk with other signaling pathways .
ar have been identified via chemical screening efforts and include 
 compounds that act on the af-2 ( figure 3b ) 
 or bf3 site ( figure 3c ) on ar to regulate its 
 activity .
the bf3 site is a hydrophobic binding pocket located adjacent to 
 af-2 on the surface of ar that can allosterically regulate binding 
 interactions between ar and coactivator proteins .
the development 
 of noncompetitive modulators ( that do not compete against dht for 
 ligand binding ) could circumvent drug resistance in ar pharmacology . 

while promising , these noncompetitive approaches have yet to yield 
 candidates for clinical implementation , likely because of the high 
 concentrations required to suppress ar activity . in the future , it may be important to utilize structure - based 
 design to generate more potent af-2 or bf3 inhibitors .
in contrast , 
 continuing interest in anti - androgen drug development 
 has led to the fda approval of enzalutamide , which targets the ar 
 ligand binding domain for the treatment of crpc ( figure 3a ) .
unfortunately , recent evidence 
 suggests that drug resistance to enzalutamide can emerge from point 
 mutations within the ar lbd , such as f876l .
additionally , drug resistance has been proposed to arise from constitutively 
 active ar splice variants lacking the ar ligand binding domain .
this has led researchers to focus on innovative 
 ways to antagonize ar splice variants and the development of n - terminal 
 domain inhibitors ( figure 3d ) .
it is important to note , however , that no structural information 
 exists for the ar n - terminal domain , complicating the design of n - terminal 
 domain antagonists . although it 
 is tempting to speculate that ar splice variants are 
 mainly responsible for drug resistance to enzalutamide , the precise 
 molecular mechanisms remain unknown .
evidence suggests that full - length 
 ar is required for signaling , although different sets of studies demonstrate 
 that er splice variants can be constitutively active in the absence 
 of ligand .
also , an intriguing report 
 has similarly suggested that the gr can become constitutively active 
 in the absence of its lbd .
future research 
 may illuminate whether other nuclear hormone receptors can exhibit 
 similar modes of action .
bioactive  hit  compounds , typically identified from 
 screening efforts , often lack the potency and selectivity required 
 for translation to a clinical setting . for this reason
compounds through 
 iterative rounds of synthesis and rigorous bioassays . while this strategy 
 remains widely utilized in both academic and industrial research programs , 
 rational design of therapeutic agents aims to streamline these issues 
 by initially identifying more potent and selective compounds .
below , 
 we describe different strategies that have been used to target ar 
 with steroid conjugates , along with preliminary evaluation of their 
 potential applications in ar pharmacology .
the 
 atp - dependent ubiquitin - proteasome pathway is a quality control mechanism 
 that conducts the programmed metabolic degradation of proteins .
ubiquitin - protein ligase ( e3 ) associates with 
 ubiquitin - conjugating enzyme ( e2 ) , providing subsequent tagging of 
 ubiquitin chains to protein substrates that results in degradation 
 by the proteasome .
rational design strategies aimed toward selectively 
 targeting proteins for degradation through e3 could establish an approach 
 to diminish the levels of aberrantly functioning proteins . the 
 crews lab has pioneered a general strategy to modulate levels of selective 
 proteins by engagement of the ubiquitin system . by use of conjugates dubbed proteolysis targeting chimeric 
 molecules ( protacs ) , the first steroid conjugate to selectively induce 
 ar degradation was developed .
protacs 
 consist of three components : a targeting moiety ( dht ) , a linker , and 
 a recognition element for e3 .
the modular synthesis of protacs establishes 
 a significant pharmacological advantage because protacs are particularly 
 amenable to chemical modification , permitting control over the physicochemical 
 features of the products .
initial ex vivo studies aimed toward 
 degrading ar yielded protac-5 ( figure 4a ) .
protac-5 was outfitted with a peptide sequence 
 ( alapyip ) as an e3 recognition domain and to induce ubiquitination 
 upon hydroxylation of the central proline residue . to assess biological activity ,
protac-5 was administered 
 to human embryonic kidney cells ( hek293 ) that stably expressed an 
 ar fluorescent fusion protein .
protein degradation was quantified 
 by a reduction in the fluorescence signal . at a concentration of 25 
 m , protac-5 successfully degraded ar without compromising normal 
 cell viability . in control studies ,
vehicle treated cells maintain 
 fluorescence , suggesting that protac-5 engages ar in the cell and 
 induces degradation .
to confirm these results , cells were treated 
 with protac-5 and immunoblotted for ar . a significant decrease in 
 ar protein level
proteolysis targeting chimeric molecules ( protacs ) for 
 ar : ( a ) 
 synthesis of protac-5 ; ( b ) chemical structure of protac - aa ; ( c ) chemical 
 structure of small molecule e3 recognition element ( left ) and cocrystal 
 structure of small molecule e3 recognition element ( blue sticks ) and 
 e3 ( orange surface rendered , right ; pdb code 3zrc ) .
figure is adapted 
 from refs ( 53 ) , ( 55 ) , and ( 56 ) .
more recently , a derivative of protac-5 , dubbed protac - aa 
 ( figure 4b ) , was administered to an ar - expressing 
 prostate 
 cancer cell line ( lncap ) to evaluate effects on cell proliferation .
protac - aa contains a shorter hydroxylated recognition 
 element for e3 and a slightly modified arginine tail to enhance cell 
 permeability .
the arginine tail enhances cell permeability through 
 an uptake mechanism mimicking the antennapedia and hiv tat proteins .
protac - aa inhibited cell growth with an inhibitory concentration 
 ( ic50 ) value of 3.8 m at 72 h and 0.217 m 
 at 144 h. a control protac lacking the arginine tail displayed ic50 values 12.5 m at 72 h and 1.5 m at 144 h. 
 western blot analysis was performed to establish that ar protein levels 
 were reduced . taken together , these results suggest that the arginine 
 tail enhances biological activity while maintaining specificity .
importantly , 
 in prostate cancer cell lines that do not express ar ( pc-3 and du-145 
 cells ) , protac - aa had no significant effect on cell viability , establishing 
 selective activity . while protacs remain promising candidates 
 for applications in ar 
 pharmacology , difficulties in large - scale production may impede rapid 
 translation into the clinic .
current efforts have focused on developing 
 more  druglike  protac molecules and the recent discovery 
 of the first small molecule targeting e3 ( figure 4c ) with an ic50 value of 4.1 m .
competitive fluorescence polarization data indicated 
 that the small molecule binds to e3 , which was confirmed by a cocrystal 
 structure .
subsequent optimization led to the first submicromolar 
 small molecule targeting e3 ( ic50 = 0.90 m ) . in the future , we may begin to see small molecule 
 protacs targeting ar , which may include , for example , enzalutamide 
 tethered to similar small molecules that are capable of recruiting 
 e3 .
apoptosis , or programmed cell death , is a physiological 
 cell suicide mechanism critical to cellular homeostasis .
inadequate activation of the apoptotic pathway 
 can play a role in the development of cancer and autoimmune diseases .
inhibitors of apoptosis proteins ( iaps ) play 
 a fundamental role in regulating apoptosis and other cellular processes . 

iaps contain a ring domain that possesses e3 activity , establishing 
 the ability to induce proteasomal degradation by tagging proteins 
 with ubiquitin chains .
the hashimoto 
 lab has developed specific and nongenetic iaps - dependent protein erasers 
 ( snipers ) .
relative to protacs , snipers 
 consist of a targeting moiety ( dht ) , linker , and a recognition element 
 for iaps .
the targeted ubiquitination of proteins by snipers relies 
 on small molecule iap recognition elements ( figure 5 ) .
the biological activity of an ar targeting compound , sniper-13 , 
 was evaluated by western blot . in human mammary tumor ( mcf-7 )
cells 
 that express ar , sniper-13 decreased ar protein levels at a concentration 
 of 30 m . the high concentration required to induce degradation 
 may be attributed to the hydrolytically unstable ester and oxime linkages .
chemical structure of specific and nongenetic 
 iaps dependent protein 
 eraser 13 ( sniper-13 ) .
the modular assembly of snipers allows for the incorporation 
 of 
 virtually any  targeting moiety  .
this characteristic , 
 and the ability to recruit e3 with a small molecule , establishes a 
 versatile molecular platform to address many protein targets . in the 
 future
, research efforts may focus on generating stable linkages between 
 the targeting moiety and the iap recognition element or altering linker 
 lengths to optimize activity of sniper conjugates against various 
 protein targets .
metallo - based 
 cytotoxic agents , 
 such as cisplatin , remain a viable option for the treatment of cancer . from a mechanistic standpoint ,
these compounds 
 exert their biological activities by binding to nucleobases in dna 
 and inducing damage to dna that ultimately triggers apoptosis .
although widely used in the clinic , these agents 
 are generally nonspecific and exhibit shortcomings that include severe 
 side effects resulting from compromised normal cell viability and 
 drug resistance .
this has led to the exploration of metallo - based 
 chemotherapeutic agents that target specific organs or tumors to minimize 
 adverse side effects .
conjugation of a  targeting moiety  
 to metallo - based cytotoxic agents could potentially circumvent nonspecific 
 interactions by selectivity targeting cells that overexpress particular 
 proteins , establishing a delivery vector to localize the effects of 
 new therapeutic agents .
recent studies 
 from the hannon group have discovered the first metallo - based chemotherapeutic 
 conjugates to target ar .
the authors 
 developed an efficient protocol to readily synthesize an array of 
 steroid conjugates to act as delivery vehicles .
ethisterone , the 17-ethynyl 
 homologue of testosterone , was conjugated to pyridines , quinolines , 
 and isoquinolines utilizing sonogashira cross - coupling conditions . 

subsequent coordination to platinum(ii ) complexes yielded metallo - based 
 bifunctional agents ( figure 6 ) .
initial evaluation of the cytotoxic effects for the two most 
 promising 
 metallo - based bifunctional agents in cell lines that express ar ( t47d 
 cells ) revealed promising biological activity for a cis conjugate 
 ( ic50 = 15.9 m ) but not the trans conjugate ( ic50 = 63 m ) , suggesting that the geometry of the platinum(ii ) 
 complexes plays a critical role . additionally , the cis conjugate exhibited 
 greater potency than cisplatin itself ( ic50 = 32 m ) . 

cell uptake studies reveal that the  targeting moiety  
 enhances drug delivery , suggesting that the hydrophobic character 
 of ethisterone facilitates molecular transport across the cellular 
 membrane .
importantly , the presence of the  targeting moiety  
 in the cis conjugate ( relative to control compounds lacking steroid 
 moieties ) led to significant structural effects on dna .
the distortion of dna upon binding the cis conjugate 
 was greater than that observed for cisplatin , suggesting that the 
 steric bulk of ethisterone promotes greater unwinding of dna to accommodate 
 binding of the complex .
metallo - based conjugates have 
 significant utility as a platform 
 for targeted drug delivery .
the work outlined above suggests that 
 metallo - based conjugates can be crafted to exert toxic effects preferentially 
 on cell types that overexpress ar .
, demonstration that 
 coadministration of a competitive ligand , such as dht , abrogates the 
 activity of the metallo - conjugate would further support the hypothesis 
 that targeting is mediated specifically through binding to ar .
ultimately , 
 similar strategies could potentially be elaborated for targeting additional 
 metallo - conjugates to a range of malignant cell types , while mitigating 
 cytotoxic effects on other tissues .
alkylating agents act through 
 dna damaging mechanisms and are commonly used in cancer therapy .
crafting alkylating therapies to specifically target malignant 
 cells could minimize cytotoxic effects to normal cells and lead to 
 the development of potent anticancer agents . in an effort to 
 block dna repair enzymes ,
the essigmann group has developed heterobifunctional 
 dna - damaging agents to specifically target prostate cancer cells ( figure 7 ) .
the alkylating agent n , n - bis-2-chloroethylaniline was linked 
 to a steroid hormone that targets ar , allowing the conjugate to simultaneously 
 bind ar and dna .
this strategy results in the blockade of dna repair 
 enzymes in prostate cancer cells that overexpress ar , subsequently 
 leading to the disruption of ar - mediated transcription and signaling . 
 using radiometric competitive binding assays , the relative binding 
 affinity of n
, n - bis-2-chloroethylaniline 
 was determined to be 20% for ar and 4.2% for pr .
this result 
 establishes that the conjugate is more selective for ar than pr . in 
 addition , only n , n - bis-2-chloroethylaniline , 
 and not the negative control ( n , n - bis-2-methoxyethylaniline ) , covalently modified dna .
administration 
 of the alkylating agent at a concentration of 10 m induced 
 apoptosis , as determined by flow cytometry and cleavage of poly adp - ribose 
 polymerase ( parp ) in western blot analysis .
as expected , the negative 
 control did not induce apoptosis at an equivalent concentration .
more 
 importantly , xenograft studies in immunocompromised mice revealed 
 90% inhibition of tumor growth through intraperitoneal injection ( daily 
 dose of 30 mg / kg ) .
these results demonstrate the effectiveness of 
 using targeted alkylating agents to selectively suppress prostate 
 cancer tumor growth .
an emerging avenue in molecular 
 pharmacology is the development of multivalent therapeutic agents . 

multivalency can be used to establish enhanced binding affinity , termed 
 avidity , and specificity for corresponding biomolecular targets through 
 multisite binding contacts . displaying 
 ligands or 
targeting moieties  on modular oligomer 
 frameworks allows chemists to precisely craft architectures capable 
 of inhibiting highly specific protein 
in addition , the ability to create monodisperse 
 molecular scaffolds enables control over important physicochemical 
 features of the products , including solubility and cellular uptake .
peptoids are a class of peptidomimetics 
 composed of n - substituted glycine units joined through tertiary amide 
 linkages .
peptoids have recently been exploited as multivalent platforms 
 to design conjugates capable of targeting different nucleic acids 
 and protein receptors .
peptoids are stable against proteases and display 
 enhanced cell permeability profiles . the incorporation 
 of over 200 different peptoid side chains has enabled numerous applications 
 in chemistry and biology , including enantioselective catalysis , molecular 
 recognition , antimicrobial activity , intracellular delivery , and antitumor 
 activity in vivo .
peptoids are compatible with solid - phase synthesis techniques and 
 can be assembled in a sequence - specific manner to afford monodisperse 
 products .
additionally , the conformation 
 of peptoid oligomers can be controlled though macrocyclic constraints 
 and side chain interactions . utilizing peptoids 
 as a versatile chemical platform , the kirshenbaum 
 lab designed multivalent ethisterone conjugates to specifically target 
 the ar ldb and modulate ar activity via different mechanisms of action .
ethisterone was conjugated at the 17- 
 position to the peptoid scaffold via highly stable triazole linakges . 

initial efforts evaluated effects of valency , spacing , and conformational 
 ordering on ar activity ( figure 8) .
fluorescence 
 polarization assays were conducted to determine if the conjugates 
 compete against dht for binding to the ar ligand binding domain .
results 
 from these studies revealed that hexavalent ( 4 ) and spaced 
 divalent conjugates ( 5 and 6 ) compete for 
 binding .
mono- , di- , and trivalent conjugates ( 13 ) and a cyclic divalent conjugate ( 7 ) did not 
 compete for ar binding . a control peptoid conjugate outfitted with 
 pr ligands did not activate ar in a luciferase reporter assay , suggesting 
 the ethisterone conjugates are selective for ar . in cell proliferation 

studies that model castration - resistant prostate cancer ( lncap - abl 
 cells ) , conjugates 6 and 7 exhibited potent 
 antiproliferative properties .
as expected , the anti - androgen bicalutamide 
 ( vide supra ) failed to suppress proliferation in this resistant cell 
 line .
importantly , cytotoxic effects of conjugates 6 and 7 were not observed in cell lines that do not express ar ( pc-3 
 and hek293 cells ) , establishing that conjugates selectively target 
 ar .
figure 
 is adapted from ref ( 90 ) . in a follow - up investigation
, 
 the authors used confocal microscopy , 
 time - resolved fluorescence resonance energy transfer , chromatin immunoprecipitation , 
 flow cytometry , and microarray analysis to gain insight into the mechanism 
 of action for conjugates 6 and 7 .
upon administration of conjugates 6 and 7 to hek293 cells transfected with an ar fluorescent 
 fusion protein , conjugate 6 did not promote ar nuclear 
 localization while conjugate 7 did , suggesting competitive 
 and noncompetitive mechanisms of action , respectively .
ar coactivator 
 recruitment assays revealed that conjugate 6 did not 
 promote binding between ar and coactivator proteins while conjugate 7 partially recruited coactivator proteins . in dna binding 
 experiments , both conjugates 6 and 7 reduced 
 the occupancy of ar to the psa enhancer ( vida supra ) .
conjugate 7 , but not conjugate 6 , induced arrest in the 
 g0/g1 phase of the cell - cycle and displayed 
 contrasting patterns in global gene expression .
intriguingly , conjugate 6 and 7 share extensive chemical similarities , 
 indicating that the disposition of the ligand presentation on the 
 scaffolds can exert a significant influence on the mechanism of action . 

conjugate 6 did not promote ar nuclear localization or 
 coactivator binding and inhibited dna binding .
in contrast , conjugate 7 promoted ar nuclear localization and induced cell - cycle 
 arrest through a noncompetitive mode of action .
the modularity 
 of peptoid synthesis establishes a versatile chemical 
 platform to generate an array of three - dimensional architectures to 
 target and modulate the activity of different biomolecular targets . 

generation of peptidomimetic conjugates capable of antagonizing ar 
 via distinct mechanisms of action could circumvent drug resistance 
 in ar pharmacology .
peptoids offer a chemical platform that can be 
 utilized to optimize biological activity and hold significant promise 
 as next generation therapeutics for prostate cancer .
the estrogen receptor ( er ) has 
 a well - characterized mechanism of 
 action .
it is known that native ligand ( estradiol ) binding to the 
 er ligand binding domain induces a conformational rearrangement that 
 promotes dimerization , as determined by site - specific mutational analysis .
additionally , x - ray crystal structures of er 
 dimers in the presence of ligand and other er modulators have been 
 reported , establishing a template for molecular design by elucidating 
 the structural parameters of the er dimer complex .
pioneering work from the katzenellenbogen lab has probed er 
 function 
 with various bivalent conjugates tethered by different linkers . using high - resolution structural information , 
 the first steroidal constructs aimed toward targeting the er dimer 

initial studies 
 focused on developing a correlation between linker length and binding 
 affinity .
the authors concluded that bivalent conjugates incorporating 
 a 35  linker were most suitable for enhancing er binding 
 affinity .
steroidal bivalent conjugates modulate estrogen receptor ( er ) activity 
 through bivalent binding interactions .
crystal structure of the er 
 ligand binding domain ( gray ribbon , pdb code 1ere ) is bound to native 
 ligand ( estradiol , red spheres ) .
nonsteroidal bivalent conjugates 
 that induce 
 agonistic and antagonistic er conformations ( figure 10 ) were designed and synthesized in order to distinguish intra- 
 from intermolecular binding events .
bivalent 
 agonist conjugates displayed weak binding affinity , presumably due 
 to burial of the hydrophilic linker within the protein interior .
in 
 contrast , it was determined that antagonist conjugates incorporating 
 a 14.4  linker induced an intramolecular binding event ( i.e. , 
 one targeting moiety optimized for competitive binding and the other 
 for binding to additional hydrophobic pockets such as activation function 
 2 , figure 11 ) .
additionally , a 29  linker 
 was found to induce an intermolecular binding event .
increases in 
 linker length above 29  resulted in reducing binding affinities , 
 presumably due to unfavorable entropic effects .
importantly , most 
 antagonistic nonsteroidal bivalent conjugates were more potent at 
 inhibiting cell proliferation in breast cancer cells ( mcf7 ) than a 
 monovalent pharmacophore control .
er conformation is dependent upon ligand 
 binding : ( a ) er bound 
 in an agonist conformation ( gray ribbon ; diethylstilbestrol , colored 
 spheres ; helix-12 in orange ; coactivator peptide in red ; pdb code 3erd ) ; ( b ) er bound in 
 an antagonist conformation ( gray ribbon ; hydroxytamoxifen , colored 
 spheres ; helix-12 in orange ; pdb code 3ert ) .
diagram depicting intra- and intermolecular er binding events that 
 are dependent on linker length .
a critical objective from a molecular design approach is 
 the ability 
 to induce different er conformations that are dependent upon ligand 
 binding .
as discussed above , the conformation induced upon agonist 
 or antagonist ligand binding to er ( figure 10 ) plays a critical role in the biological outcome . in an antagonist 

conformation , an intra- or intermolecular binding event can occur 
 between two distinct  targeting moieties  .
an open question 
 is whether structure - based design can be utilized to generate heterobifunctional 
 conjugates that target two distinct binding sites on ar ( i.e. , one 
 targeting moiety optimized for competitive binding at the ligand binding 
 domain and the other for binding to an additional hydrophobic pocket , 
 such as af-2 or bf3 ) .
over the past decade , targeting canonical or membrane - associated 
 ar with heterobifunctional or multivalent constructs displaying anti - androgen 
 drug ligands has emerged as a potential family of therapeutics .
these 
 compound classes hold great promise as effective therapeutic agents 
 due to their ability to modulate ar activity through unique mechanisms 
 of action . because of the large number of reports ,
we highlight only 
 a few representative examples of promising strategies that have been 
 used to target ar with nonsteroidal conjugates .
recently , the 
 oyelere lab reported a nonsteroidal heterobifunctional 
 conjugate outfitted with histone deacetylase inhibitors ( figure 12a ) .
histone deacetylase 
 inhibitors show great promise in preclinical cancer models , but their 
 inability to selectively target malignant tissue has restricted therapeutic 
 development . by conjugating histone deacetylase inhibitors to nonsteroidal 
 anti - androgen ligands , selective modulation of ar activity at concentrations 
 lower than clinical anti - androgens was achieved .
these results introduce 
 a novel method to antagonize the ar and pave the way for next generation 
 therapeutics .
nonsteroidal conjugates targeting ar : ( a ) chemical structure 
 of 
 heterobifunctional conjugate displaying histone deacetylase inhibitor 
 linked to a nonsteroidal antiandrogen ligand ; ( b ) chemical structure 
 of heterobifunctional conjugate displaying doxorubicin linked to a 
 nonsteroidal antiandrogen ligand ; ( c ) schematic depiction of a multivalent 
 gold nanoparticle displaying nonsteroidal antiandrogen ligands that 
 target membrane - associated ar
. figure is obtained from refs ( 98 ) , ( 99 ) , and ( 101 ) . in similar studies , the koch lab reported a nonsteroidal 
 heterobifunctional 
 conjugate containing doxorubicin , a nonselective cytotoxic therapeutic 
 dna intercalator used in the clinic ( figure 12b ) . to enhance selectivity ,
doxorubicin was 
 conjugated to a nonsteroidal anti - androgen ligand through a salicylamide 
 linker that can be hydrolyzed ( t1/2 = 
 57 min under physiological conditions ) to yield a doxorubicin  formaldehyde 
 schiff base .
the anti - androgen conjugate successfully delivered the 
 doxorubicin - formaldehyde schiff base to cells overexpressing ar , highlighting 
 the ability of this approach to enhance selectivity by releasing the 
 pharmacophore in prostate cancer cells .
lastly , the el - sayed 
 lab introduced the first nonsteroidal multivalent 
 conjugates that selectively target membrane - associated ar ( figure 12c ) .
bicalutamide 
 was conjugated to gold nanoparticles , generating architectures that 
 display approximately ( 2.25  0.02 )  10 ligands / particle . 

the multivalent compounds enhanced potency by 1 order of magnitude , 
 in comparison to the monovalent ligand , in prostate cancer cells . 

these results establish that conjugation of numerous copies of a known 
 pharmacophore to a molecular scaffold can significantly increase antiproliferative 
 effects and may overcome resistance that arises from monovalent treatment .
there is a growing appreciation for the design of potent and selective 
 therapeutic agents targeting the ar for prostate cancer patients . 

targeted drug therapy is beginning to play a pivitol role in new drug 
 discovery efforts .
classically , small molecules identified via chemical 
 screening efforts have been considered to offer a relatively straightforward 
 path for clinical implementation . in certain cases ,
extensive high - resolution 
 structural information enables structure  activity relationship 
 profiles that can be utilized for optimization , facilitating translation 
 into the clinic .
unfortunately , their therapeutic responses can be 
 short - lived because of acquired resistance .
the studies highlighted 
 in this review indicate how new chemical 
 entities are being designed to engage ar with high potency .
additional preclinical 
 studies will be required to validate their potential for clinical 
 translation . in many cases
, it will be necessary to evaluate critical 
 parameters such as selectivity , in vivo potency , and binding affinity . 

as discussed , chemical modifications at certain positions on the steroid 
 core can result in diminished binding affinities , potentially limiting 
 their utility in ar pharmacology .
these molecular architectures 
 have been demonstrated to elicit 
 potent biological responses and more importantly target the ar in 
 novel ways . in the future , we may begin to see examples of monodisperse 
 homo- and heterogeneous bivalent or multivalent displays in which 
 high - resolution structural data enable evaluation of structure  activity 
 relationships that have propelled many small molecule drug discovery 
 efforts .
more importantly , heterobifunctional displays will likely 
 be designed to target two distinct binding sites on ar , enhancing 
 potency and establishing new modes of ar antagonism .
these constructs 
 could potentially address the challenge of overcoming resistance in 
 prostate cancer patients .
 the 
 androgen receptor ( ar ) is a major therapeutic target in prostate 
 cancer pharmacology . 
 progression of prostate cancer has been linked 
 to elevated expression of ar in malignant tissue , suggesting that 
 ar plays a central role in prostate cancer cell biology . 
 potent therapeutic 
 agents can be precisely crafted to specifically target ar , potentially 
 averting systemic toxicities associated with nonspecific chemotherapies . 
 in this review , 
 we describe various strategies to generate steroid 
 conjugates that can selectively engage ar with high potency . 
 analogies 
 to recent developments in nonsteroidal conjugates targeting ar are 
 also evaluated . 
 particular focus is placed on potential applications 
 in ar pharmacology . 
 the review culminates with a description of future 
 prospects for targeting ar . 
Introduction
Prostate
Cancer: A Global Concern
Androgen Receptor: Structure
and Function
Androgen-Dependent and -Independent Prostate Cancer
Targeting AR with Steroid Conjugates
Toward Novel
AR Antagonists by Molecular Design: Taking Inspiration
from the Estrogen Receptor
Targeting AR with Nonsteroidal Conjugates
Conclusion