characterization of a new class of androgen …...small molecule therapeutics characterization of a...

Small Molecule Therapeutics

Characterization of a New Class of Androgen ReceptorAntagonists with Potential Therapeutic Application inAdvanced Prostate Cancer

Huifang Li1, Mohamed D.H. Hassona1,5, Nathan A. Lack1,2, Peter Axerio-Cilies1, Eric Leblanc1,Peyman Tavassoli1, Natalia Kanaan1, Kate Frewin1, Kriti Singh1, Hans Adomat1, Konrad J. B€ohm3,Helge Prinz4, Emma Tomlinson Guns1, Paul S. Rennie1, and Artem Cherkasov1

AbstractThe human androgen receptor plays a major role in the development and progression of prostate cancer

and represents a well-established drug target. All clinically approved androgen receptor antagonists

possess similar chemical structures and exhibit the same mode of action on the androgen receptor.

Although initially effective, resistance to these androgen receptor antagonists usually develops and the

cancer quickly progresses to castration-resistant andmetastatic states. Yet even in these late-stage patients,

the androgen receptor is critical for the progression of the disease. Thus, there is a continuing need for

novel chemical classes of androgen receptor antagonists that could help overcome the problem of

resistance. In this study, we implemented and used the synergetic combination of virtual and experi-

mental screening to discover a number of new 10-benzylidene-10H-anthracen-9-ones that not only

effectively inhibit androgen receptor transcriptional activity, but also induce almost complete degradation

of the androgen receptor. Of these 10-benzylidene-10H-anthracen-9-one analogues, a lead compound

(VPC-3033) was identified that showed strong androgen displacement potency, effectively inhibited

androgen receptor transcriptional activity, and possesses a profound ability to cause degradation of

androgen receptor. Notably, VPC-3033 exhibited significant activity against prostate cancer cells that

have already developed resistance to the second-generation antiandrogen enzalutamide (formerly known

as MDV3100). VPC-3033 also showed strong antiandrogen receptor activity in the LNCaP in vivo xenograft

model. These results provide a foundation for the development of a new class of androgen receptor

antagonists that can help address the problem of antiandrogen resistance in prostate cancer. Mol Cancer

Ther; 12(11); 2425–35. �2013 AACR.

IntroductionThere is currently no curative treatment for patientswith

castration-resistant prostate cancer (CRPC) and the corre-spondingmedian survival is estimated at�16 to 18months

(1–3). Numerous studies have showed that the androgenreceptorplaysa critical, central role in thedevelopment andprogression of CRPC (4–7) and that inhibition of thisnuclear receptor is an effective option for the treatment oflate-stage disease.

The currently marketed antiandrogens, such as niluta-mide, flutamide, and bicalutamide all act as classicalantagonists binding to the hormone binding site (HBS),resulting in conformational changes of the protein whichprevent coactivation. Although these drugs initially sup-press tumor growth, the cancer often develops treatmentresistance. Central among the factors thatmake the andro-gen receptor less sensitive to antiandrogens aremutationsthat weaken the androgen receptor–drug interactions atthe HBS. Moreover, certain mutations in the HBS such asW741L/C and T877A convert conventional antiandro-gens to agonists which can inadvertently promote can-cer growth and progression (8–10). The newly approveddrug enzalutamide and investigational drug ARN-509(11, 12), aim to address this problem, but current reportssuggest that resistance to these HBS-directed drugsinvariably develops (13). One of the underlying reasons

Authors' Affiliations: 1Vancouver Prostate Centre, University of BritishColumbia, Vancouver, BritishColumbia, Canada; 2School ofMedicine, KocUniversity, Istanbul, Turkey; 3Leibniz Institute for Age Research (FLI), Jena,Germany; 4Institute of Pharmaceutical and Medicinal Chemistry,Westf€alische Wilhelms-Universit€at, M€unster, Germany; and 5Faculty ofPharmacy, Helwan University, Cairo, Egypt

Note: Supplementary data for this article are available at Molecular CancerTherapeutics Online (http://mct.aacrjournals.org/).

H. Li, M.D.H. Hassona, and N.A. Lack contributed equally to this work.

T. Guns's, P.S. Rennie's, and A. Cherkasov's labs made equal contribu-tions to this work.

Corresponding Author: Artem Cherkasov, Vancouver Prostate Centre,University of British Columbia, 2660 Oak Street, Vancouver, British Colum-bia, Canada V6H 3Z6. Phone: 604-875-4111; Fax: 604-875-5654; E-mail:[email protected]

doi: 10.1158/1535-7163.MCT-13-0267

�2013 American Association for Cancer Research.

MolecularCancer

Therapeutics

www.aacrjournals.org 2425

on May 16, 2020. © 2013 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Published OnlineFirst August 12, 2013; DOI: 10.1158/1535-7163.MCT-13-0267

http://mct.aacrjournals.org/

is the fact that all known antiandrogens, including enza-lutamide, share common structural motifs (Supplemen-tary Fig. S1) responsible for the target binding and, there-fore, may be associated with the same risk resistance.Thus, there is a pressing need for non-cross-resistantandrogen receptor antagonists with novel molecular scaf-folds and/or alternative mechanisms of androgen recep-tor inhibition.

Materials and MethodsStructure-based virtual screening fornovel androgenreceptor antagonists

Two docking programs Glide SP (14) and eHiTs (15)were used to dock �3 million purchasable compoundsfrom the ZINC database (16). Two androgen receptorcrystal structures (PDB code:2PNU.pdb and 3L3X.pdb;refs. 17, 18) from theProteinData Bank (19)were preparedfor the docking using Maestro suite (see SupplementaryMaterials and Methods for details; ref. 20).

Molecular dynamicsWe have used the binding pose of the compound VPC-

3022 inside of the androgen receptor HBS site generatedby docking to run classical molecular dynamics simula-tion for 10 nanoseconds to evaluate the dynamic behaviorof the complex (see details in Supplementary Materialsand Methods).

ChemistryThemost common synthetic procedure for the synthesis

of benzylidene-10H-anthracen-9-ones is based on the con-densation reaction of 10H-anthracen-9-one with aromaticaldehydes. Some of these compounds have been previ-ously reported (21) to act as inhibitors of tubulin poly-merization and were obtained as described in that study.Compounds not mentioned therein were synthesized byan aldol-type condensation reaction of 10H-anthracen-9-one with appropriately substituted benzaldehydes underbasic conditions in the presence of pyridine/piperidine(see details in Supplementary Materials and Methods).The starting benzaldehydes as well as 10H-anthracen-9-one were obtained from commercial sources.

In vitro studies by cell-based and biochemical assaysCell culture. LNCaP, LAPC4, and PC3 human pros-

tate cancer cells were obtained from American TypeCulture Collection and grown in RPMI 1640 mediumsupplementedwith 5% FBS (Invitrogen). LNCaP, LAPC4,and PC3 cells were tested and authenticated by IdexxRadil (case number 14616-2011) in June 2011. The LNCaPeGFP cell line was stably transfected with an androgen-responsive probasin-derived promoter fused to an eGFPreporter (LN-ARR2PB-eGFP) using a lentiviral approach(22), and were grown in phenol-red-free RPMI 1640 sup-plementedwith 5% charcoal-stripped serum (CSS).HeLa-AR cells, stably expressing wild-type androgen receptor,were grown in DMEM supplemented with 5% FBS.

MDV3100-resistant LNCaP cells were provided by Dr.Zoubeidi (23), and were cultured in RPMI 1640 supple-mented with 5% FBS and 10 mmol/L MDV3100. All cellswere maintained at 37�C in 5% CO2.

eGFP cellular androgen receptor transcription assay.The androgen receptor transcriptional activity wasassayed as previously described (22).

Prostate-specific antigen assay. The evaluation ofprostate-specific antigen (PSA) levels secreted into themedia was conducted in parallel to the eGFP assay usingthe same plates. After cells were incubated for 3 days,150 mL of the media was taken from each well, and addedto 150 mL of PBS. PSA levels were then evaluated usingCobas e 411 analyzer instrument (Roche Diagnostics)according to the manufacturer’s instructions.

Biolayer interferometry assay. The direct reversibleinteraction between small molecules and the androgenreceptor was measured as previously described (24).

The androgen displacement assay. The androgen dis-placement was assessed with the Polar Screen AndrogenReceptor Competitor Green Assay Kit (Invitrogen) as perthe instructions of the manufacturer.

SRC2-3 peptide displacement assay. The androgenreceptor activation function 2 (AF2) specific peptide dis-placement was assayed as previously described (24, 25).

Transient transfection. HeLa-AR cells were seededin 96-well plate at a density of 20,000 cells per well inDMEM supplemented with 5% CSS. After incubation for24 hours, the cells were transfected with ARR3tk-lucifer-ase plasmid (26). Twenty-four hours after transfection,the cells were treated with 0.1 nmol/L R1881 and thetested compounds at different concentrations for 1 day.Then cells were lysed using passive lysis buffer, andassayed for luciferase activity. The luciferase activity wasnormalized based on the luminescence unit of controlwells treated with the vehicle and 0.1 nmol/L R1881by the formula: %luciferase activity ¼ (sample-vehicle)/(0.1 nmol/L R1881-vehicle) � 100.

Cell viability assay. LNCaP,MDV3100-resistant, andPC3 cells were plated at 3,000 cells per well in RPMI 1640containing 5% CSS in a 96-well plate, treated with0.1 nmol/L R1881 and compounds (0–1.5 mmol/L) for96 hours. After treatment for 4 days, cell density wasmeasured using the 3-(4, 5-dimethylthiazol-2-yl)-5-(3-car-boxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazoliumassay according to the manufacturer’s protocol (CellTi-ter 961 Aqueous One Solution Reagent; Promega). Thepercentage of cell survival was normalized to the celldensity of control wells treated by vehicle and 0.1 nmol/LR1881, and calculated as: %survival¼ (sample � vehicle)/(0.1 nmol/L R1881-vehicle) � 100.

Dose–response Western blot. LNCaP, MDV3100-resistant LNCaP, HeLa-AR, and LAPC4 cells were grownin RPMI 1640/DMEM with 5% CSS (500,000 cells/well)with increasing concentrations of compounds (0–25mmol/L) in the absence or presence of 0.1 nmol/LR1881. The plates were incubated for 24 hours and thenlysed with passive lysis buffer. The protein concentration

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Mol Cancer Ther; 12(11) November 2013 Molecular Cancer Therapeutics2426




was quantified by bicinchoninic acid protein assay kit(Thermo Scientific). Thirty-five micrograms of proteinlysate from each treatment was boiled for 5 minutes insodium dodecyl sulfate (SDS) sample buffer and run ona 12% SDS polyacrylamide gel. The protein was trans-ferred onto a polyvinylidene difluoride membrane. Themembrane was probed for androgen receptor withmouse monoclonal AR-441 primary antiboday (SantaCruz) followedby incubationwith goat anti-mouse horse-radish peroxidase conjugated secondary antibody. Asa loading control, b-actin was probed using goat poly-clonal antibody, Actin C-11 (Santa Cruz). The proteinswere visualized by a chemiluminescent detection system(GE Healthcare).Quantitative real-time PCR. LNCaP and MDV3100-

resistant LNCaP cells were grown in 12-well plates(100,000 cells/well) in RPMI1640 supplemented with5% CSS. Cells were treated with serially diluted concen-trations of compounds (0–25 mmol/L) in the presence orabsence of 0.1 nmol/L R1881. Total RNA was extractedfrom cells using Trizol total RNA isolation reagent.Reverse transcriptase reactions contained 2 mg of RNAsamples, 5� first strand reaction buffer, 0.1 M DTT, 10mmol/L dNTPmix, RandomHexamers, MMLV-RT, andRNase OUT. The reactions (30 mL) were incubated in a48-well plate for 1 hour at 37�C, 5 minutes at 95�C, andthen held at 4�C. Real-time (RT) PCR amplification ofcDNA was conducted on the ABI PRISM 7900 HTSequence Detection System (Applied Biosystems) bymixing 1 mL RT product with 9.5 mL water, 12.5 mLSYBR-GREEN and 1 mL each of the following primers:50-GCAGGCAAGAGCTGAAGATA-30 and 50-CCTTTG-GTGTAACCTCCCTTGA-30. Androgen receptor geneexpression was normalized to 18S rRNA levels in thesamples as an IS using the following primers: 50-CGAT-GCTCTTAGCTGAGTGT-30 and 50-GGTCCAAGAATTT-CACCTCT-30. Quantification of mRNA was done usingthe comparative cycle threshold (Ct) method. Each assaywas carried out in triplicate.In vitro tubulin polymerization assay. The tubulin

polymerization inhibitory activity was measured as pre-viously published (27).

In vivo studies of maximum tolerated dose, efficacy,and serum-level evaluationAll animal experiments were conducted in accor-

dance with the University of British Columbia Commit-tee on Animal Care. For the maximum tolerable dose(MTD) and serum-level evaluation, 27 6- to 8-week-oldathymic nude mice (Harlan Sprague-Dawley, Inc.) wereintravenously administered 200 to 300 mL of a VPC-3033injection solution formulated using 1:10, hydroxypro-pyl-cyclodextrin:ddH2O to represent doses of 10, 25,50, or 100 mg/kg, via the tail vein. Mice were monitoredfor 24 hours for signs of acute toxicity including death,lethargy, blindness, and disorientation. In addition, tomeasure serum drug levels, mice (n ¼ 3) were sacrificedfollowing injection at time points corresponding to 1, 4,

and 24 hours. Mice were terminated using CO2 followedby cervical dislocation and blood was immediatelycollected by cardiac puncture. Typically, 500 to 700 mLof blood was obtained and then placed into a SerumSeparator tube (Microtainer; Becton Dickinson), mixed,and placed on ice. Serum samples were centrifugedand the supernatant was placed into eppendorf tubeswhich were stored at �20�C pending analysis by liquidchromatography/mass spectrometry analysis (LC/MS).

For efficacy studies, 20 6- to 8-week-old nude mice(Harlan Sprague-Dawley, Inc.) weighing 25 to 31 g weresubcutaneously inoculated with an LNCaP cell line(106 cells in BD Matrigel; BD Biosciences) at both ananterior and posterior dorsal site. After 28 days, thedeveloping tumors were measured and mice randomlyassigned into different treatment groups based on tumorsize. Mice-bearing xenograft tumors with volumesbetween 100 and 200 mm3 qualified for entry into thestudy (28). Then 3 different treatment groups wereassigned: VPC-3033 (10 mg/kg) vehicle (1:10 hydroxy-propyl-cyclodextrin:ddH2O), and enzalutamide. Caliperswere used to measure the 3 perpendicular axes of eachtumor. The formula V¼ L�W �Hð Þp6 where L is the

length, W is the width, and H is the height, was used tocalculate the tumor volume. For 3 weeks, 250 to 350 mLvolumes of a 1 mg/mL injection solution were intrave-nously administered via the tail vein twice per week.Mice were also weighed weekly and monitored daily forsigns of acute toxicity including death, lethargy, blind-ness, and disorientation.

Serum extraction and LC/MSSerum samples generated from the in vivo studies were

thawed and 20 mL transferred to individual eppendorftubes. Internal standard [5.6 mL of 1 mg/mL deuteratedtestosterone (d3T, C/D/N Isotopes)] was then addedfollowed by 50 mL of acetonitrile after which sampleswere vortexed for 5 to 10 seconds and centrifuged as for5 minutes at 20,000 � g to sediment precipitated protein.The clarified supernatant was transferred to LC vials foranalysis. Standards were prepared in a similar fashionusing blank mouse serum and also 50% methanol assample matrix. Optimal grade (Fisher) solvents and 18MW water (Millipore) were used for sample preparationand subsequent LC/MS analysis.

Analysis was carried out with an Acquity UPLC cou-pled in series with an eLambda PDA and a QuattroPremier (Waters). A 100 mm BEH C18, 1.7m column(Waters) was used for separations with a 40% to 70%acetonitrile (ACN) gradient from0.2 to 3minutes, ramped1 minute to 95% ACN for flushing (1 minute) followedby 2 minutes re-equilibration for a 7 minute run length(0.1% formic acid present throughout).Wavelengths from210 to 800 nm at 1.2 nm resolution and 2 points/sec werecollected with the PDA. Extracted chromatograms of375 nm were used for VPC-3033 detection. All MS datawere collected in ESþ at unit resolution with the follow-ing instrument parameters: capillary, 3.0 kV; extractor

Novel Antagonists Inducing Androgen Receptor Degradation

www.aacrjournals.org Mol Cancer Ther; 12(11) November 2013 2427




and RF lens, 5 and 0.1 V; source and desolvation tem-peratures, 120 and 350oC; desolvation and cone (N2) flow,900 and 50 L/h; collision gas (Ar) flow, 0.15 mL/min(7.3e�3mbar). Compoundswere detected usingmultiplereaction monitoring with m/z 299 > 281 for VPC-3033andm/z 292 > 97 for d3T (24 V/25 V and 32 V/21 V cone/collision volt combinations, respectively) with 0.1-seconddwell each. Retention time for d3T and VPC-3033 were2.55 and 3.8 minutes, respectively.

Quanlynx (Waters) was used for analysis of data withexternal calibration for PDA data and internal standardnormalized calibration forMSdata. Calibration standardsranged from 0.01 to 0.9 mmol/L (5 points) with R2 >0.99and all % deviation from nominal <12% except for OD375at the lowest concentration. Comparison of spiked serumwith neat standards indicated little to no matrix interfer-ence, which was present with extraction efficiencies cal-culated to be 110%.

A major metabolite with retention time of 2.1 minuteswas also observed in the OD375 and m/z 299 > 281channels with a UV/Vis spectrum similar to VPC-3033and an observed m/z 475 (scan mode), consistent with aglucuronic acid minus H2O adduct. Quantification ofthis glucuronide was carried out also against the VPC-3033 calibration.

ResultsIn silico screening for chemically diverse andeffective androgen receptor binders

The androgen receptor HBS is primarily composed ofhydrophobic residues that can form strong nonpolarinteractionswith androgenic steroids such as testosteroneand dihydrotestosterone (DHT). The protein–ligandanchoring can be additionally stabilized by a network ofhydrogen bonds involving R752, Q711, N705, and T877polar residues. Details of the interactions between andro-gen receptor and its steroidal and nonsteroidal agonistshave been extensively discussed and elaborated (29–31).However, the mechanism of action of antagonists againstandrogen receptor is much less characterized, and therelevant structural information is only available for spe-cific mutated forms of the androgen receptor that interactwith antiandrogens agonistically (8–10). Combined withthe observation that residues forming the androgen recep-tor HBS are remarkably flexible and can adjust to ligandsof various sizes (17, 31), these factors make virtual screen-ing for the androgen receptor a challenging task.

Three million purchasable compounds from the ZINCdatabase (16) were docked into 2 selected crystal struc-tures of the androgen receptor (2PNU and 3L3X) usingGlide SP program. About 700,000 compounds that suc-cessfully docked with Glide SP score < �7 in both runswere then redocked into the androgen receptorHBSusingeHiTs program (15). A total of 130,000 structures wereselected that were consistently docked with both algo-rithms [i.e., having root mean square deviation (RMSD) <2 A for the docking poses produced by Glide SP and

eHiTs]. These compounds were further evaluated byseveral onsite rescoring approaches including Glide XP(14), eHiTs score (32), London dG scoring and pKi criteriacomputed by MOE program (33), and X-score (34).

With this information, a cumulative scoring of 7 differ-ent predicted parameters was generated where each mol-ecule received a binary 1.0 score for every "top 10%appearance." The final cumulative vote (with the maxi-mum possible value of 7) was then used to rank thetraining set entries. Based on the cumulative score, 50compounds were selected for experimental evaluation.

Identification of 10-benzylidene-10H-anthracen-9-ones as effective androgen receptor antagonists

The selected 50 chemicals were initially assessedwith anondestructive eGFP reporter assay to quantify levels ofandrogen receptor transcriptional activity in LNCaPeGFP cells, which stably express an androgen-responsiveprobasin promoter in front of an eGFP reporter (22).Among the tested chemicals, 6 compounds showed effec-tive transcriptional inhibition (>85%) when administeredat a single 50 mmol/L dose (Table 1). These compoundswere then tested for their ability to displace DHT from theandrogen receptor using a polar screen competitor greenassay kit, and were found to have IC50 values rangingfrom 0.63 to 50 mmol/L (Table 1). These compounds didnot affect SRC2-3 peptide displacement, suggesting theydo not interact with the AF2 coactivation site of theandrogen receptor (data not shown; ref. 24).

The most active compound VPC-3022 was further mea-sured for the transcriptional inhibition in the eGFP assay atvarious concentrations, and it showed characteristic dose-dependent behavior, with an IC50 value of 4 mmol/L (Fig.1A). It is well known that the endogenous androgen recep-tor in LNCaP cells harbors a T877A mutation. To validateVPC-3022 for its inhibitory efficacy onwild-type androgenreceptor, we usedHeLa-AR cells transfectedwith anARR3

tk-luciferase reporter (26). VPC-3022 showed inhibition ofwild-type androgen receptor in a concentration-dependentmanner with an IC50 of 1 mmol/L (Fig. S2), consistent withthe activity from our eGFP transcriptional inhibition assayin LNCaP cells. To rule out possible false positives fromthe androgen receptor transcriptional eGFP assay, we val-idated the results by quantifying the expression of PSA(35). As expected, VPC-3022 induced a dose-dependentdecrease of secreted PSA with a corresponding IC50 valueof 3.6 mmol/L (Fig. 1A). We have further investigatedinteraction of the most active compound VPC-3022 withthe androgen receptor C-terminal ligand-binding domain(LBD) using the biolayer interferometry (BLI). The BLIresults (Fig. 1B) showed direct and reversible interactionbetween the androgen receptor and VPC-3022 with anequilibrium dissociation constant between compound andprotein (KD) value of 4.36 mmol/L. To evaluate the overalleffect ofVPC-3022 onprostate cancer cell viability,wehaveconducted the cell viability (MTS) assays using 3 cell lines:LNCaP, PC3 as well as recently developed MDV3100-resistant LNCaP cells. As shown in Fig. 1C), VPC-3022

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exhibit profound concentration-dependent suppression ofcell survival, especially with those that have resistance tothe current antiandrogen enzalutamide. Although the par-ticular mechanism(s) of MDV3100-resistance in these celllines has not yet been clearly elucidated, it involves reten-tion of the androgen receptor and no additional receptormutations (23). More specifically, at a concentration of 1.5mmol/L, VPC-3022 inhibited the cell proliferation of

MDV3100-resistant cells by almost 100% and LNCaP cellsby 50%, but only a small effect was observed on androgenreceptor-negative PC3 cells.

Interestingly, the VPC-3022 compound was alsofound to induce an almost complete degradation of theandrogen receptor in various prostate cancer cell lines,including HeLa-AR and LAPC4 with wild-type andro-gen receptor, LNCaP with mutant androgen receptor

Table 1. Structures and activity profiles of the androgen receptor HBS binders identified from our in silicoscreening

VPC-ID Structure% Inhibition at50 mmol/L (eGFP)

DHT displacementactivity IC50 (mmol/L)

2055 89.9 10

3002 86.3 5.9

3008 99.3 >10

3015 92.0 >10

3018 88.2 >50

3022 106.6 0.625–2.5






(T877A), and a recently developed MDV3100-resistantLNCaP cells (Fig. 2; ref. 23). At the same time, VPC-3022did not alter androgen receptor mRNA transcript level(Fig. S3) nor did the addition of the transcriptionalinhibitor cyclohexamide affect androgen receptor deg-radation (Fig. S4), strongly suggesting that the loss ofandrogen receptor is due to degradation of the protein.The exact mechanism of androgen receptor degradationby VPC-3022 is currently being investigated. Giventhese results, VPC-3022 was selected as a parental com-pound for further structural modifications to improvethe antiandrogen receptor effect and increase the ther-apeutic potential.

Binding mode prediction of VPC-3022 by moleculardynamics study

To develop an effective strategy for structural optimi-zation of VPC-3022, its predicted interaction with theandrogen receptor HBS was investigated in greaterdetails. The docking pose of VPC-3022 reflects mainly ahydrophobic character of its anchoring to theHBS and theoverall orientation of the compound in the site resemblespositioning of the native ligand—DHT (Fig. S5). Becauseof the infeasibility of experimental evaluation of antago-nistic configurations of the androgen receptor, we haveconducted a molecular dynamics simulation of VPC-3022/HBS complex. The established 10 nanoseconds

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Figure 1. A, dose–response curves of VPC-3022 and VPC-3033 for the androgen receptor inhibition measured by the eGFP cellular androgen receptortranscription assay; PSA inhibition by the studied compounds VPC-3022 and VPC-3033; androgen competition binding curves of VPC-3022 andVPC-3033. B, BLI dose–response curves (0.09–3 mmol/L) showing direct androgen receptor binding of compounds VPC-3022 and VPC-3033 in serialdilution. C, effects of VPC-3022 and VPC-3033 on the cell growth of 3 prostate cancer cell lines. Androgen receptor-positive LNCaP and MDV3100-resistant cells, and androgen receptor-negative PC3 cells were treated with VPC-3022 and VPC-3033 in a concentration-dependent manner in thepresence of 0.1 nmol/L R1881. Error bars, SD.

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molecular dynamics trajectories (Fig. S5) reflected overallstability of the complex with a resulting RMSD valuearound 2.5A. The molecular dynamics optimized posi-tioning of VPC-3022 inside the HBS revealed possibleformation of a hydrogen bond with residue R752, whichhad not been captured by the original docking experi-ment. Based on this predicted binding mode inside thetarget site, a series of analogues (Table 2) were developedby retaining the anthracenone moiety and modifying thesubstituents on the benzylidene group.

Development of the 10-benzylidene-10H-anthracen-9-ones as antiandrogen prototypeA series of 10-benzylidene-10H-anthracen-9-ones were

synthesized by an aldol-type condensation reaction of10H-anthracen-9-one with appropriately substitutedbenzaldehydes under basic conditions in the presenceof pyridine/piperidine (Table 2; ref. 36). The createdcompounds were subsequently evaluated for their abil-ity (i) to displace androgen from the receptor; (ii) toinhibit the androgen receptor in the eGFP and PSAassays; (iii) for their direct interaction with the androgenreceptor LBD, as detected by the BLI; (iv) to inhibit thecell proliferation; and (v) to degrade androgen receptorin prostate cancer cells. In these experiments, except aweak agonist (VPC-3037), the rest of tested 10-benzyli-dene-10H-anthracen-9-ones showed potent androgendisplacement from the androgen receptor and effective,dose-dependent inhibition of its transcriptional activity,

with corresponding IC50 values estimated in 0.2 to50 mmol/L range (Table 2). Importantly, 2 particularanalogues—VPC-3033 and VPC-3045 showed 10-foldenhanced antiandrogen potency compared with the par-ental compound VPC-3022. The direct binding to andro-gen receptor LBD and cell viability were also examinedfor the analogues, as exemplified by VPC-3033 in Fig. 1,which show similar binding pattern to VPC-3022 andstrong inhibition of cancer cell proliferation.

The androgen receptor-degrading ability of these che-micals seemed to be also enhanced—the Western blotimages presented in Fig. 2 illustrate almost completeelimination of the receptor from LNCaP, MDV3100-resis-tant LNCaP, HeLa-AR, and LAPC4 cells at concentrationsof 6 to 25 mmol/L VPC-3033. Of note, 2 other synthesizedanalogues, VPC-3031 and VPC-3041, also showed signif-icant androgen receptor degradation at concentrations of6 to 25 mmol/L (Fig. S4). To confirm the androgen receptordegradation by these compounds, the mRNA expressionwas measured in LNCaP and MDV3100-resistant cellstreated at the same concentrations of the compounds as inthe Western blot assay. As shown in Fig. S3, the mRNAlevels were not affected by the treatment of these com-pounds at different concentrations. The combined evi-dence suggests that this class of compounds inducesdegradation of androgen receptor in prostate cancer cells.

As this chemical class has been reported to act as anti-microtubule agents with tubulin polymerization inhibi-tory activity (21), the tubulin polymerization activity was

Figure 2. A, VPC-3022 and VPC-3033degradeandrogen receptor inprostate cancer cells. LNCaP,MDV3100-resistant (MDV3100-res.), HeLa-AR, and LAPC4 cellswere treated with VPC-3022 andVPC-3033 for 24 hours at theindicated concentrations in thepresence of 0.1 nmol/L R1881.Whole-cell extracts were preparedfrom each treatment and subjectedto Western blot analysis with anti-androgen receptor or b-actinantibodies. B, band density ofandrogen receptor (AR) relative tob-actin measured by ImageJprogram.






examined. Importantly, as reported previously, the tu-bulin polymerization inhibitory activity for the highlyactive androgen receptor inhibitors was not very pro-nounced, even for the most potent compound VPC-3033 (IC50 ¼ 9.9 � 0.39 mmol/L; Fig. S6), further sup-porting the conclusion that the observed antiandrogenreceptor activity is not simply an artifact as a consequenceof the inhibition of tubulin polymerization.

In vivo evaluation of VPC-3033 revealed its abilityto suppress androgen receptor function

The lead compound VPC-3033 was investigated forits effect on the tumor growth using the LNCaP xeno-

graft model (28, 37). Results from preliminary acutetoxicity studies indicated that doses up to and including50 mg/kg could be tolerated by the studied mice withno decrease in body weight. The measured serum levelssuggested that the compound could be administeredeffectively via intravenous tail vein injection andthat the compound could be detected for up to 24 hours(Fig. 3). At higher doses, we estimate the serum Cmax tobe between 10 and 100 mmol/L. Although clearancewas fairly quick, based on in vitro data (Table 2, eGFPIC50 ¼ 0.3 mmol/L), the plasma concentration shouldstill be well within the predicted therapeutic windowfor a substantial duration, up to about 4 hours, given

Table 2. Structures and measured activities of benzylidene-10H-anthracen-9-one derivatives

VPC-ID R1 R2 R3 R4 R5eGFP IC50

(mmol/L)DHT displacementactivity IC50 (mmol/L)

3022 –OH 3.3 0.625–2.53023 –OCH3 –OCH3 –OCH3 1.6 103024 –COOCH3 –OH 7.9 403025 2.2 0.625–2.53026 2-Methylthiophene 6.9 0.625–2.53027 –C(CH3)3 –OH –C(CH3)3 16.2 403028 –OCH3 –OCH3 8.3 >403029 –COOCH3 9 >403030 5-Methyl-1,3-benzodioxole 4.3 2.5–103031 –NO2 3.1 >403032 –OH –OH –OH 48.8 103033 –OH 0.3 0.625–2.53034 –OCH3 –OCH3 6.9 103035 –OH –OCH3 4.6 0.625–2.53036 –CH3 –OH –CH3 1.1 2.5–103037 –OCH3 –OCH3 Weak agonist 2.5–103038 –OCH3 –OH –OCH3 1.4 10–403039 –OH –OCH3 1.2 10–403040 –OCH3 2.6 403041 –OCH3 –OCH3 2.8 10–403042 –OH –OH 2.3 >403043 –OH –OCH3 1.7 403044 –OCH3 –OH –OCH3 3.8 10–403045 –OH –OCH3 0.2 103046 –OCH2-phenyl 10.9 >403047 –CF3 1.1 103048 –Cl –Cl 6.6 403049 –OCH3 2.9 2.5

Li et al.





the PK profile and assuming reasonable absorption. Adose of 10 mg/kg was chosen based on these prelim-inary studies and previous literature reports of antian-drogens (12). At this dose, the growth of tumor waseffectively suppressed compared with the vehicle con-trol over a 3-week intravenous dosing regimen (P < 0.01from day 14 onward; Fig. 3). The results clearly indicatethat VPC-3033 could effectively inhibit androgen sen-sitive LNCaP xenograft growth in vivo, suggesting thisclass of compounds is effective as androgen receptorantagonists.

DiscussionAs the resistance invariably develops to current anti-

androgens in prostate cancer, even to the newly approvedsecond-generation enzalutamide, novel androgen recep-tor antagonists with new scaffolds and mechanism of

action are greatly needed. In this work, a large-scalevirtual screening was conducted to find small moleculeantagonists for the human androgen receptor that caneffectively interact with androgen receptor HBS and yetpossess chemical scaffolds that are significantly differentfrom currently used clinical antiandrogens, such as bica-lutamide and enzalutamide. Following experimentalevaluation of virtual hits, a number of active androgenreceptor antagonists were identified in Table 1. Surpris-ingly, the compound 4-hydroxy-10-benzylidene-10H-anthracen-9-one (VPC-3022) was found to not only effec-tively interactwith the androgen receptorHBS (Fig. 1), butalso cause degradation of androgen receptor in prostatecancer cells (Fig. 2).

Following in silico modeling work around the struc-ture of the hit compound (VPC-3022), a 3-hydroxyl sub-stituted analogue (VPC-3033) was synthesized (Table 2),characterized, and shown to be a significantly morepotent androgen receptor antagonist (Fig. 1). This com-pound showed a stronger androgen receptor degrada-tion profile (Fig. 2), and exhibited a more profoundability to inhibit the growth of prostate cancer cells. Theremarkable inhibitory effect on cells that have devel-oped resistance to the second-generation antiandrogen,enzalutamide (Fig. 1; ref. 11), indicated its potential as apossible second line therapy following the developmentof resistance to current antiandrogens. When this com-pound was evaluated in a tumor xenograft model, asignificant effect on the suppression of tumor growthwas seen compared with the vehicle and enzalutamidecontrols (Fig. 3). Although promising, work is currentlyongoing to improve the PK/PD characteristics by mod-ifying known sites of potential bioconversion.

Importantly, the chemical scaffold of 10-benzylidene-10H-anthracen-9-one is very different from the struc-tures of currently used antiandrogens, which all havea common scaffold (Fig. S1). Chemicals belonging tothe 10-benzylidene-10H-anthracen-9-one family havebeen previously implicated in some biological processesincluding inhibition of tubulin polymerization (21,38, 39). However, this does not seem to be themechanismof action observed in this work, as there was no cor-relation between antitubulin activity and either andro-gen displacement or androgen receptor transcriptionalinhibition.

Previous reports indicated that mutation of W741to leucine or cysteine will generate additional spacein the HBS that allows accommodation of the bulkyphenyl ring of bicalutamide and converts its antagonistactivity on the androgen receptor into an agonist thatstimulates transcriptional activity and cancer growth(40). It has been simulated by docking that bindingof VPC-3033 to the androgen receptor HBS occurs at anotable distance from the W741 residue (Fig. 4) andtherefore a mutation here is not likely to have an effecton the compound’s androgen receptor binding and activ-ity. Similarly, the well-documented agonist-convertingT877A mutation, as found in the androgen receptor

A10

1

0.1

0.01

0.001

300

200

100

0

B

0

0 5 10Days after starting drug injection

ControlVPC-3033 (10)

VPC-3033 (10)

VPC-3033 and glucuronide PK

(mg/kg i.v.)

Tumor shrinkage of VPC-3033

(mg/kg i.v.)

VPC-3033 (25)VPC-3033 (50)gluc (10)gluc (25)gluc (50)

Enzalutamide (10)

% C

ha

ng

e o

f c

on

tro

lS

eru

m c

on

ce

ntr

ati

on

(m

mo

l/L

)

15 20

***

****

25

4 8 12Time (h)

16 20 24

Figure 3. A, a limited PK analysis of VPC-3033 following single 10, 20,and 50 mg/kg i.v. injections displays circulating levels achieved andrelatively rapid clearance for both the compound and a majorglucuronide metabolite that was observed. Cmax would be expected tobe 1 to 2 orders of magnitude higher than the initial 1-hour pointcollected. The 24-hour point of 10 mg/kg was detectable, but beyondquantifiable limitation. B, the in vivo effect of VPC-3033 (10 mg/kg) ontumor volume. The effects of VPC-3033 were determined usingLNCaP mice xenografts (n ¼ 6). Data are presented as mean � SEM.�, P < 0.05 was considered significant change; ��, P < 0.001 wasconsidered very significant change compared with vehicle control.






present in LNCaP cells, should not influence binding ofVPC-3033 to this site, as the compound does not form anycritical contacts with T877 or its mutant(s) as do flutamideanalogues (40). In support of this, our 10-benzylidene-10H-anthracen-9-one derivatives, including VPC-3033,showed effective inhibition of the LNCaP cell line as wellas cell lines with wild-type androgen receptor. Whentested in the LNCaP xenograft model, the lead compoundVPC-3033 showed effective in vivo potency against thetumor growth (Fig. 3).

In conclusion, the established in vitro and in vivoinhibitory activity of VPC-3033 on the human androgenreceptor and the prostate cancer cell growth makes thiscompound an excellent prospective antiandrogen.The preliminary structure–activity relationship infor-mation obtained around its analogues may serve as auseful basis for the development of an entirely new classof drugs for treating antiandrogen-resistant prostatecancer.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design:H. Li, N.A. Lack, P. Axerio-Cilies, E.T. Guns, P.S.Rennie, A. CherkasovDevelopment of methodology:H. Li, N.A. Lack, E. Leblanc, P. Tavassoli,H. Adomat, E.T. Guns, P.S. RennieAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): H. Li, M.D.H. Hassona, N.A. Lack, E. Leblanc,

N. Kanaan, K. Singh, H. Adomat, K.J. Bohm, H. Prinz, E.T. Guns, P.S.RennieAnalysis and interpretation of data (e.g., statistical analysis, biostatis-tics, computational analysis): H. Li, M.D.H. Hassona, N.A. Lack,E. Leblanc, N. Kanaan, K. Singh, H. Adomat, K.J. Bohm, H. Prinz, P.S.Rennie, A. CherkasovWriting, review, and/or revision of the manuscript: H. Li, M.D.H. Has-sona, N. Kanaan, K. Singh, H. Adomat, H. Prinz, E.T. Guns, P.S. Rennie,A. CherkasovAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): H. Li, M.D.H. Hassona, E. Leblanc,P. Tavassoli, K. Frewin, H. Adomat, P.S. Rennie, A. CherkasovStudy supervision: E. Leblanc, E.T. Guns, P.S. Rennie, A. Cherkasov

AcknowledgmentsThe authors thank J. Leong and D. Ma for helping the in vitro

experiments, Ms. M. Yieng Chin for helping in vivo study, Drs. M.Gleave and A. Zoubeidi for providing us with MDV3100-resistantLNCaP cells.

Grant SupportThis work was supported by an operating grant 272111 from

Canadian Institutes of Health Research (A. Cherkasov and P.S.Rennie), a Department of Defense award PC111132 (A. Cherkasovand P.S. Rennie), and the PC-STAR Project (A. Cherkasov and P.S.Rennie) funded by Prostate Cancer Canada with the support ofSafeway.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

ReceivedApril 10, 2013; revisedAugust 2, 2013; acceptedAugust 5, 2013;published OnlineFirst August 12, 2013.

Figure 4. The docked pose of VPC-3033 (C, D) within the androgen receptor HBS site compared with current antiandrogen bicalutamide (A) and flutamide (B).Residues Trp741 and Thr877 of androgen receptor known to be involved in drug resistance are highlighted.

Li et al.





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2013;12:2425-2435. Published OnlineFirst August 12, 2013.Mol Cancer Ther Huifang Li, Mohamed D.H. Hassona, Nathan A. Lack, et al. Cancerwith Potential Therapeutic Application in Advanced Prostate Characterization of a New Class of Androgen Receptor Antagonists

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