Hybrid enzalutamide derivatives with histone deacetylase inhibitor activity decrease heat shock protein 90 and androgen receptor levels and inhibit viability in enzalutamide-resistant C4-2 prostate cancer cells

Article abstract of DOI:10.1124/mol.116.103416

Histone deacetylase inhibitors (HDACIs) can disrupt the viability of prostate cancer (PCA) cells through modulation of the cytosolic androgen receptor (AR) chaperone protein heat shock protein 90 (HSP90). However, toxicities associated with their pleiotropic effects could contribute to the ineffectiveness of HDACIs in PCA treatment. We designed hybrid molecules containing partial chemical scaffolds of enzalutamide and suberoylanilide hydroxamic acid (SAHA), with weakened intrinsic pan-HDACI activities, to target HSP90 and AR in enzalutamideresistant PCA cells. The potency of the new molecules, compounds 2-75 [4-(3-(4-cyano-3-(trifluoromethyl)phenyl)-5,5- dimethyl-4-oxo-2- Thioxoimidazolidin-1-yl)-2-fluoro-N-(7-(hydroxyamino)- 7-oxoheptyl)benzamide] and 1005 [(E)-3-(4-(3-(4- cyano-3-(trifluoromethyl)phenyl)-5,5-dimethyl-4-oxo-2- Thioxoimidazolidin- 1-yl)-2-fluorophenyl)-N-hydroxyacrylamide], as inhibitors of nuclear and cytosolic histone deacetylases was substantially lower than that of SAHA in cell-free and in situ assays. Compounds 2-75 and 1005 antagonized gene activation by androgen without inducing chromatin association of AR. Enzalutamide had no effect on the levels of AR or HSP90, whereas the hybrid compounds induced degradation of both AR and HSP90, similar to (compound 1005) or more potently than (compound 2-75) SAHA. Similar to SAHA, compounds 2-75 and 1005 decreased the level of HSP90 and induced acetylation in a predicted approximately 55 kDa HSP90 fragment. Compared with SAHA, compound 2-75 induced greater hyperacetylation of the HDAC6 substrate a- Tubulin. In contrast with SAHA, neither hybrid molecule caused substantial hyperacetylation of histones H3 and H4. Compounds 2-75 and 1005 induced p21 and caused loss of viability in the enzalutamide-resistant C4-2 cells, with efficacies that were comparable to or better than SAHA. The results suggest the potential of the new compounds as prototype antitumor drugs that would downregulateHSP90 andAR in enzalutamide-resistant PCA cells with weakened effects on nuclear HDACI targets.

Full text of DOI:10.1124/mol.116.103416

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TITLE PAGE
Hybrid Enzalutamide Derivatives with Histone Deacetylase Inhibitor Activity Decrease
HSP90 and the Androgen Receptor Levels and Inhibit Viability in Enzalutamide Resistant
C4-2 Prostate Cancer Cells
Rayna Rosati, Bailing Chen, Mugdha Patki, Thomas McFall, Siyu Ou, Elisabeth Heath, Manohar
Ratnam and Zhihui Qin
RR, MP, TM, EH, MR: Barbara Ann Karmanos Cancer Institute and Department of Oncology,
Wayne State University, Detroit, MI 48201-2013
BC, SO, ZQ: Department of Pharmaceutical Sciences, Wayne State University, Detroit, MI
4
8201-2013

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RUNNING TITLE PAGE
Running Title: Enzalutamide-histone deacetylase inhibitor hybrid molecules
Corresponding author: Manohar Ratnam, Ph.D., 4100 John R, HWCRC 840.1, Detroit, MI
4
8201. Tel: 313-576-8612. Email: ratnamm@karmanos.org
Number of text pages: 37
Tables: 0
Figures: 9
References: 70
#
#
#
of words in Abstract: 249
of words in Introduction: 992
of words in Discussion: 1107
Non-standard abbreviations; Enz, Enzalutamide; SAHA, Suberoylanilide hydroxamic acid;
HDAC, Histone deacetylase; HDACi, Histone deacetylase inhibitor; PCa, Prostate cancer;
CRPC, Castration recurrent prostate cancer; AR, Androgen receptor

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Abstract
Histone deacetylase (HDAC) inhibitors (HDACi) can disrupt viability of prostate cancer (PCa)
cells through modulation of the cytosolic androgen receptor (AR) chaperone protein HSP90.
However, toxicities associated with their pleiotropic effects could contribute to the
ineffectiveness of HDACi in PCa treatment. We designed hybrid molecules containing partial
chemical scaffolds of enzalutamide and suberoylanilide hydroxamic acid (SAHA), with
weakened intrinsic pan-HDACi activities, to target HSP90 and AR in enzalutamide-resistant
PCa cells. The potency of the new molecules (2-75 and 1005) as inhibitors of nuclear and
cytosolic HDACs was substantially lower than that of SAHA in cell-free and in situ assays. 2-75
and 1005 antagonized gene activation by androgen without inducing chromatin association of
AR. Enzalutamide had no effect on the levels of AR or HSP90 whereas the hybrid compounds
induced degradation of both AR and HSP90, similar to (1005) or more potently than (2-75)
SAHA. Similar to SAHA, 2-75 and 1005 decreased the level of HSP90 and induced acetylation
in a predicted ~55 KDa HSP90 fragment. Compared to SAHA, 2-75 induced greater hyper-
acetylation of the HDAC6 substrate alpha-tubulin but in contrast to SAHA neither hybrid
molecule caused substantial hyper-acetylation of histones H3 and H4. 2-75 and 1005 induced
p21 and caused loss of viability in the enzalutamide-resistant C4-2 cells with efficacies that were
comparable to or better than SAHA. The results suggest the potential of the new compounds as
prototype antitumor drugs that would downregulate HSP90 and AR in enzalutamide-resistant
prostate cancer cells with weakened effects on nuclear HDACi targets.

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Introduction
Prostate cancer (PCa) is the second leading cause of cancer-related deaths in men in
the United States (Siegel et al., 2015). PCa is initially managed with surgery, radiation,
androgen antagonists (e.g., bicalutamide) and surgical or chemical castration. However, the
relapsed or metastatic disease post-castration (castration-recurrent prostate cancer or CRPC)
has poor prognosis with most patients dying within 2 years (Karantanos et al., 2015). Innovative
treatment approaches are urgently needed to treat CRPC patients.
Androgen receptor (AR) signaling is a major driving force in all stages of PCa (Chen et
al., 2009). CRPC cells evolve mechanisms to re-activate AR signaling under androgen
deprivation conditions (Mitsiades, 2013); these mechanisms include overexpression and gain-
of-function mutations of AR (Joseph et al., 2013; Korpal et al., 2013), overexpression of AR
splice variants (AR-Vs) (Li et al., 2013), compensatory cross-talk between AR and other
signaling pathways (Liu et al., 2014) and enhanced intra-tumoral androgen biosynthesis
(Nakamura et al., 2005). Enzalutamide (Enz) is a newly FDA approved AR antagonist that
prolongs survival of CRPC patients (Beer et al., 2014; Scher et al., 2012). Enz competitively
binds to AR with 5-8 fold higher affinity than bicalutamide and, in contrast to bicalutamide, does
not promote AR nuclear translocation (Tran et al., 2009). Nevertheless, acquired resistance to
Enz typically develops within months and is associated with a relatively short-lived patient
survival benefit. Indeed, in vivo generated CRPC cell line models that vastly overexpress AR
(
e.g., C4-2 cells), presumably in combination with changes in other cellular signaling pathways,
are completely resistant to Enz in conditioned media while remaining addicted to AR (Patki et al.,
016).
2
A possible strategy to overcome resistance to androgen depletion is to induce
destabilization and degradation of AR and its associated proteins in CRPC cells. AR is
stabilized in the cytosol by its interaction with heat shock protein 90 (HSP90) and other
chaperone proteins. HSP90 is commonly overexpressed in many types of cancer cells and has

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been explored as a drug target for cancer treatment, including PCa (Bhat et al., 2014; Neckers
and Workman, 2012). HSP90 is an ATP-dependent molecular chaperone that aids the folding
and stability of a number of client proteins, such as steroid receptors, protein kinases,
transcription factors and proteins involved in regulating cell survival. Therefore, inhibition of
HSP90 leads to degradation of its client proteins via the ubiquitin-proteasome pathway.
Association of the AR apo-protein with HSP90 is critical for stabilizing AR in a conformation that
allows androgen binding (Fang et al., 1996; Veldscholte et al., 1992). In PCa cells, HSP90
inhibitors induce AR degradation and impair AR nuclear translocation while simultaneously
reducing the levels of other oncogenic client proteins, such as p-AKT/AKT, EGFR and IGF-IR
and survivin (He et al., 2013; Liu et al., 2015; Saporita et al., 2007; Solit et al., 2002).
Simultaneous disruption of AR and other aberrant growth/survival networks via HSP90 inhibition
is an advantageous treatment strategy for CRPC as this would silence potentially mutually
compensatory oncogenic signaling pathways. Despite this attractive scientific rationale, clinical
development of HSP90 inhibitors for PCa treatment were disappointing (Heath et al., 2008; Oh
et al., 2011; Pacey et al., 2011; Thakur et al., 2015) and was also limited by adverse toxicity to
non-target tissues.
One of the actions of histone deacetylase (HDAC) inhibitors (HDACi) is to disrupt HSP90
activity. HDACs remove acetyl groups from lysine residues of histone and non-histone proteins.
Eighteen HDACs categorized into four classes have been identified in mammalian cells. Among
them, HDAC6 is a zinc-dependent, class-IIb HDAC and is localized in the cytoplasm (Kramer et
al., 2014). HDAC6 deacetylates HSP90 (Bali et al., 2005; Kovacs et al., 2005). Inhibition of
HDAC6 could result in hyper-acetylation of HSP90, loss of ATP binding and dissociation and
degradation of its client proteins, including AR. The HDACi LAQ-824 (Chen et al., 2005), PDX-
1
01 (i.e. Belinostat) (Gravina et al., 2013), suberoylanilide hydroxamic acid (SAHA or vorinostat)
(Marrocco et al., 2007; Sato et al., 2012) and natural products sulforaphane (Gibbs et al., 2009)

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and genistein (Basak et al., 2008) have all been reported to reduce AR protein levels in PCa
cells by disrupting the HDAC6-HSP90 chaperone function.
Clinical use of HDACi is now confined to hematological malignancies. Although nuclear
HDACs (e.g., HDAC1 and HDAC3) are indispensable for AR transcriptional activity (Welsbie et
al., 2009) and increased HDAC levels have been reported in clinical CRPC samples and
positively correlated to Gleason scores (Burdelski et al., 2015; Weichert et al., 2008), they are
not likely to serve as effective drug targets to treat PCa. HDACi, such as vorinostat (SAHA)
(
Bradley et al., 2009), romidepsin (FK-228) (Molife et al., 2010), panobinostat (LBH-589)
Rathkopf et al., 2013) and pracinostat (SB-939) (Eigl et al., 2015) have been tested in CRPC
(
patients but have resulted in modest outcomes. Toxicities associated with their pleiotropic
effects could contribute to the ineffectiveness of HDACi in PCa treatment. Moreover, recent
studies have associated the pleiotropic effects of HDACi, particularly epigenetic modifications of
chromatin-associated proteins, with induction of epithelial to mesenchymal transition (EMT) in
prostate, endometrial and nasopharyngeal cancer cells (Jiang et al., 2013; Kong et al., 2012;
Tam and Weinberg, 2013; Uchida et al., 2012). Therefore clinical translation of the extensive
and promising pre-clinical findings of the efficacies of HDACi in treating solid tumors must
address the issue of toxicities that prevent application of effective HDACi treatment regimens in
the clinic.
We sought to develop enzalutamide derivatives armed with HDACi activity to antagonize
AR and HSP90 actions in AR-overexpressing and enzalutamide resistant CRPC cells with
reduced pleiotropic effects typical of strong HDACis. Accordingly, we have designed,
synthesized and tested the prototype compounds 2-75 and 1005. The 2-75 and 1005 chemical
scaffolds are designed to retain AR binding affinity. The compounds are also designed to have
lower intrinsic HDACi activity compared to SAHA, thus reducing the HDACi activity against non-
target proteins. Nevertheless, the HDACi activity is expected to produce effective disruption of

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AR as well as other HSP90 client proteins, resulting in loss of viability in Enz-resistant CRPC
cells.

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Materials and Methods
Compound Synthesis. Detailed procedures for the synthesis of compounds 2-75, 1005, 3-52
and 1002 are described under Supplemental Materials. The chemical identities of the
1
13
compounds were confirmed by using H, C-NMR and high resolution mass spectrometry.
HDAC activity assay. In vitro HDAC inhibition was measured by using the HDAC fluorimetric
assay/drug discovery Kit (Enzo Life Sciences, BML-AK500) and the HDAC6 fluorometric drug
discovery kit (Enzo Life Sciences, BML-AK516) following the manufacturer’s protocols and
instructions. IC50 values were calculated from nonlinear aggression plots using GraphPad
Prism5 software.
Cell Culture and Reagents. LNCaP and PC3 cell lines were from American Type Culture
2
Collection (Manassas, Va). LNCaP and C4-2 cells were routinely grown at 37°C in 5% CO in
RPMI 1640 medium supplemented with 10% FBS (Invitrogen); 100units/ml penicillin, 100 µg/ml
streptomycin, 2mM L-glutamine mixture (Invitrogen); and sodium pyruvate (1mM) (Invitrogen).
PC3 cells were grown in RPMI 1640 medium supplemented with 10% FBS (Invitrogen);
1
00units/ml penicillin, 100 µg/ml streptomycin, 2mM L-glutamine mixture (Invitrogen). Affinity-
purified rabbit anti-human antibody to AR (sc-816), mouse anti-human antibody to GAPDH (sc-
7724) and affinity-purified mouse anti-human antibody to alpha tubulin (sc-8035) were
4
purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Affinity-purified rabbit anti-human
antibody to HSP90 (C45G5) #4877, rabbit anti-human antibody to acetylated lysine #9441, and
affinity-purified rabbit anti-human antibody to p21 Waf1/Cip1 #2947S were purchased from Cell
Signaling Technology. Affinity-purified rabbit anti-human antibody to acetyl-tubulin (#ABT241),
acetyl Histone H4 (#07-328), acetyl Histone H3 (#ABE18) were purchased from Millipore.
R1881 was kindly provided by Dr. Stephan Patrick (Karmanos Cancer Institute). Cycloheximide

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was from Sigma. All experiments were conducted using phenol-red free growth media. For
hormone depletion, cells were grown in phenol-red free RPMI 1640 medium supplemented with
1
0% charcoal stripped FBS (Sigma-Aldrich) which was heat inactivated at 56°C for thirty
minutes, and a mixture of 100units/ml penicillin, 100 µg/ml streptomycin and 2 mM L-glutamine
for 96 h.
Cell Viability Assay. Cells were trypsinized and 6000 cells/well were seeded in 96-well plates
coated with poly-D-lysine. The cells were seeded in phenol red-free medium supplemented with
1
0% FBS, 100units/ml penicillin, 100 µg/ml streptomycin, 2mM L-glutamine mixture and sodium
pyruvate (1mM) for C4-2 cells and phenol red-free medium supplemented with 10% FBS,
00units/ml penicillin, 100 µg/ml streptomycin and 2mM L-glutamine mixture for PC3 cells. The
1
cells were grown at 37°C in 5% CO2. Twenty four hours after seeding in the 96-well plates, the
cells were treated with indicated compound or DMSO (vehicle). The culture medium was not
changed during the time course of the assay. On Day zero and on Day 3, cell viability was
determined using the MTT assay. MTT (10µL, 5mg/mL) was added to each well and incubated
for 2h at 37°C. The formazan crystal sediments were dissolved in 100µL of DMSO, and the
absorbance at 570nm was measured using the BioTek Synergy 2 Microplate Reader (BioTek,
Winooski, VT). The assay was conducted in sextuplicate wells and values were normalized to
day zero (Patki et al., 2014). IC50 values were calculated from nonlinear aggression plots using
GraphPad Prism5 software.
Western Blot Analysis. Cells were washed once with phosphate buffered saline (PBS) and
then lysed with RIPA buffer (150mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1%
SDS, 50mM Tris of pH 8.0) containing a protease inhibitor cocktail (Pierce, Thermo Fisher
Scientific). The cell lysates were then incubated on ice for 40 minutes. Total protein
concentrations were estimated using the Bradford Assay (Bio-Rad). Protein samples (10-40 ug)

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were heated at 95°C for 5 minutes and resolved by electrophoresis on 8% polyacrylamide-SDS
gels and electrophoretically transferred to PVDF membranes (Millipore, Billerica, MA). The
membranes were then probed overnight at 4°C with the appropriate primary antibody followed
by the appropriate horseradish peroxidase-conjugated secondary antibody. The blots were then
developed to visualize the protein bands using the HyGLO Chemiluminescent HRP Antibody
Detection Reagent (Denville Scientific, Metuchen, NI) (Salazar et al., 2011).
RNA isolation, Reverse Transcription, and Real Time PCR. Total RNA was isolated from
cells using the RNeasy Mini Kit (Qiagen). Reverse transcription PCR was then performed using
5
00 ng of total RNA with random primers and using the high-capacity complementary DNA
Archive kit (Applied Biosystems). The complementary DNA from this reaction was measured
using quantitative real time PCR using the StepONE Plus Real Time PCR system (Life
Technologies Corporation, Carlsbad, CA). All reactions were performed in triplicate and
normalized to glyceraldehyde-3-phosphate-dehydrogenase values in the same samples. All
primers and Taqman probes were purchased from the applied Biosystems inventory (Invitrogen)
(Patki et al., 2015).
Chromatin Immunoprecipitation (ChIP). C4-2 cells were treated with either vehicle, R1881 (1
nM or 10 nM), or 10uM of each indicated compound for 2 h and then subjected to ChIP using
anti-AR antibody (sc-816 from Santa Cruz, CA). The ChIP assay was performed using the EX
ChIP chromatin immunoprecipitation kit (catalogue number 17-371 from Millipore, Temecula,CA)
according to the vendor’s protocol. The ChIP signals were measured by quantitative real time
PCR analysis of the immunoprecipitated products. Each sample was tested in triplicate (Patki et
al., 2013).

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Statistical Analysis. All experiments were performed in triplicate groups and repeated at least
three times. The error bars in all graphs represent the standard deviation. Statistical analysis
was performed using one-way ANOVA with post-hoc and LSD (Least Square Differences)
and/or T-test (McFall et al., 2015).
Results
Design and synthesis of compounds 2-75 and 1005 with partial chemical scaffolds of Enz
and SAHA. Compounds 2-75 and 1005 were designed to retain partial functional scaffolds of
Enz and SAHA (Figure 1A). Upon binding to AR, the cyano group of Enz/Enz derivatives forms
a critical hydrogen bond with Arg752, and the conformationally restricted thiohydantoin ring in
the middle forces the rest of the molecule to the “H11 pocket”, a region near the C terminus of
helix 11 and the loop connecting helices 11 and 12 (Balbas et al., 2013). To design derivatives
with both AR-binding and HDACi activities, different linkers connecting Enz and a zinc binding
group (ZBG) were introduced to retain the above structural features that are required for AR
binding in an antagonist-related conformation. 1005 is a cinnamyl hydroxamic acid derivative
with a three-carbon linker. A relatively longer carbon chain in 2-75 was used to more closely
mimic the chemical structure of SAHA. Compound 7 (3-52), a close structural analogue of 2-75
using methyl ester to replace ZBG was synthesized as a control compound without an HDACi
functional group (Figure 1B). Compound 7 (3-52) and synthetic intermediate compound 2 (i.e.
1
002, an ethyl ester analogue of 1005, Figure 1A, B) were used to investigate AR antagonist
properties of 2-75 and 1005 scaffold, respectively.
Both 2-75 and 1005 were synthesized from a 4’-iodo substituted intermediate (Figure 1B)
1
2
. Acrylate linker of 1005 was introduced via Pd(OAc) -catalyzed Heck reaction to afford
compound 2, followed by hydrolysis of ethyl ester, coupling to THP (tetrahydropyranyl acetal)-
protected hydroxylamine and the final acidic deprotection. To synthesize 2-75, carboxylation of
aryl iodine 1 was performed using a palladium-catalyzed carboxylation reaction (Cacchi et al.,

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2
003), the resulted carboxylic acid then coupled with primary amines to attach the alkyl chain
with protected hydroxamic acid (compound 6) or methyl ester (compound 7). Removal of THP
protecting group gave the final product 2-75. The detailed synthetic procedures are described in
the Supplemental Material.
Compounds 2-75 and 1005 possess intrinsically weak inhibitor activity against nuclear
HDACs and cytosolic HDAC6. To measure HDAC inhibitory activities of 2-75 and 1005, cell-
free enzymatic assays were performed against a nuclear extract of Hela cells and also against
human recombinant HDAC6. HDAC1 and HDAC2 are enriched in nuclear extracts whereas
HDAC6 is a cytosolic enzyme that is the principal modulator of the acetylation status of HSP90.
The HeLa cell nuclear extract was used to evaluate inhibitory activity against nuclear HDACs. 2-
7
5 and 1005 dose-dependently inhibited HDACs enriched in the Hela nuclear extract with IC50
values of 1.08 µM and 2.41 µM compared to a value of 0.30 µM for SAHA (Figure 2A). In
contrast, compounds 3-52 and 1002, which share the chemical scaffolds of 2-75 and 1005
respectively, but lack the HDACi functional group, did not inhibit nuclear HDAC activity even up
to a concentration of 25 µM (Figure 2B). As expected, Enz also lacked any HDACi activity in
contrast to a pan-HDACi, trichostatin A (TSA), which was used as a positive control (Figure 2B).
2
-75 and 1005 were also weaker inhibitors of recombinant HDAC6, with IC50 values of 2.0 µM
and 6.93 µM, respectively compared with the IC50 of 0.85 µM for SAHA (Figure 2C). As
expected, the negative control compounds 3-52 and 1002 as well as Enz did not show
significant inhibitory activity against HDAC6 even at a concentration of 25 µM (Figure 2D). Again,
TSA served as the positive control in Figure 2D.
The HDACi activities of 2-75 and 1005 were also compared with those of their parent
compounds in situ in both LNCaP and C4-2 prostate cancer cells using activation of the DLC1
tumor suppressor gene as the readout. SAHA induces histone acetylation at the DLC1 promoter
and effectively increases DLC1 mRNA expression in prostate cancer cells (Zhou et al., 2012).

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Accordingly, DLC1 mRNA was strongly up-regulated by SAHA in both LNCaP cells (Figure 2E)
and in C4-2 cells (Figure 2F). As expected from the fact that DLC1 is not an AR-regulated gene,
Enz had no effect on DLC1 mRNA expression. Consistent with the results of the cell-free HDACi
assays above, compounds 2-75 and 1005 were poor inducers of DLC1 mRNA compared with
SAHA, both in LNCaP cells and in C4-2 cells (Figure 2E and 2F). Further, similar to the results
from cell-free assays, 1005 was a much weaker HDACi than 2-75 in the in situ assays (Figure
2
E and 2F). Taken together, the results indicate that compounds 2-75 and, to a greater degree,
005 have much less potent HDACi activity in the cellular context reflecting their intrinsically
1
weak inhibitor activities compared with SAHA.
The partial Enz chemical scaffold confers AR targeted antagonist activity without ligand-
induced chromatin association of AR. Although C4-2 cells are not growth-inhibited by Enz
due to hormone-independent actions of AR, the canonical androgen target genes KLK3 and
TMPRSS2 are activated by androgen and their activation is inhibited by androgen antagonists
(Ratnam et al., 2013). HDACi are also potent inhibitors of the androgen signaling axis as they
cause degradation of AR in the cytosol in addition to other cellular effects (Chen et al., 2005;
Gravina et al., 2013; Gryder et al., 2013). To test whether the Enz moiety could enable 2-75 and
1
005 to target to AR, we tested the ability of compound 3-52 to inhibit activation of KLK3 and
TMPRSS2 by androgen. Compound 3-52 shares the chemical scaffold of 2-75 but lacks the
HDACi functional group; therefore 3-52 should depend on the partial Enz chemical scaffold to
antagonize gene activation by androgen. SAHA partially inhibited activation of KLK3 (Figure 3A)
and TMPRSS2 (Figure 3B) by the synthetic androgen R1881 in C4-2 cells whereas Enz showed
progressive inhibition at higher doses. Compounds 2-75 and 1005 were both better inhibitors of
gene activation by androgen compared with either Enz or SAHA (Figure 3A and 3B). On the
other hand, compound 3-52 inhibited activation of KLK3 and TMPRSS2 to a degree that was
comparable to Enz suggesting that the partial Enz chemical scaffold in compounds 2-75 and

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1
7
005 retained an Enz-like AR binding property. The superior androgen antagonist activities of 2-
5 and 1005 compared with Enz may be explained by their additional HDACi activities.
AR ligands including agonists and classical androgen antagonists such as bicalutamide
promote nuclear translocation of AR and the binding of AR to canonical hormone (androgen)
response elements associated with androgen-regulated genes. In contrast, Enz does not
stimulate AR nuclear translocation and DNA binding (Guerrero et al., 2013; Tran et al., 2009).
To test whether the partial Enz chemical scaffold would mobilize AR to the chromatin, we
employed chromatin immunoprecipitation using C4-2 cells treated with androgen, Enz, SAHA
and compound 2-75. As a target site for the ChIP assay, we chose the well-established AR
binding enhancer elements located 4kb upstream of the transcription initiation site of the KLK3
gene. As seen in Figure 3C, androgen treatment strongly stimulated chromatin association of
AR whereas Enz, SAHA and compound 2-75 all gave the basal ChIP signal corresponding to
the vehicle treatment control. These results suggest that the new compounds must antagonize
AR in the cytosolic rather than in the nuclear compartment.
Compounds 2-75 and 1005 induce enhanced degradation of AR and HSP90 and hyper-
acetylation in a putative 55 KDa HSP90 fragment. Previous observations using potent non-
targeted HDACi have shown that the compounds directly affect the AR signaling axis by hyper-
acetylation of the AR chaperone complex, through inhibition of HDAC6, leading to degradation
of HSP90 as well as release and degradation of AR. We therefore hypothesized that despite
their intrinsically weak HDACi activities, the Enz moiety may enable compounds 2-75 and 1005
to more effectively target AR in its chaperone complex, leading to relatively efficient degradation
of AR. To test this possibility, we treated C4-2 cells with Enz, SAHA, 1005 and 2-75 at doses
ranging from 1µM to 10µM for 24h. Western blots of the cell lysates were probed for AR and
GAPDH (loading control) and the AR band intensities relative to GAPDH were quantified using
ImageJ software (Figure 4A). Whereas Enz did not cause an appreciable change in the AR

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protein level, SAHA did cause a decrease in AR level in a dose-dependent manner (Figure 4A).
Compared to SAHA, both 2-75 and 1005 decreased the AR level to a greater extent with 2-75
being more effective than 1005 at each dose (Figure 4A). To determine whether the decrease in
AR was due to increase in the rate of AR degradation we tested the effects of the compounds
after blocking de novo protein synthesis using cycloheximide. We monitored degradation of p21
to confirm the activity of cycloheximide. As expected there was a rapid decrease in p21 upon
treatment with cycloheximide confirming that the treatment efficiently blocked de novo protein
synthesis (Figure 5). In the presence of cycloheximide the AR protein level was decreased by
approximately half at the end of 24h indicating a relatively slow turnover of the AR protein.
Under these conditions treatment with SAHA, 1005 and 2-75 all caused greater declines in the
AR level with 2-75 showing the strongest effect (Figure 5). The results indicate that the
decrease in AR caused by 1005 and 2-75 is due to increased degradation of AR. The extent of
degradation of AR in C4-2 cells appeared adequate to offset the high level of overexpression of
AR that is necessary to support growth in these cells.
To explore a possible link between decreased AR levels and effects of the compounds
on the AR chaperone complex, we examined whether compounds 2-75 and 1005 decreased the
level of HSP90. Probing of the lysates from the treated cells (48h treatment) for HSP90 by
western blot and quantification of HSP90 was conducted by procedures similar to that used
above for AR. Enz had no effect on the level of HSP90 whereas in the SAHA-treated cells, a
decrease in HSP90 was evident at the higher doses (5µM and 10µM) (Figure 4B). On the other
hand, cells treated with 1005 and 2-75 showed more marked reduction in HSP90, with 2-75
being more efficient than 1005. Probing identical western blots with an antibody against
acetylated lysine showed that SAHA as well as 1005 and 2-75, but not Enz, showed hyper-
acetylation of a ~55 KDa polypeptide (Figure 4C), similar to one that has previously been
identified as a fragment HSP90 produced by SAHA treatment (Park et al., 2015). Taken
together, the above results are consistent with the view that the ability of the compounds to

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induce acetylation and reduction of HSP90, and consequently AR degradation, underlies the
ability of the compounds to attenuate AR signaling.
The hybrid molecules selectively inhibit cytosolic HDAC6 in situ. As HSP90 in the AR
chaperone complex is a target of the cytosolic HDAC6, the hyper-acetylation of degradation of
HSP90 induced by 2-75 and 1005 is likely to occur through inhibition of HDAC6. If this were the
case, we may expect that 2-75 and 1005 would also induce hyper-acetylation of -tubulin which
is diagnostic of HDAC6 inhibition. To test this possibility, we treated C4-2 cells with Enz, SAHA,
1
005 and 2-75 at doses ranging from 2.5µM to 10µM for 24h. Western blots of the cell lysates
were probed for acetyl-tubulin, as well as total -tubulin. The band intensities for acetyl-tubulin
relative to total -tubulin were quantified using ImageJ software (Figure 6). 2-75 induced a
greater degree of hyper-acetylation of -tubulin (relative to total tubulin) compared to SAHA,
whereas 1005 produced a similar effect albeit to a somewhat lesser degree than SAHA (Figure
6
). When the same cell lysates were probed using antibodies against acetylated histones H3
and H4, it was clear that SAHA alone induced a strong induction of histone acetylation (Figure
). The results clearly demonstrate strong and selective in situ activity of 2-75 and 1005 on
cytosolic HDAC6.
6
2
-75 and 1005 up-regulate p21 and inhibit viability of Enz-resistant prostate cancer cells.
HDACi activate transcription of p21. However, as compounds 2-75 and 1005 exhibited weak
intrinsic HDACi activity against nuclear HDACs and as their apparent major cellular HDACi
activity was related to targeting of the AR axis within the cytosolic compartment, it was of
interest to examine their ability to induce p21.
Enz had no effect on p21 mRNA expression in either the Enz-sensitive LNCaP cells
(Figure 7A) or in the Enz-insensitive C4-2 cells (Figure 7B), whereas SAHA induced p21 mRNA
in both cell lines (Figure 7A and 7B). Compounds 2-75 and 1005 both induced p21 to a greater
extent than SAHA in the two cell lines (Figure 7A and 7B). Moreover, combined treatment with

Molecular Pharmacology Fast Forward. Published on July 5, 2016 as DOI: 10.1124/mol.116.103416
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equimolar concentrations of Enz and SAHA did not induce p21 to a greater extent than SAHA
alone, indicating the importance of the hybrid scaffold of 2-75 and 1005 (Figure 7C).
To expect therapeutic effects from 2-75 and 1005, it is important to establish that, similar
to SAHA, they can induce loss of viability in Enz-resistant CRPC cells, rather than mere growth
inhibition. Therefore, the effects of 2-75, 1005 and SAHA on cell viability were assessed in the
well-established C4-2 model of Enz-resistant CRPC.
In C4-2 cells Enz could not appreciably affect viability even at a concentration of 10 µM,
whereas SAHA caused loss of viability in a dose dependent manner (Figure 8A). Compounds 2-
7
5 and 1005 both caused greater loss of viability compared with SAHA with compound 2-75
being more effective than 1005 (Figure 8A). As a control, compound 1002, which has a
chemical scaffold similar to 1005 but lacks the HDACi activity (Figure 2B and 2D), was unable to
affect C4-2 cell viability (Figure 8A). As another experimental control, at the lower drug
concentration (2.5 uM) although SAHA, 2-75 and 1005 caused growth inhibition, combining Enz
with SAHA (each at 2.5 uM) did not enhance the ability of SAHA to inhibit cell growth (Figure
8
B). In the AR-negative PC3 PCa cells, SAHA induced loss of viability in a dose-dependent
manner (Figure 8C). However, in contrast to C4-2 cells, neither 2-75 nor 1005 affected viability
of PC3 cells within the duration of the assay (Figure 8C). As expected, the AR-positive and
hormone-dependent LNCaP cells were sensitive to SAHA, 2-75 and 1005 as well as Enz
(Supplemental Figure 1).
The results indicate that despite the weaker inherent HDACi activities of 2-75 and 1005
compared with SAHA, the compounds could be as good or better at reducing viability of Enz-
resistant and AR-overexpressing PCa cells.

Molecular Pharmacology Fast Forward. Published on July 5, 2016 as DOI: 10.1124/mol.116.103416
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Discussion
The success of clinical interventions in prostate cancer, including surgical or chemical
castration and treatment with androgen antagonists and androgen synthesis inhibitors support
the view that the majority of prostate tumors are addicted to AR to support PCa growth and
progression (Zegarra-Moro et al., 2002). Nevertheless, the current interventions that target
androgen/AR signaling are circumvented by the tumors, most commonly through mechanisms
that restore functional AR (Feldman and Feldman, 2001; Yuan and Balk, 2009), resulting in
short-lived clinical benefit from the treatments. The goal of this study was to develop a class of
compounds that may overcome this manner of resistance to the conventional treatments by
efficiently disrupting both AR and HSP90 in the AR-HSP90 complex with minimal effects on
most other cellular targets. To accomplish this, we synthesized compounds that would
incorporate properties of two well-known drugs, an HDACi (SAHA) that efficiently modifies and
disrupts the cytosolic AR chaperone complex and an AR ligand (Enz), which is a high affinity AR
antagonist. We additionally sought to substantially weaken the intrinsic HDACi activity of the
drug to minimize its ability to affect many targets. The studies described above suggest that
compounds 2-75 and 1005 may be prototype molecules that fit this paradigm.
The HDACi functional groups in 2-75 and 1005 conferred only weak HDACi activity in
cell-free assays using either nuclear HDACs or the cytosolic HDAC6 compared with SAHA; the
shorter carbon chain in 1005 resulted in even weaker HDACi activity than 2-75. The relative
potencies of HDACi inhibition of 2-75 and 1005 was clearly reflected in their relatively poor
ability to induce DLC1, an established nuclear target gene of HDACi (Guan et al., 2006; Zhou et
al., 2010; Zhou et al., 2012), that is strongly induced by SAHA. 2-75 and 1005 were also poor
modulators of histone acetylation in situ compared with SAHA. Therefore, the new molecules
may have less toxic effects than those associated with the potent pan-HDACi activity of SAHA
(Bradley et al., 2009).

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SAHA partially inhibited gene activation by androgen but did not produce a further dose-
dependent inhibition between 1µM and 10 µM concentrations. At this time, we do not have a
clear explanation for why this effect of SAHA was only partial except that it may be related to the
pleiotropic cellular effects of SAHA including its effects on cross-talking molecular pathways.
More important, 2-75 and 1005 produced a dose-dependent inhibition of gene activation by
androgen similar to Enz. The stronger inhibition observed for the compounds compared to Enz
may be attributed to their HDACi moieties. However, the close parallel between the control
compound 3-52 and Enz in their dose-dependent antagonism of gene activation by androgen,
despite the lack of a HDACi functional group in 3-52, indicates that the Enz moiety in 2-75 and
1
005 is functional in enabling binding to AR. Additionally, ChIP analysis showed that the
modified Enz scaffold retained the inability of Enz to mobilize AR to its chromatin binding sites in
the nucleus in contrast to conventional androgen antagonists.
In the context of targeted delivery to AR via their Enz moiety, the weak intrinsic HDACi
activities of 2-75 and 1005 were adequate to mimic or surpass the effects of SAHA on AR
protein levels. The efficiency of degradation of AR by 1005 was comparable to SAHA but 2-75
clearly induced AR degradation to a greater degree at each dose. This difference between 2-75
and 1005 may be related to the fact that the HDACi activity of 1005 was less than that of 2-75,
despite their common Enz moiety. To test the mechanism by which 2-75 and 1005 may cause
AR degradation, we relied on literature reports that HDACi destabilize and degrade AR by
hyper-acetylating and inducing degradation of the cytosolic AR chaperone protein, HSP90
(Chen et al., 2005; Gravina et al., 2013; Gryder et al., 2013). It has also been reported that the
hyper-acetylation and degradation of HSP90 coincides with the appearance of a ~55 KDa
HSP90 polypeptide fragment. As a diagnostic test of this mechanism, we observed that similar
to SAHA, 2-75 and 1005 did indeed cause a decrease in HSP90, with 2-75 being more efficient
than either SAHA or 1005. We were able to observe the predicted ~55KDa fragment in cells
treated with SAHA, 2-75 or 1005 using an antibody against acetylated lysine; however our

Molecular Pharmacology Fast Forward. Published on July 5, 2016 as DOI: 10.1124/mol.116.103416
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antibody against HSP90 was unable to detect this fragment, possibly because the levels of the
cleaved HSP90 fragment were too low to be in the detectable range of the antibody.
Nevertheless, all indications point to the AR chaperone complex in the cytosol as the mediating
the action of 2-75 and 1005. Consistent with this view HDAC6 which is associated with the AR-
HSP90 complex was more strongly and selectively inhibited in situ by 2-75 compared to SAHA.
Indeed 1005 which had weaker intrinsic HDACi activity than 2-75 also strongly inhibited HDAC6
in situ with virtually no effect on histone acetylation.
An increase in the expression of the cyclin-dependent kinase inhibitor p21 (Gartel and
Tyner, 2002) is a hall mark of the antiproliferative effects of HDACi (Gui et al., 2004; Marks,
2
004). Potent inhibition of the nuclear HDAC1 at the promoter of the p21 gene is associated
with induction of p21 by HDACi (Lagger et al., 2003). Inhibitors of HSP90 also increase p21
expression in PCa cells (Chen et al., 2005). Therefore it is significant that p21 mRNA was
induced by both 2-75 and 1005 more strongly than SAHA and that combination with Enz did not
further increase p21 induction by SAHA. The inability of Enz to induce p21 in the hormone-
dependent LNCaP cells despite the sensitivity of the AR signaling in these cells to Enz suggests
that induction of p21 by 2-75 and 1005 may not be directly related to disruption of AR; rather, it
may be due to their effects on additional HSP90 client proteins through AR-mediated targeting
of HDACi activity to HSP90. Notably, AKT and GR are also HSP90 client proteins and
upregulated AKT or GR signaling were reported to result in Enz resistance (Arora et al., 2013;
Bitting and Armstrong, 2013; Montgomery et al., 2015; Toren et al., 2015),. Therefore, these
pathways could be involved in the loss of viability induced by the compounds.
HDACi pharmacophores have previously been linked to a chemical scaffold of
cyanonilutamide, which is another nonsteroidal AR antagonist (Gryder et al., 2013). However,
the antiproliferative effects of cyanonilutamide-HDACi were related to their ability to induce AR
nuclear localization, enabling elevated local concentrations of HDACi activities in the nucleus. In

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contrast, our prototype drug molecules were designed to limit nuclear HDACi activities as an
approach to limiting toxicity. Our working model that would need further testing is that in C4-2
cells, 2-75 and 1005 bind to cytosolic AR and inhibit HDAC6 associated with the AR chaperone
complex, resulting in HSP90 acetylation and degradation, AR degradation, suppression of
ligand-insensitive gene activation by AR and inhibition HSP90 interactions with additional client
proteins (Schematic in Figure 9). We propose that this mechanism may address some of the
limitations of strong pan-HDAC inhibitors related to toxicity.

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Acknowledgements
We thank Yanfang Huang for technical support.
Authors’ Contributions
Participated in research design: Rosati, Chen, Patki, Ou, McFall, Ratnam, Qin
Conducted experiments: Rosati, Chen, Patki, Ou, Qin
Contributed new reagents or analytic tools: Rosati, Chen, Ou, Heath, McFall, Ratnam, Qin
Performed data analysis: Rosati, Chen, Ou, McFall, Ratnam, Qin
Wrote or contributed to the writing of the manuscript: Rosati, Chen, Ratnam, Qin

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