Development of Protein Degradation Inducers of Androgen Receptor by Conjugation of Androgen Receptor Ligands and Inhibitor of Apoptosis Protein Ligands

Article abstract of DOI:10.1021/acs.jmedchem.7b00168

Targeted protein degradation using small molecules is a novel strategy for drug development. We have developed hybrid molecules named specific and nongenetic inhibitor of apoptosis protein [IAP]-dependent protein erasers (SNIPERs) that recruit IAP ubiquitin ligases to degrade target proteins. Here, we show novel SNIPERs capable of inducing proteasomal degradation of the androgen receptor (AR). Through derivatization of the SNIPER(AR) molecule at the AR ligand and IAP ligand and linker, we developed 42a (SNIPER(AR)-51), which shows effective protein knockdown activity against AR. Consistent with the degradation of the AR protein, 42a inhibits AR-mediated gene expression and proliferation of androgen-dependent prostate cancer cells. In addition, 42a efficiently induces caspase activation and apoptosis in prostate cancer cells, which was not observed in the cells treated with AR antagonists. These results suggest that SNIPER(AR)s could be leads for an anticancer drug against prostate cancers that exhibit AR-dependent proliferation.

Full text of DOI:10.1021/acs.jmedchem.7b00168

Article
Development of protein degradation inducers of
androgen receptor by conjugation of androgen receptor
ligands and inhibitor of apoptosis protein (IAP) ligands
Norihito Shibata, Katsunori Nagai, Yoko Morita, Osamu Ujikawa, Nobumichi Ohoka, Takayuki Hattori,
Ryokichi Koyama, Osamu sano, Yasuhiro Imaeda, Hiroshi Nara, Nobuo Cho, and Mikihiko Naito
J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 08 Jun 2017
Downloaded from http://pubs.acs.org on June 9, 2017
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Journal of Medicinal Chemistry is published by the American Chemical Society. 1155
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Development of protein degradation inducers of androgen receptor by conjugation of
androgen receptor ligands and inhibitor of apoptosis protein (IAP) ligands
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Norihito Shibata , Katsunori Nagai , Yoko Morita , Osamu Ujikawa , Nobumichi Ohoka ,
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Takayuki Hattori , Ryokichi Koyama , Osamu Sano , Yasuhiro Imaeda , Hiroshi Nara ,
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Nobuo Cho , Mikihiko Naito
†Divisions of Molecular Target and Gene Therapy Products, National Institute of Health
Sciences, 1ꢀ18ꢀ1 Kamiyoga, Setagayaꢀku, Tokyo 158ꢀ8501, Japan.
‡
Pharmaceutical Research Division, Takeda Pharmaceutical Co. Ltd., 26ꢀ1,
MuraokaꢀHigashi 2ꢀchome, Fujisawa, Kanagawa 251ꢀ0012, Japan
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ABSTRACT
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Targeted protein degradation using small molecules is a novel strategy for drug
development. We have developed hybrid molecules named Specific and Nonꢀgenetic
inhibitor of apoptosis protein [IAP]ꢀdependent Protein Erasers (SNIPERs) that recruit IAP
ubiquitin ligases to degrade target proteins. Here, we show novel SNIPERs capable of
inducing proteasomal degradation of the androgen receptor (AR). Through derivatization of
the SNIPER(AR) molecule at AR ligand, IAP ligand and linker, we developed 42a
(SNIPER(AR)ꢀ51), which shows effective protein knockdown activity against AR.
Consistent with the degradation of the AR protein, 42a inhibits ARꢀmediated gene
expression and proliferation of androgenꢀdependent prostate cancer cells. In addition, 42a
efficiently induces caspase activation and apoptosis in prostate cancer cells, which was not
observed in the cells treated with AR antagonists. These results suggest that SNIPER(AR)s
could be leads for an antiꢀcancer drug against prostate cancers that exhibit ARꢀdependent
proliferation.
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INTRODUCTION
Prostate cancer (PC) is the secondꢀleading cause of cancer deaths, after lung cancer, in
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American males. Since the progression of PC is primarily stimulated by androgens,
inhibition of the secretion and/or action of androgens is highly effective in controlling the
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growth of PC. Besides surgical castration (orchiectomy) or androgen synthesis inhibitors,
androgen receptor (AR) antagonists are widely used to treat PC. These antagonists suppress
androgen signaling by inhibiting the binding of androgens to AR. Currently, AR antagonists
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such as flutamide, bicalutamide and enzalutamide are used clinically. In combination, AR
antagonists and androgen synthesis inhibitors show significant synergistic effects by
blocking androgen signaling as well as by suppressing transient increase in testosterone
levels. Although such therapy is initially effective, a considerable population of patients
develops castrationꢀresistant prostate cancer (CRPC) after the prolonged use of an AR
6ꢀ8
antagonist, primarily because of an increased level of AR expression in clinical situations.
Therefore, downꢀregulation of the AR level is regarded as a promising strategy to treat
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castrationꢀresistant PCs.
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Downꢀregulation of the AR protein can be achieved by genetic knockdown
technologies, such as antisense oligonucleotides, RNA interference and genome editing.
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Despite its great therapeutic potential, genetic knockdown remains clinically challenging,
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because oligonucleotides are not efficiently taken up into cells and delivery to the desired
target tissues is technically difficult. An alternative approach is protein knockdown, which
induces the degradation of target proteins via the ubiquitin proteasome system (UPS).
Based on this approach, molecules named Specific and Nonꢀgenetic inhibitor of apoptosis
11ꢀ22
protein [IAP]ꢀdependent Protein Erasers (SNIPERs)
and Proteolysis Targeting
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3ꢀ32
Chimeras (PROTACs)
have been developed. Both classes represent hybrid molecules
composed of two different ligands connected by a linker; one ligand is for the protein of
interest (POI) and the other is for E3 ubiquitin ligases. Accordingly, these molecules are
expected to crosslink the POIs and E3 ubiquitin ligases in cells, resulting in the
ubiquitylation and subsequent degradation of the POIs via the UPS. To date, we have
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developed several SNIPERs by connecting POI ligands to 79 (bestatin) a ligand for
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cellular IAP 1 (cIAP1), which induces the degradation of the POIs comprising cellular
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13, 21
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retinoic acid binding proteinꢀII,
estrogen receptor, transforming acidic coiledꢀcoilꢀ3
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and BCRꢀABL.
In this study, we designed and synthesized SNIPER(AR)s that induce proteasomal
degradation of the AR protein by conjugating an AR antagonist to an IAP ligand, 24
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(LCLꢀ161 derivative). We also showed that SNIPER(AR)s not only inhibit the growth but
also induce the cell death of PC cells, which exhibit ARꢀdependent proliferation.
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RESULTS AND DISCUSSION
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CHEMISTRY
The syntheses of 4aꢀd (SNIPER(AR)ꢀ2, 8, 23 and 26, respectively) were performed
according to Scheme 1. Removal of the Boc group of bestatin analog 1, followed by
condensation with the AR ligand part 2aꢀd afforded the corresponding coupling products
3aꢀd, which were treated with dimethylamine to give the bestatinꢀbased SNIPER(AR)s
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aꢀd.
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Scheme 1. Synthesis of 4aꢀd
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a
Reagents and conditions: (a) (1) aq. HCl, THF, rt; (2) 2a, 2b, 2c, or 2d, EDC,
HOBtꢀH O, DIPEA, THF, rt, 41–76%; (b) Me NH, MeOH, rt, 68–98%.
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Compound 4e (SNIPER(AR)ꢀ27) having a different pyrazoleꢀbased AR ligand was
prepared by the route shown in Scheme 2. Protection of the pyrazole nitrogen atom of 5
with 2ꢀ(trimethylsilyl)ethoxymethyl (SEM) group and subsequent alkaline hydrolysis of the
ester group yielded the benzoic acid derivative 7, which was condensed with the
Bocꢀprotected diamine 8 to give 9. Treatment of 9 with hydrochloride–cyclopropyl methyl
ether (CPME) provided the fullyꢀdeprotected intermediate, which was reacted with excess
diꢀtertꢀbutyl dicarbonate under basic conditions, and subsequently treated with sodium
methoxide to furnish the pyrazole analog 10. Nucleophilic substitution of the fluorine atom
of 11 with 10 gave 12. Bocꢀdeprotection of 12 followed by condensation with 13 produced
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e, which was converted to the target compound 4e by Fmocꢀdeprotection.
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Scheme 2. Synthesis of 4e
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a
Reagents and conditions: (a) SEMCl, NaH, DMF, 0 °C to rt, 89%; (b) aq. NaOH, THF,
MeOH, rt, 84%; (c) 8, EDC, HOBt, DMF, rt, 97%; (d) (1) 4 M HCl in CPME, MeOH, THF,
reflux; (2) Boc O, aq. NaHCO , THF, rt, then NaOMe, MeOH, rt, 77%; (e) 11, NaH, DMF,
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0 °C to rt, 97%; (f) (1) 4M HCl in CPME, THF, dioxane, rt; (2) 13, EDC, HOBt, DIPEA,
DMF, rt, 73%; (g) Me NH, MeOH, rt, 81%.
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Compound 18 (SNIPER(AR)ꢀ12) was synthesized by the route depicted in
Scheme 3. The carboxylate 2a was condensed with the Bocꢀprotected diamine 14 to
produce 15. Removal of the Boc group of 15 and subsequent condensation of the obtained
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amine with 16 yielded the coupling product 17. Final Bocꢀdeprotection of 17 afforded the
desired compound 18.
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Scheme 3. Synthesis of 18
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THF, rt; (2) 16, HATU, DIPEA, DMF, 0 °C, 95%; (c) 4 M HCl in CPME, THF, rt, 84%.
The synthetic route to 23 (SNIPER(AR)ꢀ31) is presented in Scheme 4. Mitsunobu
reaction of the compound 19 with azidoꢀalcohol 20 gave the corresponding coupling
product, which was subjected to Staudinger reduction to provide the primary amine 21.
Coupling of 21 with the carboxylic acid 2a afforded 22, which was converted to the target
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compound 23 by Bocꢀdeprotection. The reference compound 24 was obtained from 19
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under Mitsunobu reaction conditions.
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Scheme 4. Synthesis of 23 and 24
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(1) MeOH, DEAD, Ph P, THF, 0 °C to rt; (2) 4 M HCl in CPME, rt, 42%.
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moieties were synthesized according to Scheme 5. Sonogashira coupling of the
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bromobenzene analog 25 with the acetylene 26 produced 27, which was sulfonylated with
pꢀtoluenesulfonyl chloride to give 28. Reaction of 28 with 19 under basic conditions,
followed by removal of the Boc group of 31a furnished the desired product 32a. Palladium
catalyzed hydrogenation of the alkyne derivative 27 produced 29, which was converted to
the target compound 32b via the threeꢀstep procedure, i.e. tosylation, substitution with 19,
and final deprotection.
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Scheme 5. Synthesis of 32a and 32b
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DMF, 50 °C, 67–72%; (e) TFA, rt, 78–86%.
The syntheses of 42a–e (SNIPER(AR)ꢀ51, ꢀ53, ꢀ52, ꢀ171, and ꢀ65, respectively) having
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different substitution pattern and linker length were carried out by the procedure depicted in
Scheme 6. Various tosylated precursors (36a–e) were prepared by the three protocols: (i)
reaction of the phenol analogs 33a,b with the diol 34 under Mitsunobu reaction conditions,
followed by tosylation; (ii) coupling of 33a with the diꢀtosylated diol 37a or 37b; (iii)
coupling of 38 with 37c, mesylation of the benzyl alcohol 39, and subsequent reaction with
the pyrrole 40. The tosylates 36a–e were converted to the desired products 42a–e in a
similar procedure described for the preparation of 32 (Scheme 5).
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Scheme 6. Synthesis of 42a–e
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m
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36c (n = 2)
3
6d (n = 4)
O
O
OTs
3
OH
O
OTs
N
d
3
e
NC
HO
HO
O
TsO
OTs
NH
NC
3
3
8
39
36e
37c
NC
40
NC
N
O
R2
f
o
N
N
O
N
N
Boc
O
H
m
S
N
O
N
H
N
NC
N
O
O
O
S
p
n
O
HO
O
19
41a (p-, n = 3, R = Boc)
1b (m-, n = 3, R = Boc)
41c (p-, n = 2, R = Boc)
4
NC
4
4
1d (p-, n = 4, R = Boc)
1e (o-, n = 3, R = Boc)
g
4
2a (p-, n = 3, R = H), SNIPER(AR)-51
42b (m-, n = 3, R = H), SNIPER(AR)-53
4
4
2c (p-, n = 2, R = H), SNIPER(AR)-52
2d (p-, n = 4, R = H), SNIPER(AR)-171
42e (o-, n = 3, R = H), SNIPER(AR)-65
a
Reagents and conditions: (a) 34, DEAD, Ph P, THF, 0 °C to rt, 73–94%; (b) TsCl,
3
pyridine, 0 °C to rt, 68–75%; (c) 37a or 37b, K CO , DMF, 50 °C, 46–51%; (d) 37c,
2
3
K CO , DMF, 40 °C, 46%; (e) (1) MsCl, Et N, THF, 0 °C; (2) 40, NaH, DMF, 0 °C, 83%;
2
3
3
(
f) 36a, 36b, 36c, 36d, or 36e, K CO , DMF, rt to 60 °C, 67–81%; (g) TFA, rt, 70–90%.
2 3
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6
Scheme 7 shows the procedure used for the preparation of 48a–c (SNIPER(AR)ꢀ66, ꢀ67,
and ꢀ68, respectively) having a benzene ring embedded in the linker moiety.
Monoꢀtosylation of the diol 43a–c, followed by reaction with 33a under basic conditions
yielded 45a–c, which were tosylated to give 46a–c. Reaction of 46a–c with 19 and final
Bocꢀdeprotection of 47a–c produced the desired products 48a–c.
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9
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9
0
a
Scheme 7. Synthesis of 48a–c
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a
Reagents and conditions: (a) TsCl, pyridine, 0 °C, 52–56%; (b) 33a, K CO , DMF, 60
2
3
°
C, 82–99%; (c) TsCl, pyridine, 0 °C to rt, 83–87%; (d) 19, K CO , DMF, rt to 50 °C, 74–
2 3
8
2%; (e) TFA, rt, 85–89%.
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48
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53
54
55
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58
59
60
Induction of AR protein degradation by SNIPER(AR)
We designed a hybrid molecule 4a, composed of 79 and 76 (AR antagonistꢀ1), which
3
5
possesses strong binding affinity to AR. Based on the coꢀcrystal structure of AR
ligandꢀbinding domain (W741L mutant) complexed with bicalutamide (Protein Data Bank
3
6
(PDB) ID: 1Z95), the fluorophenyl moiety of bicalutamide is located at the AR surface.
Therefore, the PEG (polyethylene glycol) linker was ligated to the corresponding benzyl
moiety of 76 at one end, and 79 at the other end, as shown in Figure 1A.
To examine whether 4a reduces the AR protein level, human PC 22Rv1 cells
were treated with graded concentrations of 4a, and the wholeꢀcell lysates were analyzed by
western blot. The AR protein level was significantly decreased by treatment with 4a at 30
ꢁ
M for 6 h (Figure 1B). Combination treatment with 30 ꢁM 76 and 30 ꢁM 80 (methyl
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3
3
bestatin) did not decrease the AR protein level, indicating that linking the two ligands into
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0
a single molecule is essential for the reduction of the AR protein (Figure 1B).
3
5, 37
We next conjugated 79 to various AR ligands,
whose binding affinities to AR
−
9
−6
ranged from 10 to 10 M. Compounds 4a, 4c and 4d, which contain AR ligands with the
binding affinity of 0.0014, 0.0014 and 0.053 ꢁM, showed IC values of 0.027, 0.19, and
5
0
0.19 ꢁM in binding affinity to AR, and DC50 values of 21.6, 11.7 and 20.5 ꢁM in the
reduction of AR protein expression, respectively (Figure 1C, D). However, 4b and 4e,
which contain AR ligands with IC values of 0.054 and 1.5 ꢁM, had IC values of 1.1 and
5
0
50
>
10 ꢁM, and DC50 values of >30 and >30 ꢁM, respectively. These results suggest that
incorporation of an AR ligand with a higher binding affinity is preferable to develop a
SNIPER(AR) with effective protein knockdown activity.
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Figure 1
Reduction of AR protein levels by bestatinꢀconjugated SNIPER(AR)s.
(A) Chemical structures of the SNIPER(AR)s. (B) Reduction of AR protein by 4a
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6
(SNIPER(AR)ꢀ2). (C) Reduction of AR protein by SNIPER(AR)s containing various AR
ligands. 22Rv1 cells were incubated with the indicated concentrations of SNIPER(AR)s or
a mixture of the two chemically separate ligands (76 and 80, 30 ꢁM each) for 6 h. Numbers
below the AR panel represent AR/GAPDH ratio normalized by vehicle control as 100. Data
in the graphs are means ± standard deviation (n = 3). *, P<0.05 compared with vehicle
control (B, C). (D) Binding affinity and protein knockdown activity of the
bestatinꢀconjugated SNIPER(AR)s. IC50 values and DC50 values (concentrations of the
SNIPER(AR)s required to reduce the protein expression of AR by 50%) of SNIPER(AR)s
were obtained from three separate experiments. The binding affinities of the AR ligands
were cited from References 34 and 36.
0
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9
0
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9
0
Optimization of SNIPER(AR)
We selected 4a as a lead, and derivatized it at several locations in turn – the E3 ligase ligand,
the connection point (joint) between the AR ligand and linker, and finally the linker itself –
to improve the protein knockdown efficacy against AR. First, we replaced bestatinꢀmoiety
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with
two
other
specific
IAP
antagonists,
methyl
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22
23
24
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42
43
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48
49
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52
53
54
55
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59
60
(
2S)ꢀ2ꢀ(((2S)ꢀ1ꢀ((2S)ꢀ2ꢀcyclohexylꢀ2ꢀ(((2S)ꢀ2ꢀ(methylamino)propanoyl)amino)acetyl)pyrro
3
8
lidineꢀ2ꢀcarbonyl)amino)ꢀ3,3ꢀdiphenylpropanoate (MVꢀ1) and 24, to generate 18 and 23,
3
9, 40
respectively (Figure 2A), since 79 is not a specific ligand for cIAP1.
For 4a and 18, the
minimum concentrations necessary to reduce the AR protein levels were 30 and 20 ꢁM,
respectively, while 23 was effective at 3 ꢁM and above. Thus, among the three tested IAP
ligands, incorporation of 24 conferred the greatest AR protein degradation activity on the
corresponding SNIPER(AR).
Compound 23 was further derivatized by modification of the joint (the functional
group connecting 76 and the linker), i.e., amide (23), ether (42a), acetylene (32a) or alkyl
groups (32b). These compounds all showed comparable abilities to reduce the AR protein
level (Figure 2B). From 42a, we further derivatized the molecule by varying the position at
which the linker was connected to 76: either the o-, m- or p-position on the benzyl moiety.
The m-analog (42b) showed the same ability to reduce the AR protein level as did the
p-analog (42a) (Figure 2C). However, the o-analog (42e) showed no activity against the AR
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protein at concentrations of 1 or 3 ꢁM (Figure 2C). Since the benzyl moiety of 76 is likely
to be located on the surface of the AR protein, conjugation of the linker at the oꢀposition
may have hindered the binding of 42e to the AR protein.
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Figure 2
Derivatization of SNIPER(AR) by changing the cIAP1 ligand (A), joint
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(functional group between 76 and linker) (B), and position of joint (C). 22Rv1 cells were
incubated with the indicated concentrations of SNIPER(AR)s for 6 h. Numbers below the
AR panel represent AR/GAPDH ratio normalized by vehicle control as 100. Data in the
graphs are means ± standard deviation (n = 3). *, P<0.05 compared with vehicle control.
From 42a, we further synthesized analogs with different linker lengths.
SNIPER(AR)s with different lengths of PEG linker chain (n = 3, 4, 5) substantially reduced
the AR protein levels at 3 ꢁM, while combination treatment with 3 ꢁM 76 and 3 ꢁM 24 did
not affect the AR protein level (Figure 3A). Next, we examined the effect of linker
flexibility on the protein knockdown activity. In contrast to 42a, which contains a flexible
PEG linker, the series 48a–c, with rigid phenyl linkers, showed no activity against AR
protein (Figure 3B), suggesting that flexibility of the linker is important for the reduction of
AR protein levels by SNIPER(AR)s. On the basis of these results, we chose 42a for further
biological study.
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Figure 3
Derivatization of SNIPER(AR) by changing linker length (A) and
linker molecules (B). 22Rv1 cells were incubated with the indicated concentrations of
SNIPER(AR)s or 3 ꢁM of the two chemically separate ligands (76 and 24) for 6 h.
Numbers below the AR panel represent AR/GAPDH ratio normalized by vehicle control as
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6
100. Data in the graphs are means ± standard deviation (n = 3). *, P<0.05 compared with
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9
0
vehicle control.
Biological Evaluation
SNIPER(AR)s are presumed to induce the ubiquitylation and subsequent degradation of AR
via the UPS. Treatment of 22Rv1 cells with 42a at 3 ꢁM reduced the AR protein level in a
timeꢀdependent manner, while this reduction of AR by 42a was abrogated by a proteasome
inhibitor,
benzyl
Nꢀ((2S)ꢀ4ꢀmethylꢀ1ꢀ(((2S)ꢀ4ꢀmethylꢀ1ꢀ(((2S)ꢀ4ꢀmethylꢀ1ꢀoxopentanꢀ2ꢀyl)amino)ꢀ1ꢀoxopent
4
1
anꢀ2ꢀyl)amino)ꢀ1ꢀoxopentanꢀ2ꢀyl)carbamate 77 (MG132) (Figure 4A). These results
indicate that 42a induces the proteasomal degradation of AR. The proteasomeꢀdependent
reduction of AR protein by 42a was similarly observed in another PC cell line, VCaP,
which has an endogenous AR gene amplification and exhibits androgenꢀdependent
42
proliferation (Figure 4B).
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Figure 4
SNIPER(AR) induces the proteasomal degradation of AR protein. (A,
B) Effect of proteasome inhibitor, 77, on protein knockdown activity of 42a in 22Rv1 (A)
and VCaP (B) cells. Cells were incubated with 3 ꢁM 42a with or without 10 ꢁM 77 for the
indicated time. Numbers below the AR panel represent AR/GAPDH ratio normalized by
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vehicle control as 100. Data in the graphs are means ± standard deviation (n = 3). *, P<0.05
0
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compared with vehicle control.
Next, we tested the effect of 42a on ARꢀmediated gene expression and
proliferation in androgenꢀdependent PC VCaP cells. When cells were treated with 1 nM
dihydrotestosterone (DHT), the expressions of ARꢀregulated genes (PSA, TMPRSS2,
43ꢀ45
KLK2 and NKX3.1)
were upꢀregulated. Compound 42a at 3 ꢁM suppressed the
expression of these ARꢀregulated genes (Figure 5A), which is consistent with the reduction
of AR protein. In line with this, 42a at 3 ꢁM dramatically inhibited the growth of VCaP
cells (Figure 5B). A similar growthꢀinhibitory effect of 42a was also observed in another
PC cell line, LNCaP (Figure 5C), which expresses the AR protein and exhibits
androgenꢀdependent proliferation. However, 42a did not inhibit cell growth in the
androgenꢀindependent cell lines PCꢀ3, A549 or HT1080 under the same conditions (Figure
5
C). Treating the cells with 42a at 100 ꢁM for 6 h seriously reduced the viability of all the
cell lines, suggesting the toxicity of this compound. These data indicate that AR
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degradation by 42a results in the inhibition of (i) ARꢀmediated gene expression and (ii)
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androgenꢀdependent PC cell proliferation.
Figure 5
SNIPER(AR) inhibits ARꢀmediated gene expression and proliferation
of androgenꢀdependent PC cells. (A) Expression of androgenꢀresponsive genes in VCaP
cells. Cells were incubated with 1 nM DHT in the absence or presence of 3 ꢁM 42a or 76
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