In Vivo Knockdown of Pathogenic Proteins via Specific and Nongenetic Inhibitor of Apoptosis Protein (IAP)-dependent Protein Erasers (SNIPERs)*

Many diseases, especially cancers, result from aberrant or overexpression of pathogenic proteins. Specific inhibitors against these proteins have shown remarkable therapeutic effects, but these are limited mainly to enzymes. An alternative approach that may have utility in drug development relies on selective degradation of pathogenic proteins via small chimeric molecules linking an E3 ubiquitin ligase to the targeted protein for proteasomal degradation. To this end, we recently developed a protein knockdown system based on hybrid small molecule SNIPERs (Specific and Nongenetic IAP-dependent Protein Erasers) that recruit inhibitor of the apoptosis protein (IAP) ubiquitin ligases to specifically degrade targeted proteins. Here, we extend our previous study to show a proof of concept of the SNIPER technology in vivo. By incorporating a high affinity IAP ligand, we developed a novel SNIPER against estrogen receptor α (ERα), SNIPER(ER)-87, that has a potent protein knockdown activity. The SNIPER(ER) reduced ERα levels in tumor xenografts and suppressed the growth of ERα-positive breast tumors in mice. Mechanistically, it preferentially recruits X-linked IAP (XIAP) rather than cellular IAP1, to degrade ERα via the ubiquitin-proteasome pathway. With this IAP ligand, potent SNIPERs against other pathogenic proteins, BCR-ABL, bromodomain-containing protein 4 (BRD4), and phosphodiesterase-4 (PDE4) could also be developed. These results indicate that forced ubiquitylation by SNIPERs is a useful method to achieve efficient protein knockdown with potential therapeutic activities and could also be applied to study the role of ubiquitylation in many cellular processes.

Pharmacological inhibitors of oncogenic kinases, such as imatinib and crizotinib, have demonstrated remarkable therapeutic activities against malignant cells expressing the respective target proteins (1). However, there are many pathogenic proteins without enzymatic activity to which pharmacological inhibitors can hardly be developed (2,3). An alternative approach is to down-regulate the expression of the pathogenic proteins, which is usually achieved in vitro by genetic methods using oligonucleotides, such as antisense DNA and doublestranded RNA. Oligonucleotides, however, are scarcely incorporated into cells without transfection reagents. When systemically administered in vivo, they are mostly taken up by hepatocytes, and the delivery to the desired target tissues cannot be easily achieved, which complicates the clinical application of this technology (4,5).
As a new method to down-regulate pathogenic proteins in a nongenetic manner, we and others have devised a protein knockdown system to induce selective degradation of target proteins by using small molecules, called Proteolysis Targeting Chimeras (PROTACs) 3 and Specific and Nongenetic IAP-dependent Protein Erasers (SNIPERs), which possess sufficient membrane permeability (6,7). These compounds are chimeric molecules containing two different ligands connected by a linker; one is a ligand for an E3 ubiquitin ligase and the other is for the target protein, which are designed to cross-link these proteins to induce polyubiquitylation and proteasomal degradation of the target proteins in the cells. To recruit von Hippel-Lindau (VHL) E3 ligase complex and cereblon (CRBN) E3 ligase complex, a VHL inhibitor based on the HIF-1␣ peptide and a phthalimide moiety were respectively conjugated in PROTACs (8 -10), whereas bestatin was incorporated into SNIPERs to recruit cellular inhibitor of apoptosis protein 1 (cIAP1) E3 ligase (11).
To date, several SNIPER compounds have been developed targeting cellular retinoic acid-binding proteins (CRABPs), nuclear receptors such as estrogen receptor ␣ (ER␣), and spindle regulatory protein-transforming acidic coiled-coil-3 (TACC3), which specifically down-regulate the respective target proteins (12)(13)(14)(15). These SNIPERs contain bestatin as a ligand for cIAP1, which possesses modest binding affinity but induces autoubiquitylation and proteasomal degradation of cIAP1 E3 ligase (16), which may limit the protein knockdown efficacy. To demonstrate a proof of concept of this technology in vivo, SNIPERs with more potent activity are required.
IAPs are a family of antiapoptotic proteins containing one to three baculoviral IAP repeat (BIR) domains (17)(18)(19). Some family members, such as cIAP1, cIAP2, and X chromosome-linked IAP (XIAP), directly interact with and regulate caspases, executioners of apoptosis, via the BIR domain (20 -23). These IAPs are attractive targets for tumor therapy because IAPs are overexpressed in multiple human malignancies and implicated in promoting tumor progression, treatment failure, and poor prognosis (24 -26). SMAC/DIABLO is an endogenous inhibitory protein of the IAPs, which binds to the proteins via its N-terminal IAP-binding motif. Based on the IAP-binding tetrapeptides of SMAC, many potent and cell-permeable peptidomimetic IAP antagonists (also known as SMAC mimetics) have been developed, some of which are under evaluation in clinical phase studies as antitumor drugs (17,27). These IAP antagonists interact with the BIR domains of IAP proteins to induce autoubiquitylation and proteasomal degradation of IAPs (28 -30). Because these IAP antagonists show higher affinity to IAPs than bestatin, we reasoned that novel SNIPERs with potent protein knockdown activity could be developed by incorporating IAP antagonists into SNIPERs.
Here, we demonstrated that the incorporation of an LCL161 derivative as an IAP ligand into SNIPERs allowed us to develop potent SNIPERs against several target proteins. We also showed in vivo protein knockdown of ER␣ and growth inhibition of ER␣-positive human breast tumors in a xenograft model by SNIPER(ER).

Development of Potent SNIPER(ER)s by Incorporation of IAP
Antagonists-We previously developed a SNIPER(ER) by conjugating bestatin to 4-hydroxytamoxifen (4-OHT) (SNIP-ER(ER)-14 in this paper), which induced proteasomal degradation of ER␣ mediated by cIAP1 in MCF-7 breast tumor cells at 10 M (14). To improve the protein knockdown activity, we replaced the bestatin moiety with an IAP antagonist, MV1, which shows higher affinity to IAPs than bestatin; the resulting SNIPER(ER)-19 reduced the ER␣ protein at 30 nM (Fig. 1, A  and B). Then we synthesized a series of SNIPER(ER)s containing different ER ligands or different linker lengths, and SNIPER(ER)-20 showed the most potent activity among them (Fig. 1, A-C).
To further improve the activity, we incorporated several IAP antagonists and found that SNIPER(ER)-87 with an LCL161 derivative reduces the ER␣ level more potently than SNIP-ER(ER)-20 (Fig. 2, A and B). The effective knockdown (50% reduction) of ER␣ by SNIPER(ER)-87 was observed at a concentration as low as 3 nM, and maximum activity was observed at around 100 nM. SNIPER(ER)-87 and -88, the latter of which has a longer linker, rapidly reduced the ER␣ protein within 1 h after treatment, and the reduction was sustained for 48 h (Fig. 2C). SNIPER(ER)-89 with a shorter linker than SNIPER(ER)-87 showed attenuated knockdown activity (Fig. 2D). Combination treatment with the ER ligand (4-OHT) and the IAP antagonist (LCL161 derivative) did not decrease the ER␣ protein, indicating that linking the two ligands into a single molecule is critical for protein knockdown (Fig. 2, C and D). SNIPER(ER)-87 also showed protein knockdown activity in other human breast tumor T47D and ZR75-1 cells (Fig. 2E). In addition to ER␣ protein, SNIPER(ER)-87 effectively reduced the level of cIAP1, but only slightly reduced that of XIAP (Fig. 2B), indicating that SNIPER(ER)-87 simultaneously activates autoubiquitylation and proteasomal degradation of cIAP1 as observed with other SNIPERs and IAP antagonists (11-17, 28 -30).

SNIPER(ER)-87 Specifically Induces Degradation of the ER␣ Protein by the Ubiquitin-Proteasome System (UPS)-
To explore the mechanism of SNIPER(ER)-87-induced reduction of the ER␣ protein, we first examined the effect of UPS inhibitors. The decrease in the ER␣ protein by SNIPER(ER)-87 detected by Western blotting and immunocytochemical analysis was abrogated by the proteasome inhibitor MG132 (Fig. 3, A and B). Similar results were obtained by treatment with another proteasome inhibitor, bortezomib, and a ubiquitin-activating enzyme inhibitor, MLN7243, indicating that SNIPER(ER)-87 induces UPS-dependent degradation of the ER␣ protein (Fig.  3C). MLN4924, an inhibitor of NEDD8-activating enzyme, did not affect the activity, suggesting that cullin-based ubiquitin ligases are not involved in the ER␣ degradation (Fig. 3C). Next, we performed the ubiquitylation assay. MCF-7 cells were transfected with an expression vector of HA-tagged ubiquitin and then treated with SNIPER(ER)-87 or control compounds in the presence of MG132. The cell lysates were immunoprecipitated with anti-HA (ubiquitin) antibody, and the precipitates were analyzed by Western blotting with an anti-ER␣ antibody to detect ubiquitylated ER␣ protein. SNIPER(ER)-87, but not the LCL161 derivative with 4-OHT, greatly induced the polyubiquitylation of ER␣ protein, as did fulvestrant, a clinically approved ER␣ degrader (Fig. 3D). Thus, SNIP-ER(ER)-87 induces polyubiquitylation of the ER␣ protein in cells.
To understand the selectivity to the target protein, the effect of SNIPER(ER)-87 on the levels of various proteins was examined. SNIPER(ER)-87 induced degradation of the ER␣ protein, but not short-lived proteins, and the proteins degraded in a cell cycle-dependent manner, whereas a protein synthesis inhibitor, cycloheximide, rapidly reduced these proteins (Fig. 3E). In addition, SNIPER(ER)-87 did not degrade other nuclear recep-  tors and the proteins targeted by different SNIPERs. This indicates that SNIPER(ER)-87 selectively degrades ER␣ and cIAP1.
XIAP Is Required for ER␣ Degradation by SNIPER(ER)-87-SNIPER(ER)-87 shows binding affinity to cIAP1, cIAP2, and XIAP, which reflects the ability of LCL161 to bind these IAPs. To determine which IAP is recruited to ER␣ by SNIPER(ER)-87, we carried out a coprecipitation assay. We focused on XIAP and cIAP1, because cIAP2 is not expressed in MCF7 cells. Cells were treated with or without SNIPER(ER)-87 in the presence of MG132; the cell lysates were immunoprecipitated with anti-ER␣ antibody, and the precipitates were analyzed by Western blotting to detect the IAPs (Fig. 4A). XIAP and cIAP1 were coprecipitated only when cells had been treated with SNIP-ER(ER)-87, indicating that these IAPs interact with ER␣ in a manner dependent on SNIPER(ER)-87. Compared with the IAP protein levels in total lysates, XIAP was more efficiently recruited to ER␣ than was cIAP1. Depletion of XIAP by siRNA greatly increased the SNIPER(ER)-mediated interaction between ER␣ and cIAP1, whereas that of cIAP1 did not affect the interaction of ER␣ with XIAP (Fig. 4B). These results indicate that SNIPER(ER)-87 preferentially recruits XIAP to ER␣. The SNIPER(ER)-mediated recruitment of XIAP to ER␣ was confirmed by reciprocal precipitation with anti-XIAP antibody (Fig. 4C). In line with this, depletion of XIAP significantly suppressed the SNIPER(ER)-87-induced degradation of the ER␣ protein, whereas depletion of cIAP1 minimally suppressed it (Fig. 5A). As a control experiment, ER␣ degradation induced by fulvestrant and ␤-estradiol was similarly analyzed, but depletion of XIAP did not abrogate the ER␣ degradation induced by these agents (Fig. 5B). These results indicate that SNIPER(ER)-87 preferentially cross-links ER␣ with XIAP, and XIAP is the primary E3 ligase responsible for the ER␣ degradation, which is mechanistically distinct from ER␣ degradation induced by fulvestrant and ␤-estradiol.
We next studied the role of the RING domain of XIAP. The ER␣ degradation by SNIPER(ER)-87 was suppressed by siRNA-mediated depletion of XIAP as above. When siRNAresistant wild-type XIAP (WT) was added back to the cells by infecting them with a lentiviral expression vector, the SNIP-ER(ER)-87-induced ER␣ degradation was restored (Fig. 5C). However, adding back XIAP RING mutants (⌬Ring and H467A) did not restore the degradation activity. In addition, overexpression of XIAP ⌬Ring inhibited ER␣ degradation by SNIPER(ER)-87 (Fig. 5D). These results suggest that the XIAP E3 ligase activity associated with the RING domain is required for the SNIPER(ER)-87-induced ER␣ degradation.
The involvement of IAPs in the SNIPER(ER)-87-induced ER␣ degradation was further studied by an approach based on chemical biology. SNIPER(ER)-143, in which an N-methylated analog of the LCL161 derivative was conjugated, showed binding affinity to ER␣ comparable with that of SNIPER(ER)-87 but lost the ability to bind IAP proteins ( Figs. 2A and 6A). Consequently, SNIPER(ER)-143 did not cross-link ER␣ with XIAP and cIAP1 in MCF-7 cells (Fig. 6B) nor induce the degradation of ER␣ (Fig. 6C). These findings indicate that the IAP binding ability is critical for the ER␣ knockdown activity of SNIPER(ER)-87.
In Vivo Protein Knockdown by SNIPER(ER)-87-To evaluate the knockdown activity of SNIPER(ER)-87 in vivo, we first measured the level of ER␣ in ovary. When female BALB/c mice were intraperitoneally injected with SNIPER(ER)-87 (10 or 30 mg/kg body weight), the ER␣ protein levels in ovary were significantly reduced (Fig. 7A). Then, to explore the in vivo protein knockdown in a tumor model, we next developed MCF-7 breast tumor xenografts in nude mice. Again, SNIPER(ER)-87 reduced the ER␣ protein level in ovary (Fig. 7B). In addition, the ER␣ protein levels in the orthotopic tumors were significantly reduced to ϳ50% in SNIPER(ER)-87-treated mice compared with those in vehicle-treated mice (Fig. 7C). Meanwhile, administration of inactive SNIPER(ER)-143 did not affect the ER␣ levels in tumors (Fig. 7D). Thus, SNIPER(ER)-87 shows in vivo protein knockdown activity in tumors.

SNIPER(ER)-87 Inhibits Estrogen
Signaling and Estrogen-dependent Tumor Growth-Because ER␣ plays an essential role in estrogen signaling and the growth of certain ER␣-positive breast tumor cells (31,32), we next examined the effect of SNIP-ER(ER)-87 on estrogen-dependent gene expression and tumor growth. In luciferase assays with an estrogen-response element reporter, SNIPER(ER)-87 effectively inhibited the ER␣-dependent transcriptional activation by ␤-estradiol (Fig. 8A), which is in good agreement with the ER␣ knockdown activity (Fig. 2B). In addition, SNIPER(ER)-87 efficiently suppressed the growth of ER␣-positive breast tumor cells (IC 50 values were 15.6 nM in MCF-7 and 9.6 nM in T47D), but not that of ER␣-negative breast tumor cells (MDA-MB-231) (Fig. 8B), which is consistent with the results of cell cycle distribution analyzed by flow cytometry (Fig. 8C). Thus, SNIP-ER(ER)-87 shows activities to inhibit estrogen signaling and growth of ER␣-positive tumor cells. To demonstrate the therapeutic significance of these findings, we also evaluated the in vivo antitumor activity of SNIPER(ER)-87 in an MCF-7 tumor xenograft mouse model. As pharmacokinetic studies indicated that intraperitoneally administered SNIPER(ER)-87 was eliminated in 24 h (Fig. 8, D and E), tumor-bearing mice were treated daily with SNI-PER(ER)-87 by intraperitoneal injection (30 mg/kg body weight) for 14 days. Treatment with SNIPER(ER)-87 attenuated tumor progression as assessed by measuring tumor volume (Fig. 8F). The inhibition of tumor cell proliferation by SNIPER(ER)-87 was confirmed by immunohistochemistry stained with S phase-related proliferating cell nuclear antigen (PCNA) (Fig. 8G). Notably, no obvious toxicities, including body weight changes, were observed throughout the 2 weeks of administration of SNIPER(ER)-87 (Fig.  8H). These results imply the potential utility of SNIPER(ER)-87 in the treatment of ER␣-positive breast tumors.  MARCH 17, 2017 • VOLUME 292 • NUMBER 11

Development of LCL161-based SNIPERs against Different
Target Proteins-To investigate the utility of the LCL161 derivative in the development of SNIPERs, we conjugated the LCL161 derivative to dasatinib, JQ-1, and a PDE4 inhibitor, ligands for BCR-ABL, BRD4, and PDE4 proteins, respectively (Fig. 9A). These LCL161-based SNIPERs showed efficient protein knockdown activities against target proteins at nanomolar concentrations (Fig. 9, B and C), which were suppressed by MG132 and MLN7243 (Fig. 9D). These results suggest that the LCL161 derivative is a useful IAP ligand in SNIPER to degrade a variety of target proteins.

Discussion
In this study, we incorporated an LCL161 derivative with the capacity to bind with high affinity to cIAP1, cIAP2, and XIAP into SNIPERs, and we successfully developed potent SNIPERs against ER␣, PDE4, BRD4, and BCR-ABL, which induce the degradation of respective target proteins via UPS in a highly specific manner. SNIPER(ER)-87 showed activity of reducing the ER␣ protein at nanomolar concentrations in an in vitro cell culture system and also in tumor xenografts in vivo. Analysis of the mechanism involved in this revealed that SNIPER(ER)-87 preferentially recruits XIAP to ER␣ in the cells, and XIAP, but not cIAP1, is the primary E3 ubiquitin ligase responsible for the SNIPER(ER)-87-induced ER␣ degradation. It is not clear why SNIPER(ER)-87 preferentially recruits XIAP as compared with cIAP1, despite its binding affinity to cIAP1 being 10-fold higher than that to XIAP. It is likely that the relative amounts and subcellular localization of ER␣ and IAPs or the latency of the IAP-BIR3 domains to which the LCL161 moiety binds could influence the SNIPER(ER)-87-induced recruitment of IAPs to ER␣ in the cells.
SNIPER(ER)-87 shows fairly good metabolic stability in serum. When SNIPER(ER)-87 was administered to mice intraperitoneally, a significant concentration was retained in the blood (Fig. 8, D and E), and ER␣ proteins in tumor xenografts and ovary were effectively reduced. In addition, daily adminis-tration of SNIPER(ER)-87 inhibited the growth of ER␣-positive human breast tumor xenografts in mice. Similar in vivo protein knockdown and potential therapeutic activities were recently reported with PROTACs and dBET1 targeting BRD4 protein by recruiting VHL and CRBN E3 ligases, respectively (10,33). These observations imply the potential utility of the protein knockdown technology to clinical applications. Because these E3 ligases, including cIAP1 and XIAP, are ubiquitously expressed in various types of cell, targeted degradation of proteins with this class of molecule could be applicable in a variety of cells. It should be noted that IAPs are frequently overexpressed in tumor cells, which is involved in resistance to tumor therapy (17,34). Therefore, the ability of SNIPERs to degrade cIAP1, and XIAP to some extent, simultaneously with the target proteins suggests that they could be particularly advantageous in killing tumor cells.
In addition to SNIPER(ER)s against ER␣ protein, we also developed potent SNIPERs targeting BRD4, PDE4, and BCR-ABL proteins by incorporating the LCL161 derivative as an E3 ligand. These SNIPERs effectively degrade respective target proteins, indicating the utility of the LCL161 derivative for the development of various SNIPERs. The protein knockdown efficacy by most SNIPER molecules, as well as that by PROTACs, was suppressed at higher concentrations (Figs. 1B, 2B, and 9B) (8, 10), which is known as a hook effect. This effect is explained by the inhibition of ternary complex formation (E3-SNIPERtarget) by an excess amount of bivalent compounds such as SNIPERs and PROTACs.
With respect to the PROTACs against BCR-ABL protein, CRBN-based but not VHL-based PROTACs can degrade BCR-ABL protein (35), suggesting that an appropriate E3 ligase should be recruited to the target proteins. It is likely that correct exposure of the lysine residues on the surface of target proteins to an appropriate E3 ligase is critically important for the efficient ubiquitylation and subsequent degradation of the target proteins. Therefore, for each target, finding the most appropriate E3 ligase recruited to the target proteins by PROTACs or SNIPERs might be important for the maximum protein knockdown activity.
Although we focused on the proteasomal degradation of target proteins by SNIPER-mediated forced ubiquitylation in this study, ubiquitylation plays a role in a variety of cellular processes, such as autophagy (36), DNA repair (37), generation of peroxisome (38), and sorting membrane proteins in recycling endosomes (39). Because of its modular structure, SNIPER-mediated forced ubiquitylation can be applied to other target proteins to study the role of ubiquitylation in such cellular processes. In addition, endogenous proteins can be ubiquitylated by SNIPERs without involving genetic engineering, which implies a potential utility of SNIPERs in drug development that induces a particular cellular response, not limited to proteasomal degradation, by forced ubiquitylation.

Chemistry
The chemical synthesis and physicochemical data on SNIPER compounds are provided in the supplemental material.  MARCH

Biology
Cell Culture-Human breast carcinoma MCF-7, T47D, and ZR-75-1 cells were maintained in RPMI 1640 medium containing 10% fetal bovine serum (FBS) and 100 g/ml kanamycin with or without 10 g/ml insulin (Sigma). Human myeloid leukemia K562 and human prostate carcinoma LNCaP cells were maintained in RPMI 1640 medium containing 10% FBS and 100 g/ml kanamycin. Human fibrosarcoma HT1080 cells were maintained in Dulbecco's modified Eagle's medium containing 10% FBS and 100 g/ml kanamycin. Cells were treated with various concentrations of the compounds for the indicated times.
Immunostaining-MCF-7 cells were treated with the indicated compounds in combination with 10 M MG132 for 3 h. They were then fixed in 100% methanol on ice for 10 min, washed four times with PBS, and blocked in PBS containing 3% BSA and 0.1% Triton X-100 (PBS-TB) for 1 h at room temperature. Next, the cells were incubated for 2 h with anti-ER␣ rabbit mAb (Cell Signaling, 8644) or anti-␣-tubulin mouse mAb (Sigma, T-5168) as the primary antibodies, and for 1 h with Alexa Fluor 488-conjugated anti-rabbit IgG or Alexa Fluor 568conjugated anti-mouse IgG (Life Technologies, Inc.) as the secondary antibodies with Hoechst 33342 (Life Technologies, Inc.). Fluorescent images were obtained using a BZ-9000 (Keyence, Osaka, Japan).
Immunoprecipitation-MCF-7 cells were treated with the indicated concentrations of the indicated compounds in combination with 10 M MG132 for 3 h. Cells were lysed using IP lysis buffer (10 mM HEPES, pH 7.4, 142.5 mM KCl, 5 mM MgCl 2 , 1 mM EGTA, and 0.1% Triton X-100), containing protease inhibitor mixtures, rotated for 15 min at 4°C, and centrifuged at 15,000 rpm for 10 min at 4°C to obtain the supernatants. The lysates that had been precleared with naked protein G-Sepharose were immunoprecipitated with protein G-Sepharose beads preincubated anti-ER␣ rabbit pAb (Santa Cruz Biotechnology, sc-543) or anti-XIAP mouse mAb (MBL, Nagoya, Japan; catalog no. M044-3) for 2 h at 4°C. The precipitates were washed with IP lysis buffer four times and analyzed by Western blotting.
Ubiquitylation Assay-MCF-7 cells were transfected with pcDNA3-HA-ubiquitin for 24 h. The cells were then incubated with the indicated compounds in the presence of MG132 (10 M) for 3 h before being harvested and lysed in SDS lysis buffer. The cell lysates were boiled for 10 min, diluted 10 times with 0.1 M Tris-HCl, pH 7.5, and immunoprecipitated with anti-HA agarose-conjugated beads (Sigma, catalog no. E6779). The precipitates were extensively washed and analyzed by Western blotting using anti-ER␣ antibody (Cell Signaling Technology, catalog no. 8644).
Luciferase Assay-MCF-7 cells were transfected with firefly luciferase reporter plasmid containing three tandem copies of estrogen-response element and control Renilla luciferase plasmid-SV40 using Lipofectamine LTX (Life Technologies, Inc.) in phenol red-free medium containing 4% charcoal/dextrantreated FBS. After 24 h, cells were treated with the indicated concentrations of SNIPER(ER)-87 in the presence or absence of 0.1 nM ␤-estradiol in phenol red-free medium containing 0.2% charcoal/dextran-treated FBS for 24 h. The firefly luciferase activity in cell lysates was measured and normalized with Renilla luciferase activity. The data represent means Ϯ S.D. (n ϭ 3).
Cell Viability Assay-Cell viability was evaluated by crystal violet staining. Cells were treated with graded concentrations of the compounds for 72 h, and then stained with 0.1% crystal violet (Wako, Osaka, Japan) in 1% ethanol for 15 min at room temperature. The cells were rinsed thoroughly with distilled water and then lysed in 1% SDS. The absorbance of cell lysate at 600 nm was measured using EnVision Multilabel Plate Reader (PerkinElmer Life Sciences).
In Vivo Protein Knockdown-Mice were housed in pathogenfree animal facilities with 12-h light/dark cycles and were fed rodent chow and water ad libitum at the National Institute of Health Sciences. All experiments were conducted in accordance with the guidelines approved by the National Institute of Health Sciences. For Fig. 7A, female 6-week-old BALB/c mice (Clea Japan, Tokyo, Japan) were randomized and divided into six treatment groups as follows: 1) vehicle treatment for 6 h (n ϭ 3); 2) 10 mg/kg SNIPER(ER)-087 treatment for 6 h (n ϭ 2); 3) 30 mg/kg SNIPER(ER)-087 treatment for 6 h (n ϭ 2); 4) vehicle treatment for 24 h (n ϭ 4); 5) 10 mg/kg SNIPER(ER)-087 treatment for 24 h (n ϭ 3); and 6) 30 mg/kg SNIPER(ER)-087 treatment for 24 h (n ϭ 3). For Fig. 7, B-D, each suspension of 1 ϫ 10 7 MCF-7 cells was mixed with an equal volume of Matrigel (Corning Life Sciences) and injected (100 l total) into the left and right mammary fat pads of 6-week-old female BALB/c nude mice (Clea Japan). After cell inoculation, ␤-estradiol solution was subcutaneously injected into the neck twice at intervals of 6 days. Fourteen days after the last ␤-estradiol injection, tumor-bearing mice were randomized and divided into two or three treatment groups as follows: for Fig. 7, B and C: 1) vehicle treatment for 24 h (n ϭ 5); and 2) 30 mg/kg SNIPER(ER)-087 treatment for 24 h (n ϭ 5); for Fig. 7D: 1) vehicle treatment for 24 h (n ϭ 4); 2) 30 mg/kg SNIPER(ER)-087 treatment for 24 h (n ϭ 4); and 3) 30 mg/kg SNIPER(ER)-143 treatment for 24 h (n ϭ 4). Compounds were administered via intraperitoneal injection. After the indicated times, the mice were sacrificed, and tissues were excised. Total lysates from the ovaries and tumors were analyzed by Western blotting.
In Vivo Tumor Growth Inhibition-Each suspension of 1 ϫ 10 7 MCF-7 cells was mixed with an equal volume of Matrigel (BD Biosciences) and inoculated (100 l total) into the left and right mammary fat pads of 6-week-old female BALB/c nude mice (Clea Japan) that had received an ␤-estradiol pellet (6 g per day) (Innovative Research of America, Sarasota, FL) under the neck skin. After 4 days, mice bearing ϳ100-mm 3 tumors were randomized and divided into two groups (n ϭ 9). One group served as a control for dosing vehicle, whereas the other group was administered SNIPER(ER)-087 (30 mg/kg, intraperitoneally, every 24 h). Tumor volumes were measured every 2 days using a caliper and calculated according to the standard formula: (length ϫ width 2 )/2. At 2 weeks, mice were sacrificed, and the tumors were excised.
Immunohistochemistry-Tumor tissues were fixed in 10% buffered formalin (Wako, Osaka, Japan) and embedded in paraffin, and 6-m sections were prepared. Sections were deparaffinized and stained with hematoxylin and eosin (40). To detect PCNA, antigens were retrieved, and colorimetric detection was performed with anti-PCNA rabbit mAb (Cell Signaling Technology, catalog no. 13110).
Cell Cycle Analysis-After treatment, cells were gently trypsinized and washed with serum-containing medium. Cells were collected by centrifugation, additionally washed with PBS, and fixed in 70% ice-cold ethanol for 1 h on ice. The cells were then washed, treated with 1 mg/ml RNase A for 1 h at 37°C, and stained in propidium iodide solution (50 g/ml in 0.1% sodium citrate, 0.1% Nonidet P-40). The stained cells were analyzed in a FACScan flow cytometer (BD Biosciences).  MARCH 17, 2017 • VOLUME 292 • NUMBER 11

JOURNAL OF BIOLOGICAL CHEMISTRY 4567
Statistical Analysis-Student's t test was used to determine the significance of differences among the experimental groups. Values of p Ͻ 0.05 were considered significant.
Measurement of Binding Affinity-Binding experiments were performed in white 384-well plates (PerkinElmer Life Sciences, catalog no. 6007290). A total of 5 l of His-tagged IAP proteins (40 nM XIAP_BIR3, 15 nM cIAP1_BIR3, and 35 nM cIAP2_BIR3) and 5 l of increasing concentrations of compounds were added to wells in the assay buffer (20 mM HEPES, 150 mM NaCl, 0.1% BSA, 0.01% Tween 20, 0.1 mM DTT, pH 7.5). After shaking at room temperature, 5 l of biotinyl-SMAC (20 nM XIAP_ BIR3, 40 nM cIAP1_BIR3, and 120 nM cIAP2_BIR3 dissolved in assay buffer) was added to the well, followed by the addition of 5 l of a mixture of anti-His 6 cryptate and SA-XLent, and 160ϫ dilution with HTRF detection buffer. After overnight incubation at room temperature in the dark, HTRF measurement was carried out on a multilabel reader (EnVision, PerkinElmer Life Sciences) with the following settings: measurement mode, time-resolved fluorescence; excitation, 320 nm; emission donor, 615 nm; and emission acceptor, 665 nm.
Fluorescence at 615 nm (F 615 nm ) represents the total europium cryptate signal, and fluorescence at 665 nm (F 665 nm ) represents the FRET signal. The ratio ϭ (F 665 nm /F 615 nm ) ϫ 10,000 was calculated, and IC 50 values were determined using this ratio by nonlinear regression curve fitting with the program XLfit.
The binding between test compounds and the human ER␣ protein was determined using the PolarScreen TM Estrogen Receptor-␣ Competitor Assay Green (Thermo Fisher Scientific, catalog no. A15882), containing recombinant ER␣ fulllength protein, Fluormone ES2 Green2, and ES2 screening buffer. Purified ER␣ and Fluormone ES2 were diluted with assay buffer to final concentrations of 25 and 4.5 nM, respectively, and 4 l of the dilution was added to each well of a 384-well black low-volume assay plate (Greiner catalog no. 784076). Then, 2 l of ES2 screening buffer containing test compounds or DMSO was added to the well. The plate was subjected to centrifugation and incubated at room temperature for 1 h, and the intensity of the fluorescence polarization signal was measured by a plate reader (Envision, PerkinElmer Life Sciences). The wells containing ER␣ and Fluormone ES2 were used as a positive control, and the wells containing only Fluormone ES2 were used as a negative control. IC 50 values were calculated by XLfit (ID Business Solutions, fit model 204) from the data expressed as % control inhibition.
Pharmacokinetics-Female BALB/c mice were purchased from Charles River Japan, maintained with 12-h light/dark cycles, and allowed free access to a chow diet (CE-2; Clea Japan) and drinking water. Animal study was conducted with a 7-day acclimation period after arrival. SNIPER(ER)-87 was dissolved in 10% DMSO, 10% Cremophor EL, 20% PEG 400, 60% distilled water, and mice were injected with 10 mg/kg SNIPER(ER)-87 intraperitoneally. After 0.5, 1, 2, 4, 8, and 24 h, blood was obtained in a tube with heparin under isoflurane-induced anesthesia and centrifuged at 12,000 rpm for 5 min to collect the plasma for measurement of compound concentrations. The care and use of the animals and the experimental protocols used in this research were approved by the Experimental Animal Care and Use Committee of Takeda Pharmaceutical Co., Ltd.