Development of stabilized peptide-based PROTACs against estrogen

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Development of stabilized peptide-based PROTACs against estrogen receptor # Jiang Yanhong, Qiwen Deng, Hui Zhao, Mingsheng Xie, Longjian Chen, Feng Yin, Xuan Qin, Weihao Zheng, Yongjuan Zhao, and Zigang Li ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.7b00985 • Publication Date (Web): 22 Dec 2017 Downloaded from http://pubs.acs.org on December 23, 2017

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ACS Chemical Biology

Development of stabilized peptide-based PROTACs against estrogen receptor α Yanhong Jiang,

†,#

Qiwen Deng,†,# Hui Zhao,‡ Mingsheng Xie,† Longjian Chen,† Feng Yin,† Xuan Qin,† Weihao ,†

Zheng,† Yongjuan Zhao,* and Zigang Li*

,†

†Key Lab of Chemical Genomics, School of Chemical Biology and Biotechnology, Shenzhen Graduate School, Peking University, Shenzhen, 518055 (China) ‡ Division of Life Sciences, Clarivate Analytics, Beijing, 100190 (China) #

Co-first author

*Correspondence: [email protected] (Z.L.), [email protected] (Y.Z.)

ABSTRACT Peptide modulators targeting protein-protein interactions (PPIs) exhibit greater potential than small-molecule drugs in several important aspects including facile modification and relative large contact surface area. Stabilized peptides constructed by variable chemistry methods exhibit improved peptide stability and cell permeability than the linears. Herein, we designed a stabilized peptide-based proteolysis-targeting chimera (PROTAC) targeting estrogen receptor α (ERα) by tethering an N-terminal aspartic acid cross-linked stabilized peptide ERα modulator (TD-PERM) with a pentapeptide that binds the Von Hippel-Lindau (VHL) E3 ubiquitin ligase complex. The resulting heterobifunctional peptide (TD-PROTAC) selectively recruits ERα to the VHL E3 ligase complex, leading to the degradation of ERα in a proteasome-dependent manner. Compared with the control peptides, TD-PROTAC shows significantly enhanced activities in reducing the transcription of the ERα-downstream genes and inhibiting the proliferation of ERα-positive breast cancer cells. In addition, in vivo experiments indicate that TD-PROTAC leads to tumor regression in the MCF-7 mouse xenograft model. This work is a successful attempt to construct PROTACs based on cell-permeable stabilized peptides, which significantly broadens the chemical space of PROTACs and stabilized peptides. INTRODUCTION Since its initial design by Sakamoto et al in 2001, proteolysis-targeting chimera (PROTAC) technology has been successfully applied in the development of heterobifunctional molecules that can prompt target protein degradation.1.2 Such heterobifunctional molecules generally consist of one end that recruits the E3 ligase complex and one end that engages the target protein. Upon complex formation, the recruited E3 ligase 1

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complex ubiquitinates the target protein, leading to the subsequent degradation of the target protein in a proteasome-dependent manner. PROTAC technology has the potential to target a broader range of proteins than standard small-molecule strategies that focus on binding site occupancy and can catalytically degrade 3

target the target protein. This technology has been successfully utilized to develop peptide-based and small molecule-based PROTACs and to selectively degrade various protein targets, including estrogen receptor α4-8, estrogen-related receptor α9, cellular retinoic acid-binding proteins10, and BET proteins11-13. So far, PROTACs are mainly based on small molecules, while peptide-based PROTACs are much less explored, probably due to the poor physiochemical properties of the unmodified peptides, such as low intracellular stability and poor cell permeability.4,14 However, peptide modulators exhibit greater potential than small-molecule drugs in several important aspects. Peptide modulators could significantly facilitate the modification of drugs and target protein-protein interactions (PPIs), which are difficult to accomplish via small molecules. In addition, they have significant advantages in binding mutated drug targets via distinct epitopes. 15.16

Thus, exploring peptide-based PROTACs could tremendously extend this technology’s chemical space

and provide new possibilities for the development of peptide-based therapeutics. However, short peptides have inherent drawbacks, as mentioned above. For the past two decades, scientists have applied different methods, 17-22

including side chain crosslinking

, nucleation

23-26

and the incorporation of β or γ amino acids

27,28

, to

constrain peptides in a desired conformation. These constrained peptides exhibit improved peptide stability and cell permeability and have been applied in a host of biological systems.29.30 ERα is a key member of the nuclear receptor ER protein family, which controls a variety of physiological and developmental processes.31 ERα is often overexpressed in breast cancer cells and promotes their estrogen-dependent proliferation32, which makes it a good drug target for breast cancer treatment. The traditional approach to inhibiting transcriptional activity of ERα is generally based on modulating the conformational states of ERα with various unnatural ligands. One of the main drawbacks of this classic strategy is drug resistance.33-35 PROTAC technology, as a strategy to directly degrade the target protein, can potentially solve this problem, and several PROTAC molecules targeting ERα have been reported.4-8 However, the reported peptide-based PROTACs have limited cell penetrating ability: additional cell-penetrating peptides are required to achieve more intracellular function. Recently, we have developed a facile N-terminal aspartic acid crosslinking strategy (TD strategy) to constrain peptides into a helical structure with good stability and cell permeability.

26

Peptidomimetic estrogen

receptor modulators (PERMs) constructed using this strategy (TD-PERMs) showed improved therapeutic 2


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properties in targeting ERα. However, TD-PERMs, which show no obvious antiproliferative effects at concentrations below 40 µM in T47D cells, has limited biological abilities. To enhance the biological activity of TD-PERMs, we applied PROTAC technology to develop stabilized peptides targeting ERα because molecules based this technology can behave catalytically in their ability to degrade proteins. We proposed that PROTACs conjugating TD-PERMs with the recruiting peptide of the Von Hippel-Lindau (VHL) E3 ligase complex might effectively target and degrade ERα and selectively prompt the apoptosis of ERα-positive cancer cells. RESULTS Design and Evaluation of Stabilized Peptide-based PROTACs Stabilized peptide-based PROTACs were constructed from three components: the stabilized peptide (TD-PERM), which can bind with ERα; the linker; and the hydroxyproline-containing pentapeptide (HIF) from hypoxia-inducible factor-1α, which can be recognized by the VHL E3 ubiquitin ligase. First, we designed three peptides with different lengths of linker, that is TD-PROTAC, TD-PROTAC-1 and TD-PROTAC-2 (see in as Table S1). The 6-aminohexanoic acid linker stood out as optimal with respect to cell viability in the ERα-positive breast cancer cell line MCF-7 (Figure S1). The peptide using this linker was named TD-PROTAC. The peptides TD-PERM, HIF, TD-PROTACsc (produced by scrambling the sequence of TD-PERM), TD-PROTACmut (produced by mutating the sequence of HIF) and PROTAClinear (produced by conjugating the HIF sequence 36

with the linear PERM ) were prepared as controls, and their structure was given in Table 1. First, we evaluated the helicity of those peptides using circular dichroism spectroscopy. The helicity of TD-PROTAC was retained (Figure 1A). In addition, the helicity of TD-PROTACsc, TD-PROTACmut constructed by TD strategy was also remained and was better than that of the linear analog PROTAClinear and HIF (Figure 1A). To test the biological activity of these stabilized peptide-based PROTACs, we then evaluated the binding affinity between the fluorescein isothiocyanate (FITC)-labeled peptides and the ERα ligand binding domain using a fluorescence polarization (FP) assay. affinities to

FITC

FITC

TD-PERM21.

TD-PROTAC,

FITC

TD-PROTACmut, and

FITC

PROTAClinear showed similar binding

FITC

TD-PROTACsc and HIF showed no binding affinity to ERα, demonstrating the

binding specificity between PERM and ERα. The positive control

FITC

PERM-2 also shows moderate affinity to

ERα (Figure 1B). 26,36 To investigate the ability of TD-PROTAC to induce ERα degradation, its cell permeabiltiy should be verified. By immunofluorescence assay and flow cytometry analysis, we proved TD-PROTAC showed good cell permeability in the ERα-positive breast cancer cell lines T47D and MCF-7 (Figure 2 and Figure S2). TD-PROTAC Induces ERα Degradation 3



To assess the ability of PROTAC-mediated degradation, we analyzed the protein level of ERα by immunoblotting. We found that TD-PROTAC induced ERα degradation in T47D cells in a dose- and time-dependent manner with a DC50 (the drug concentration that results in 50% protein degradation) lower than 20 µM (Figure 3A and 3B). Obviously, TD-PERM without the E3 ligase-recruiting peptide at the same concentration could not decrease the level of the ERα protein (Figure 3C). The two control peptides TD-PROTACsc and TD-PROTACmut did not show the ability to degrade ERα, confirming that the combination of the ERα- and VHL-binding moieties is necessary and sufficient for degrading ERα (Figure 3C). The control peptides HIF and PROTAClinear likewise could not degrade ERα under the same conditions (Figure 3C). Moreover, TD-PROTAC could also induce the degradation of ERα in MCF-7 cells (Figure S3A). We also evaluated the effect of TD-PROTAC on other nuclear receptors progesterone receptor (PR) and vitamin D receptor (VDR). As shown in Figure S4, TD-RPTOAC showed less effect on PR degradation than ERα and had negligible effects on VDR in T47D cells. As is the case for other ERα-targeting PROTACs, the TD-PROTAC-dependent decrease in ERα involves ubiquitination. In both the T47D and MCF-7 cells, the proteasome inhibitor MG-132 could retard the down-regulation of ERα induced by TD-PROTAC, providing evidence that the degradation is proteasome dependent (Figure 3D and S3B). To further confirm that TD-PROTAC induces the ubiquitination of ERα, which serves as a degradation signal, ERα was immune-precipitated from TD-PROTAC/MG-132-treated T47D cell lysates with an anti-ERα antibody and blotted with both anti-ubiquitin and anti-ERα antibodies. ERα ubiquitination increased in the presence of TD-PROTAC and MG-132 compared with the level in the presence of MG-132 alone (Figure 3D). This difference suggests that TD-PROTAC-induced ERα degradation occurs via the ubiquitin-proteasome pathway. TD-PROTAC Treatment Inhibits Receptor Signaling To further confirm the degradation of ERα and to study the downstream effects of TD-PROTAC treatment, we measured the mRNA level of pS2, a gene transcriptionally regulated by ERα, by quantitative polymerase chain reaction (qPCR) analysis. T47D cells treated with TD-PROTAC showed dramatic down-regulation of pS2 gene expression compared with cells treated with TD-PERM (Figure 4A). All other control peptides showed negligible effects on the regulation of this gene expression. TD-PROTAC Selectively Kills Breast Cancer Cells We next examined the effect of TD-PROTAC on cell proliferation. First, MTT assays were pepromed on breast cancer cells with different peptides at different concentration for 24 hours. TD-PROTAC showed obvious 4


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growth inhibition of both T47D (Figure 4B, IC50 ~ 24 µM) and MCF-7 (Figure S5A, IC50 ~ 30 µM) cells compared with TD-PERM. At the same concentration, the control peptides, TD-PROTACsc, TD-PROTACmut, HIF, and PROTAClinear had minimal effects. Notably, TD-PROTAC showed negligible cytotoxicity toward the ERα-negative breast cancer cell line MDA-MB-231 and the embryonic kidney cell line HEK 293T (Figure S5B), which indicates that the effect is ERα-dependent. Apoptosis assays were then performed to examine the cause of the cytotoxic effect of TD-PROTAC on cancer cells. Annexin V and propidium iodide (PI) were used to stain T47D cells treated with TD-PROTAC at different concentrations for 12 hours. Flow cytometry revealed that more cells showed early apoptosis features after treatment with higher doses of TD-PROTAC (Figure 4C). The effect of TD-PROTAC on the cell cycle of T47D was analyzed by flow cytometry after PI staining. After treatment with TD-PROTAC for 12 hours, more cells were arrested in the S phase (Figure 4D and S5C) in a dose-dependent manner, indicating that TD-PROTAC inhibited proliferation by inducing S-phase arrest. TD-PROTAC Inhibits Breast Tumor Growth and Reduces ERα Levels in vivo To study the anticancer activity of TD-PROTAC in vivo, we performed xenograft studies in nude mice with MCF-7 cells by administering daily intraperitoneal injections of TD-PROTAC (10 mg/kg), tamoxifen (4 mg/kg, as the positive control) or the vehicle control for 42 days. Compared with the vehicle-treated control group, the tumor volumes of the TD-PROTAC group were dramatically reduced by 75% with no significant variation in mouse body weight (Figure 5A and 5B). The histological staining of representative organs, including the heart, liver, spleen, lungs, kidneys and brain from the treated animals, revealed no pathological changes (Figure S6), which further proved that the animals tolerated the dose of TD-PROTAC. To test whether TD-PROTAC can cause ERα down-regulation in the xenografts, the ERα levels of the tumor tissues were measured by immunohistochemistry. As shown in Figure 5C, the ERα levels (brown) in the TD-PROTAC-treated tumor tissues were significantly lower than in the vehicle-treated control group. The tamoxifen-treated group, as the positive control, showed results similar to those of the TD-PROTAC-treated group except in the immunohistochemical assay, which is easily understandable as tamoxifen does not function by degrading ERα (Figure 5). These results indicate that TD-PROTAC has potential as an alternative treatment to tamoxifen. Based on these data, we conclude that TD-PROTAC can significantly inhibit breast tumor growth via reducing the protein levels of ERα. DISCUSSION PROTAC technology has gain series of success in drug development and peptide drugs achieve much 5



attention in pharmaceutical field because of their relative low molecular weights and biocompatibility as we mentioned above. Herein, we applied PROTAC technology to develop peptide modulator targeting ERα and got the peptide name as TD-PROTAC with potentiality as a drug for the breast cancer with ERα highly expressed. We conclude that TD-PROTAC selectively induces the ubiquitination and degradation of ERα by a proteasome-dependent pathway, whereas the control peptides cann't degrade ERα. TD-PROTAC can decrease the transcription of ERα-related genes, and inhibits the proliferation and prompts the apoptosis of ERα-positive cancer cells with negligible cytotoxicity toward ERα-negative cells. In addition, in vivo experiments indicate that TD-PROTAC leads to tumor regression in the MCF-7 mouse xenograft model. Particularly, our peptide can cause ERα down-regulation in the xenografts, whereas tamixifen doesn't have this ability. These results indicate that TD-PROTAC has potential as an alternative treatment to tamoxifen. Successful small molecule-based PROTACs targeting the estradiol binding site, the same binding site as tamoxifen were reported previously by Crews et al.4-6 However, peptide-based PROTACs targeting the ERα-coactivator site only showed limited cellular functions, partly due to their poor cellular uptakes.8 The TD-PROTAC approach utilized a peptide stabilization strategy developed by our own group26 to provide peptide conjugates with satisfying stability and cellular uptake. TD-PROTAC targeted at the ERα-coactivator binding site and showed satisfying cellular and in vivo activities in ERα positive breast cancer cells. Notably, small molecule-based PROTACs and TD-PROTAC targeted at different sites of the same target, which might be developed into therapeutics for breast cancers at different stages. This proof-of-concept study represents a successful application of stabilized peptides in the construction of PROTAC molecule and clearly shows the potential of stabilized peptide-PROTAC hybrids in targeting various PPIs. This strategy will certainly be extended to targets with peptide ligands that have good binding affinity but insufficient cellular functions, significantly increasing the chemical space of both constrained peptides and PROTACs. Further investigations of using the constrained-peptide-PROTAC strategy to target “undruggable” targets are under way in our laboratory and will be reported in due course. METHODS Peptide Synthesis18 Peptide was synthesized on Rink amide MBHA resin by standard Fmoc (9-fluorenylmethyloxycarbonyl) -based solid-phase peptide synthesis. Fmoc deprotection was performed with morpholine (50% in DMF) for 20 minutes twice. Coupling reactions were performed using HCTU and DIPEA for 3 hours with N2 bubbling. The allyl ester and allyl carbamate were removed using Pd(PPh3)4 (0.1 eq) and N, N-dimethylbarbituric acid (4 eq) 6


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in DCM for 2 hours twice. Cyclization was performed on the resin using PyBOP (2eq)/HOBt (2eq)/NMM (2.4 eq) in DMF for 5 hours twice. FITC labeling was performed on resins with the solution of fluorescein isothiocyanate (isomer I, 7 eq) and DIPEA (14 eq) in DMF overnight. Peptides were incorporated a beta-alanine linker before FITC labeling. Final resins were treated with 95% (v/v/v) TFA/TIS/H2O (95:2.5:2.5) for 2 hours. After air removal of most TFA, products were precipitated with cold Et2O, dissolved in CH3CN/H2O. Crude peptides were purified on semi-preparative RP-HPLC and confirmed by LC-MS. Purified peptides were resolved in DMSO for subsequent assays. Concentrations of peptides were determined by 280 nm absorption of -1

tyrosine

-1

with an extinction coefficient of 1490 M cm ; Concentrations of FITC-labeled peptides were determined by 494 nm absorption with an extinction coefficient of 1490 M-1cm-1. (By Nano-Drop ND-2000) 18,23

Circular Dichroism Spectroscopy

CD spectra were acquired using circular dichroism spectrometer (Chirascan-Plus) equipped with a temperature controller using 1 mm cell at a scan speed of 20 nm/sec in room temperature. Peptide samples were dissolved in H2O. Each sample was scanned twice and the averaged spectrum was smoothed. Final concentrations of the peptides were determined by 280 nm absorption of Tyr. Percent helicity was calculated based on the equation: Helicity% = [θ]222/[θ]max. [θ] = (-44000 + 250T) (1 - k/n) (T = 20 ˚C, k = 4 and n = number of amino acid residues in the peptide) 37-39

Fluorescence Polarization Assay

DNA encoding human ERα LBD (301-553) were cloned into pET23b (GE life sciences) and overproduced in E.coli BL21 (DE3). Cultures were grown in 2YT medium at 310 K till an OD600 of 0.8 before being transferred to 293 K for 18 hours. Cells were harvested by centrifugation (6,000 g, 277 K, 15 minutes) and lysed by sonication (273 K) in 100 mM Tris (pH 8.1, 300 mM KCl, 5 mM EDTA, 4 mM DTT and 1 mM PMSF). Cell debris was removed by centrifugation (15,000 g, 277 K, 30 minutes) and then the supernatant was purified on a 1 mL E2 affinity column (PDI technology) and eluted with 100 µM E2 (20 mM Tris pH 8.1, 0.25 M NaSCN). High molecular weight species and excess salts were removed on a Superdex 200 column equilibrated in 50 mM Tris (pH 7.4, 150 mM NaCl, 10% glycerol, 1 mM DTT). Finally, the eluted proteins were concentrated to 1-5 mg mL-1. Fluorescence polarization experiments were performed in 96-well plates. Purified ERα LBD at increasing concentrations and FITC-labeled peptides (10 nM) in assay buffer (10 µM E2, 20 mM Tris-HCl pH 8.0, 25 mM NaCl, 10% glycerol, and 1 mM TCEP) were mixed and incubated at 277 K for 1 hour in the dark. The fluorescence polarization of the labeled peptides was measured at 298 K using plate reader (Perkin Elmer) 7



with excitation at 485 nm and emission at 520 nm and then plotted against the concentrations of the LBD. The binding affinity (Kd) values were determined by fitting the data using Prism 6.0. (Phillips et al., 2011; Vaz et al., 2009; Wang et al., 2006) Flow Cytometry Analysis 5 × 104 adherent cells were seeded in 24-well plates and allowed to grow for 48 hours in complete growth medium. After treated with FITC-labeled peptides in medium with 5% fetal bovine serum (FBS) for 12 hours at 310 K, cells were digested with 0.25% trypsin for 10 minutes. After that, cells were incubated with 0.05% Trypan Blue for 3 minutes.

40

Then the cells were analyzed by flow cytometry (FACSCalibur™). A minimum of

10,000 gated events were acquired and analyzed. Experiments were performed in triplicate. Confocal Microscopy Imaging 5 × 104 adherent cells were seeded in 24-well plates on coverslips and allowed to grow for 48 hours in complete growth medium. Cells were then treated with FITC-labeled peptides in medium with 5% FBS for 12 hours at 310 K followed by washing with PBS and fixed in 4% PFA (in PBS) for 15 minutes at room temperature. After fixation, cells were treated with 0.25% triton X-100 for 5 minutes and then blocked in 1% BSA in PBST for 30 minutes at 310 K followed by washing with PBST. Then cells were incubated with anti-ERα antibody (8644S; Cell Signaling Technology) at a dilution rate of 1: 300 in 0.1% BSA in PBST for 1.5 hours at 310 K. Cells were then washed with PBST for 3 × 10 minutes and incubated with Cy5-conjugated goat anti-rabbit IgG antibody (Santa Cruz) at a dilution rate of 1:1000 at 310 K for 40 minutes. Then stained with DAPI for 5 minutes at 310 K and washed with PBST for 3 × 10 minutes. Then coverslips were mounted on slides upside down with mounting medium and were imaged by confocal laser scanning microscope (LSM510 META). Immunoblotting 5

3 × 10 adherent cells were seeded in 6-well plates and allowed to grow for 24 hours in complete growth medium. Then cells were incubated with the indicated peptides in medium with 5% FBS for the indicated period of time. To isolate protein, cells were washed with PBS and lysed in RIPA buffer supplemented with protease inhibitor (Roche). Then the lysates were centrifuged at 12,500 rpm and the supernatants were used for SDS/PAGE analysis. Western blotting was carried out following standard protocols. For Immunoprecipitation, T47D cells with or without TD-PROTAC treatment were treated with MG-132 at the indicated concentrations for 6 hours and then the cells were lysed. Briefly, anti-ERα antibody (8644S; Cell Signaling Technology) was coupled to the Protein-G agarose beads (GE healthcare), roughly 1 mg total protein from the cell lysate was incubated with the coupled beads overnight at 277 K. Beads were washed 3 times with 8


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RIPA buffer and suspended in 35 µL 2 × SDS loading buffer and boiled for 10 minutes for SDS/PAGE. Immunoblotting are performed with ubiquitin antibody (3933S; Cell Signaling Technology) and anti-ERα antibody. Quantitative Polymerase Chain Reaction 3 × 105 cells were seeded in 6-well plates and allowed to grow for 24 hours in complete medium and then were incubated with the indicated compounds in medium with 5% FBS for 24 hours. Then cells were lysed, and RNA was extracted using Trizol reagent (Invitrogen) according to the manufacturer’s protocol. The amount of RNA was quantified by a spectrophotometer (Nano-Drop ND-2000). RNA was reverse-transcribed using SuperScript III Reverse Transcriptase (Invitrogen), on which qPCR was performed using SYBR Green (Promega) according to the manufacturer’s protocols in an ABI Prism 7500 real-time PCR system (Applied Biosystems). The relative changes in transcription levels for each sample were determined by normalization to GAPDH expression. Cell Viability by MTT Assay 4 × 104 cells were placed in each well of the 96-well plates and allowed to grow overnight. Cells were incubated with 100 µL serial dilutions of peptides in medium containing 5% FBS at 310 K for 24 or 72 hours. At the end of the peptide exposure, 20 µL of MTT reagent (5mg/ml in PBS) was added and incubated at 310 K for 4 hours. The absorbance of formazan product was measured at 490 nm by a microplate reader (Perkin Elmer, Envision, 2104 Multilabel Reader). Cells without peptides were treated as control.The cell viability was analyzed using Prism 6.0. Cell Apoptosis and Cell Cycle Assay T47D cells were treated with TD-PROTAC at different concentrations in medium with 5% FBS for 12 hours. Before flow cytometry assay, cells were washed with PBS and harvested using 0.25% Trypsin (Gibco) and centrifuged at 1,000 g for 5 minutes and rinsed with PBS. For cell apoptosis analysis, cells were treated according to the FITC Annexin V apoptosis detection kit (BD Biosciences). For cell cycle profile comparison, cells were fixed with 70% ice-cold ethanol, kept at -293 K overnight. Right before running the samples, the samples were centrifuged at 2,200 g for 5 minutes and washed with PBS for 2 times. Stained the samples with 50 µl of a 100 µg ml-1 sock of RNase A and 200 µl of 0.1 mg/ml PI for 10 minutes. Samples were tested using flow cytometer (CytoFlex, Beckman Coulter) and analyzed using FlowJo 7.6.1. For each individual experiment, 4

2 × 10 cells were counted. Xenograft Tumor Model 9



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Balb/c nude mice were purchased from Shanghai Super -- B&K laboratory animal Corp.Ltd. Mice were treated 7

with estrogen sustained release tablets two days before the injection of MCF-7 cells. Approximately 1 × 10

MCF-7 cells were injected in 0.2 mL PBS with matrigel. Mice bearing tumors around 100-150 mm3 in volume were randomly divided into three groups. Mice were administered via intraperitoneal injection with vehicle control solvent (30% PEG400 + 0.5% Tween80 + 5% propylene glycol + 64.5% saline), TD-PROTAC (10 mg kg-1) or tamoxifen (4 mg kg-1) every day. Tumor size was monitored and measured by caliper measurements over a period of 42 days. The volume was calculated using the formula: V = 1/2 ab2 (where a is the largest diameter and b is the smallest diameter). Tumor xenografts were excised, routine fixed, paraffin-embedded and sliced for H&E staining and immumohistochemical staining assays. For histological experiments, organ tissues were collected on the final day and fixed in 4% buffered formalin-saline at room temperature for 24 hours. Following this, tissues were embedded in paraffin blocks and paraffin sections of 4 mm thickness were mounted on a glass slide for hematoxylin and eosin (H&E) staining. The H&E staining slices were examined under a light microscopy (Olympus BX51). For immunohistochemistry assay, tumor tissue microarray slides were immersed in 3% H2O2 for 5 minutes to inactivate the endogenous peroxidase. Nonspecific binding sites were blocked using 5% BSA for 15 minutes. Antibodies against ERα were diluted as the primary antibodies and were incubated with slides at 277 K overnight, washed and then incubated with Rabbit-Probe MACH3 HRP-polymer detection system according to the supplier’s instructions. Slides were developed with 3, 3’-diaminobenzidine substrate using the ImmPACT DAB Peroxidase Substrate Kit (Vector Laboratories) for 1-5 minutes, counterstained with hematoxylin. (All reagents were obtained from Biocare Medical.) AUTHOR CONTRIBUTIONS Y.J., H.Z. and Z.L. put forward the idea. Y.J., Q.D., Y.Z. and Z.L. designed the experiments. M.X. provided support in FP assay. M.X. and F.Y. provided support in mouse xenograft studies. L.C. helped synthesis the unnatural amino acids. X.Q. and W.Z. also provided help in the experiments and research design. Y.J., Q.D., Y.Z. and Z.L. wrote the manuscript. ACKNOWLEDGMENTS We acknowledge financial support from the Natural Science Foundation of China Grants 21372023 ， 21778009 and 81701818; MOST 2015DFA31590, the Shenzhen Science and Technology Innovation Committee,

JCYJ20170412150719814,

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，

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JCYJ20160301111338144,

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and

GJHS20170310093122365; China Postdoctoral Science Foundation 2017M610704. SUPPORTING INFORMATION The Supporting Information is available free of charge on the ACS Publications website at DOI: xxxxx Figure S1-S6, Table S1, S2, S3, and HPLC Traces and MS Spectra of synthesized peptides. REFERENCES 1.

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GRAPHICAL ABSTRACT

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Table 1. Structure of peptides used in this research Peptide HIF

Sequence Leu-Ala-Pro(OH)-Tyr-Ile-NH2 Arg-HN

O NH

TD-PERM O Arg-HN

Ile-Leu-Dap-Arg-Leu-Leu-Gln-NH2 O NH

TD-PRTOAC O Leu-HN

Ile-Leu-Dap-Arg-Leu-Leu-Gln-AHX-Leu-Ala-Pro(OH)-Tyr-Ile-NH2 O NH

TD-PRTOACsc O Arg-HN

Ile-Arg-Dap-Leu-Gln-Arg-Leu-AHX-Leu-Ala-Pro(OH)-Tyr-Ile-NH2 O NH

TD-PRTOACmut O

Ile-Leu-Dap-Arg-Leu-Leu-Gln-AHX-Ala-Ala-Ala-Tyr-Ile-NH2

TD-PRTOAClinear XHis-Lys-Ile-Leu-His-Arg-Leu-Leu-Gln-AHX-Leu-Ala-Pro(OH)-Tyr-Ile-NH2 S S PERM-2 Arg-cys-Ile-Leu-Cys-Arg-Leu-Leu-Gln-NH2 For the peptides, the green part was the sequence targeting ER , the black part was the linker group and the red part was the sequence binding VHL E3 ubiquitin ligase. Peptides without tyrosine were labeled with a tyrosine at the N-terminus and acetylated with a Ala linker to determine its concentration. For fluorescence polarization and cellular uptake assays, fluorescein isothiocyanate (FITC) was labeled at the N-terminus with a Ala linker. Dap, 2,3-diaminopropionic acid. AHX, 6-aminohexanoic acid. Pro(OH), cis-4-hydroxy-L-proline.

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Figure 1. (A) CD spectra and calculated helicity (%) of peptides in H2O (298 K, concentration normalized). (B) Binding affinities of FITC-labeled peptides with ERα. Datas are presented as means ± SEM of triplicate experiments.

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Figure 2. TD-PROTAC showed good cell permeability in T47D cells. (A) Quantification of cellular uptake of T47D cells treated with different concentration of

FITC

TD-PROTAC and the cell penetrating peptide

FITC

TAT (as

the positive control) by flow cytometry analysis. All mean fluorescence intensities were normalized to that of 10µM FITCTD-PROTAC. Measurements were performed in triplicates. (B) Confocal microscopy images of T47D cells treated with 10 µM

FITC

TD-PROTAC at 310 K for 12 hours and stained with ERα antibody. (Scale bar, 10

µm). Datas are presented as means ± SEM of triplicate experiments. *, P < 0.05. **, P < 0.01.

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Figure 3. TD-PROTAC induces ERα degradation in a proteasome-dependent manner. (A) ERα levels in T47D cells after treatment with TD-PROTAC at the indicated concentrations for 24 hours. (B) ERα levels in T47D cells after treatment with 20 µM TD-PROTAC for different lengths of time. (C) ERα levels in T47D cells after treatment with different peptides at 20 µM for 24 hours. (D) ERα levels in T47D cells after treatment with 20 µM TD-PROTAC with or without 10 µM MG-132 for 6 hours and immunoprecipitation-western blot analysis of the ubiquitinated ERα levels.

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Figure 4. TD-PROTAC treatment resulted in down-regulation of the ERα-related gene pS2 and cell death in ERα-positive cell lines. (A) Expression levels of endogenous ERα-related genes pS2 in T47D cells treated with different peptides at 20 µM after 24 hours. (Normalized to the mRNA levels of GAPDH). (B) Antiproliferative effects of peptides in the T47D cells, measured by MTT assay after 24 hours of treatment with different concentrations of peptides. (C) Early apoptosis of T47D cells, analyzed by Annexin V/PI staining after treatment with different concentrations of TD-PROTAC for 12 hours. (D) Cell cycle of T47D cells, analyzed by PI staining combined with flow cytofluorometry after 12 hours of TD-PROTAC treatment. Datas are presented as means ± SEM of triplicate experiments. *, P < 0.05. **, P < 0.01.

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Figure 5. Antitumor activity of TD-PROTAC in MCF-7 xenograft animal model. (A) Tumor volume curve of MCF-7 xenografts over time when treated with TD-PROTAC (10 mg/kg), tamoxifen (4 mg/kg) or vehicle. Tumor volumes were measured by calipers. Vehicle: 30% PEG400 + 0.5% Tween80 + 5% propylene glycol + 64.5% saline. (B) Body weight curves of mice treated with TD-PROTAC, tamoxifen or vehicle. Relative tumor volume was defined as (V-V0)/V0, where V and V0 indicate the tumor volume on a particular day and on day 0, respectively. Values represent the means ± SEM, n=4-6 tumors. (C) TD-PROTAC down-regulated ERα protein levels. The MCF-7 xenografts were immunohistologically stained with anti-ERα. Scale bar, 100 µm. Focusing objective, 40X. *, P < 0.05. **, P < 0.01 vs Vehicle.

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75x30mm (300 x 300 DPI)


Development of stabilized peptide-based PROTACs against estrogen

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