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European Journal of Medicinal Chemistry

Development of cell-permeable peptide-based PROTACs targeting estrogen receptor α

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Article Details
Authors
Yuxuan Dai, Na Yue, Junni Gong, Chunxia Liu, Qifei Li, Jiaqi Zhou, Wenlong Huang, Hai Qian
Journal
European Journal of Medicinal Chemistry
DOI
10.1016/j.ejmech.2019.111967
Table of Contents
Abstract
Graphical Abstract For
Development Of Cell-Permeable Peptide-Based PROTACs Targeting Estrogen
Receptor α
Yuxuan Dai A, B, Na Yue A, Junni Gong A, Chunxia Liu A, Qifei Li A, Jiaqi Zhou A,
Wenlong Huang A, C, Hai Qian A, C∗
Development Of Cell-Permeable Peptide-Based PROTACs Targeting Estrogen
Receptor α
Abstract
Keywords
1. Introduction
2. Results And Discussion
2.1 Design And Synthesis Of Cell-Permeable Peptide-Based PROTACs
I-6
I-5
I-4
I-3
I-2
I-1
2.2 Circular Dichroism Spectroscopy
2.3 Cytotoxicity Assay
2.4 The Compounds Induce ERα Degradation
2.5 Cell Membrane Permeability Of The Compounds.
2.6 Pro-Apoptotic Assay
2.7 Compound I-6 Induces ERα Degradation Pathway
2.8 In Vivo Antitumor Efficacy
3. Conclusion
4. Experimental Section
4.1 Materials And Animals
4.2 Synthesis And Purification Of The Compounds
4.3 Circular Dichroism Spectroscopy
4.4 Cell Culture
4.5 Cytotoxicity Assay
4.6 Flow Cytometry Analysis Cellular Uptake
4.7 Flow Cytometry Analysis Of Apoptosis
4.8 Western Blot Analysis
4.9 Immunofluorescence Assay
4.10 Animal Model
4.11 Statistical Analysis
PANNIFER A D, PICKFORD A R, PRIOR S H, READ C M, SCOTT A, BROWN D G, XU B,
Highlights
Abstract
Proteolysis-targeting chimera (PROTAC) could selectively degrade target protein and may become a promising strategy for treating estrogen receptor α (ERα) positive breast cancers. Here, we designed penetrated peptide-based PROTACs by constructing an N-terminal lactam cyclic to improve proteolytic stability and cell penetration. We used a lactam cyclic peptide as ERα binding ligand, 6-aminocaproic acid as a linker, and a hydroxylated pentapeptide structure for recruiting E3 ligase to obtain heterobifunctional compounds. The resulting optimized compound I-6 selectively recruited ERα to the E3 ligase complex for promoting the degradation of ERα. Compound I-6 possessed strong effect on MCF-7 cell toxicity (IC50 ~9.7μM) and significantly enhanced activities in inducing ERα degradation. Meanwhile, I-6 performed much stronger potency in inhibition of tumors growth than tamoxifen. This work is a successful template to construct PROTACs based on cell-permeable peptides, which could extend the chemical space of PROTACs. ∗ Corresponding author: Hai Qian, Centre of Drug Discovery, State Key Laboratory of Natural Medicines, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, China. Tel: +86-25-83271051; Fax: +86-25-83271051. E-mail: qianhai24@163.com (H. Qian).
Journal Pre-proof Development of cell-permeable peptide-based PROTACs targeting estrogen receptor α Yuxuan Dai, Na Yue, Junni Gong, Chunxia Liu, Qifei Li, Jiaqi Zhou, Wenlong Huang, Hai Qian PII: S0223-5234(19)31119-5 DOI: https://doi.org/10.1016/j.ejmech.2019.111967 Reference: EJMECH 111967 To appear in: European Journal of Medicinal Chemistry Received Date: 17 October 2019 Revised Date: 11 December 2019 Accepted Date: 11 December 2019 Please cite this article as: Y. Dai, N. Yue, J. Gong, C. Liu, Q. Li, J. Zhou, W. Huang, H. Qian, Development of cell-permeable peptide-based PROTACs targeting estrogen receptor α, European Journal of Medicinal Chemistry (2020), doi: https://doi.org/10.1016/j.ejmech.2019.111967. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Masson SAS.
Graphical Abstract for
Development of Cell-permeable Peptide-Based PROTACs targeting Estrogen
Receptor α
Yuxuan Dai a, b, Na Yue a, Junni Gong a, Chunxia Liu a, Qifei Li a, Jiaqi Zhou a,
Wenlong Huang a, c, Hai Qian a, c∗
a Center of Drug Discovery, State Key Laboratory of Natural Medicines, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, PR China b Shaanxi Key Laboratory of Brain Disorders & Institute of Basic and Translational Medicine, Xi’an Medical University, Xi’an, Shaanxi 710021, PR China c Jiangsu Key Laboratory of Drug Discovery for Metabolic Disease, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, PR China ∗ Corresponding author: Hai Qian, Centre of Drug Discovery, State Key Laboratory of Natural Medicines, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, China. Tel: +86-25-83271051; Fax: +86-25-83271051. E-mail: qianhai24@163.com (H. Qian).
Development of Cell-permeable Peptide-Based PROTACs targeting Estrogen
Receptor α
Yuxuan Dai a, b, Na Yue a, Junni Gong a, Chunxia Liu a, Qifei Li a, Jiaqi Zhou a, Wenlong Huang a, c, Hai Qian a, c∗ a Center of Drug Discovery, State Key Laboratory of Natural Medicines, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, PR China b Shaanxi Key Laboratory of Brain Disorders & Institute of Basic and Translational Medicine, Xi’an Medical University, Xi’an, Shaanxi 710021, PR China c Jiangsu Key Laboratory of Drug Discovery for Metabolic Disease, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, PR China
Abstract
Proteolysis-targeting chimera (PROTAC) could selectively degrade target protein and may become a promising strategy for treating estrogen receptor α (ERα) positive breast cancers. Here, we designed penetrated peptide-based PROTACs by constructing an N-terminal lactam cyclic to improve proteolytic stability and cell penetration. We used a lactam cyclic peptide as ERα binding ligand, 6-aminocaproic acid as a linker, and a hydroxylated pentapeptide structure for recruiting E3 ligase to obtain heterobifunctional compounds. The resulting optimized compound I-6 selectively recruited ERα to the E3 ligase complex for promoting the degradation of ERα. Compound I-6 possessed strong effect on MCF-7 cell toxicity (IC50 ~9.7µM) and significantly enhanced activities in inducing ERα degradation. Meanwhile, I-6 performed much stronger potency in inhibition of tumors growth than tamoxifen. This work is a successful template to construct PROTACs based on cell-permeable peptides, which could extend the chemical space of PROTACs. ∗ Corresponding author: Hai Qian, Centre of Drug Discovery, State Key Laboratory of Natural Medicines, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, China. Tel: +86-25-83271051; Fax: +86-25-83271051. E-mail: qianhai24@163.com (H. Qian).
Keywords
Proteolysis-targeting chimera, Ubiquitin proteasome pathway, Estrogen receptor α, Membrane penetration, Stabilized peptide
1. Introduction
Since its initial design was reported by Sakamoto et al. in 2001, proteolysis-targeting chimeras (PROTACs) has been effectively applied in the development of compounds capable of inducing target protein degradation[1-3]. PROTACs are heterobifunctional compounds which generally consist of a ligand for target protein and a recognition motif for E3 ubiquitin ligase recruitment[4-6]. PROTACs utilize the ubiquitin-proteasome system (UPS) by recruiting an E3 ligase to target protein, leading to the polyubiquitination of target protein and the subsequent degradation of the target protein[2,5]. The heterobifunctional molecules cleave and eliminate target protein by chemical recruitment, which are characterized by suitability for a broader range of protein than standard small-molecule strategies of binding site occupancy[3,7]. Peptide-based and small molecule-based PROTACs have been successfully utilized to selectively degrade various protein targets, containing estrogen receptor α (ERα)[8,9], androgen receptor[10], cellular retinoic acid-binding proteins[11], and BET proteins[12]. To date, PROTACs are primarily based on small molecules, while peptide-based PROTACs are restricted, probably due to the inferior physiochemical properties of the peptides without chemical modification, such as low intracellular stability and low cell penetration efficiency[13]. However, Protein–protein interactions with either large or shallow interfaces are stubborn for small-molecule in general, but easily targetable by peptide modulators[14,15]. Peptide modulators have advantages over small molecules in some respects, such as facilitation the modification of drugs and binding mutated drug targets via clear epitopes[16]. Thus, exploring peptide-based PROTACs could enormously expand chemical space in the territory and it is possible to provide novel chance for the development of peptide-based therapeutics. Nevertheless, as mentioned above, unmodified peptides have some intrinsic shortcomings. In recent years, side chain cross-linking[17,18] or the incorporation of the nonessential amino acids[19] is used to constrain peptides conformation to improve stability and cell permeability. These constrained peptides have been applied in biological systems[20]. Breast cancer is the most common malignant disease in women worldwide. About two thirds of breast tumors are overexpressed of estrogen receptor α (ERα)[20]. ERα is a significant member of the nuclear receptor ER protein family and influences various physiological and pathological processes. Breast cancer cells with overexpression of ERα present estrogen-dependent proliferation[20,21]. ERα could serve as a good drug target for ERα positive breast cancer therapy[22,23]. The traditional method to inhibiting transcriptional activity of ERα gene is based on regulating the conformation of ERα，but drug resistance produced by long-term medication limit its application[24]. PROTACs could directly degrade the target protein and potentially solve this problem. Some based-peptide PROTACs targeting ERα have been reported, while these PROTACs have limited cell penetrating ability and target protein degradation ability[1,8,25]. In this work, we developed that a series of PROTACs based on an unnatural cross-linked aspartic acid at the N-terminus to constrain peptide into a helical conformation, leading to improved intracellular stability and cell penetration[26]. We introduced a lactam cyclic peptide as ERα binding ligand, 6-aminocaproic acid as a linker, and a hydroxylated pentapeptide structure in hypoxiainducible factor-1α (HIF-1α) protein (Leu-Ala-Pro (OH)-Tyr-Ile) for recruiting E3 ligase to design and synthesis six compounds. The compounds were estimated for secondary structures, the antiproliferative activity in ERα-positive and ERα-negative cell lines and the ability of Inducing ERα Degradation. Furthermore, the cellular uptake, membrane permeability, pro-apoptosis, and the pathway of ERα Degradation in ERα positive MCF-7 cells of the compounds were characterized.. Finally, we researched the most optimized compound I-6 in vivo antitumor efficacy compared with tamoxifen.
2. Results and discussion
2.1 Design and synthesis of Cell-permeable Peptide-Based PROTACs
Cell-permeable peptide-based PROTACs were constructed from three components: the peptide fragment, which can bind to ERα; the linker; and the peptide fragment, which can recognize by E3 ubiquitin ligase[16,27]. Zhao et al. reported peptides 12a (sequence: His-Lys-Ile-Lys-His-Arg-Leu-Leu-Gln ) and 13 (sequence: Arg-cyclo(isoAsp-Ile-Leu-Dap)-Arg-Leu-Leu-Gln ) showed good binding affinity with ERα (the value of KD: 118 nM and 85 nM respectively)[26]. The peptides 12a and 13 were chosen as ERα recognition domain. The pentapeptide (HIF sequence: Leu-Ala-Pro(OH)-Tyr-Ile ) from HIF-1α that binds to the Von Hippel−Lindau (VHL) E3 ubiquitin ligase complex was served as E3 ubiquitin ligase recognition domain[4]. 6-aminocaproic acid (AHX)[28] was selected as the biologically-free linker (Fig. 1 and Table 1). Fig.1 Schematic diagram structure of PROTACs. According the strategy, we designed the I-1 and I-2 to achieve degraded ERα (see in Table 1). Cross-linked aspartic acid strategy was used in the construction of I-2, which could restrict helical conformational of peptide by forming an i, i+3 lactam bond between isoAsp (Lisoaspartic acid) and Dap (2,3-diaminopropionic acid), leading to improved intracellular stability and cell penetration. Speltz et al. reported that introduction of γ-methyl group in the side chain of a cyclic peptide may constrain the helical conformation[29]. Neopentyl glycine (Npg) is considered to be the ideal choice with a side chain of Npg containing a γ-methyl group and the same length as Leu to increase the helical conformation. Thus, as shown in Table 1, we designed I-3, I-4, I-5, I-6 by retaining the lactam bond of I-2 and the substitution of Leu4, Leu7 and Leu8 by Npg sequentially. The compounds were synthesized using a standard solid phase peptide synthesis approach with Fmoc/tBu chemistry as previous reported. The synthetic routes of the cyclic peptides were illustrated in supporting information (Fig. S1). The purities of the compounds were all above 95%. The observed multiply charged ions of the conjugates were shown in Table S1 and Table S2, by ESI mass spectrometry.
I-6
I-5
I-4
I-3
I-2
I-1
2.2 Circular Dichroism Spectroscopy
To study the influence of chemical modification on the secondary structure of peptide, we assessed the solution helicity of peptides using circular dichroism spectroscopy (CD)[30]. The helical structure of the peptide showed two negative absorption peaks at 208 nm and 222 nm, and one positive absorption peak near 190 nm. As shown in Fig. 2, linear peptide I-1 was a randomly coiled while I-2-I-6 containing i,i+3 lactam ring exhibited helical conformation in ddH2O. Subsequently, the effects of compound I-6 on the level of other nuclear receptors were tested of I-4, I-5 and I-6 was enhanced compared to I-2, meanwhile, I-6 displayed the highest helicity, suggesting that the incorporation of Npg in appropriately positioned may increase the stabilized helicity of peptide.
2.3 Cytotoxicity assay
Fig. 3 Cellular activities of the compounds. (A) and (B) ERα positive MCF-7, A2780 viabilities treated by different compounds for 48 h were shown. Tamoxifen was used as positive control. (C) and (D)The viabilities of ERα negative MDA-MB-231 and normal cell line HUVEC treated by different compounds for 48 h were shown. To evaluate their inhibition of cell proliferation, all of the compounds were screened against the MCF-7, A2780 cancer cell lines (ERα positive), MDA-MB-231 cancer cell line (ERα negative) and HUVEC cell line (ERα negative). According to the results shown in Fig. 3A, the compounds I-2-I-6 and tamoxifen possessed anti-proliferative activity in MCF-7 cells while the linear peptide I-1 showed little toxicity at a concentration of 20 µM. Compared to I-2, the compounds I-3-I-6 exhibited higher cytotoxicity in MCF-7 cells treated with 20 µM compounds. Among them, compound I-6 presented the strongest cytotoxicity in a dose-dependent manner with the IC50 of ~9.7µM. Compound I-6 also showed anti-proliferative effects on ERα positive A2780 cells (Fig. 3B). In contrast, Compound I-6 showed negligible toxicity toward ERα negative MDA-MB-231 cells and normal HUVEC cells (Fig. 3C and Fig. 3D), which indicated that the effect was ERα-dependent.
2.4 The compounds Induce ERα Degradation
To assess the ability of compounds could induce ERα degradation, the protein level of ERα was measured by western blot. As shown in Fig. 4A and Fig. 4B, in MCF-7 cells, linear peptide I-1 barely decreased the level of the ERα protein at a concentration of 10 µM while I-2-I-6 possessed ability of inducing ERα degradation in varying degrees, wherein the compound I-6 displayed the strongest effect. MCF-7 cells were then treated with various concentrations (0, 3, 6, 10, 20 µM) of I-6 for 12 h. I-6 induced ERα degradation in a concentration-dependent manner (Fig. 4C and Fig. 4D). Next, the same cell line was treated with 10 µM I-6 for different time periods (0, 1, 4, 8, 12, 24 h) (Fig. 4E and Fig. 4F). As expected, I-6 induced ERα degradation was also time-dependently. Meanwhile, I-6 could induce the degradation of ERα as well as in A2780 cells (Fig. 4G and Fig. 4H). These results demonstrated that compound I-6 potently induced ERα degradation in MCF-7 cells in a dose- and time-dependent manner. Thus, compound I-6 may inhibit tumor cell proliferation by inducing ERα degradation. for 12 h. (C) ERα levels in MCF-7 cells after treatment with compound I-6 at the indicated concentrations for 12 h. (E) ERα levels in MCF-7 cells after treatment with 10 µM compound I-6 for different lengths of time. (G) ERα levels in A2780 cells after treatment with different compounds (I-1-I-6) at 10 µM for 12 h. GAPDH was probed as loading control. (B), (D), (F), (H) Band intensity was analyzed by Image J software and protein expression was presented as the ratio of target protein's band intensity to that of GAPDH.
2.5 Cell membrane permeability of the compounds.
In order to investigate cell permeability of the compounds, FITC labeled compounds were synthesized (Table S2). The endocytosed compounds in ERα positive MCF-7 cancer cells could be tracked by immunofluorescence assay and flow cytometry analysis. The respective high fluorescent intensities implied high accumulated concentration of the dye or drug within cells. Concurrently, the increase in fluorescence intensity for labelled compounds suggested that an active cellular uptake mechanism may be involved. It was evident that the endocytosis of I-2FITC -I-6FITC containing lactam ring were higher than linear peptide I-1FITC at the same concentration, visibly suggested that the formation of lactam ring has a significant influence on the cellular uptake (Fig. 5A and Fig. 5B). Npg-introduced compounds I-4FITC, I-5FITC, I-6FITC showed better cell penetration compared to I-2FITC and I-6FITC displayed the highest cellular uptake efficiency. The results demonstrated that a stable helical conformation of the compounds and hydrophobic properties of Npg side chain may contribute to cell membrane penetration. Next, we chose I-6 as optimized compound for obtaining quantitative information on the concentration- and time-gradient cellular uptake. Compound I-6 was taken up by MCF-7 cells in a dose(Fig. 5C and Fig. 5D) and time-dependent manner (Fig. 5E and Fig. 5F). To further validated, visualization of immunofluorescence assay was utilized to track I-6FITC. The picture showed that the compound I-6FITC could enter the ERα positive breast cancer cell MCF-7 effectively and appeared diffuse intracellular localization (Fig. 6)
2.6 Pro-apoptotic Assay
To further study the mechanism of cancer cell death induced by compounds, the effect on ERα positive MCF-7 cancer cells apoptosis analysis was conducted with Annexin-FITC/PI staining. As revealed by flow cytometry analysis, the activation of apoptosis and necrosis of MCF-7 cells were insignificant in the control group (Fig. 7A and Fig. 7C). Compounds I-2-I-6 had pro-apoptosis and pro-necrosis activities in varying degrees, and the I-6 exhibited the strongest effect. The percentage of late apoptosis cells reached 13.6% after treatment with 10 µM I-6, which was 5-and 2.3-fold higher than the I-1 and I-2 group. Moreover, the percentage of necrotic cells 26.2%, which was 9-and 3.3-fold higher than the I-1 and I-2 group. At the same time, late apoptosis and necrosis ratio appeared concentration-dependent increase after MCF-7 cells were treated with I-6 with concentration of 5 µM to 20 µM (Fig. 7B and Fig. 7D). described in (A) and (B), respectively. *P < 0.05. **P < 0.01. ***P < 0.001 vs I-2. Data are presented as the mean ± SD (n = 3).
2.7 Compound I-6 induces ERα degradation pathway
To investigate that ERα degradation depend on strengthening the polyubiquitination and proteasome pathway, we assessed I-6-induced ERα degradation as the presence of proteasome inhibitor MG-132 which was widely applied in the inhibition of the proteasome activity. As shown in Fig.8A and Fig. 8B, ubiquitinated-ERα increased in the presence of I-6 and MG-132 compared with the protein level in the presence of MG-132 alone or in the presence of I-6 alone. This result indicated that I-6 induced ERα degradation depended on the proteasome pathway. Subsequently, the effects of compound I-6 on the level of other nuclear receptors were tested, such as progesterone receptor (PR) and vitamin D receptor (VDR). The data presented I-6 had little effect on the protein levels of PR and VDR (Fig.8C and Fig. 8D). Thus, I-6 utilized the ubiquitin-proteasome pathway to selectively degrade ERα.
2.8 In Vivo Antitumor Efficacy
To study the anticancer activity of I-6 in vivo, we made tumor-bearing mice model with 4-T1 cells by administering once a day peritumoral injection of I-2 (10 µmol/kg), I-6 (10 µmol/kg), Tamoxifen (10 µmol/kg) as the positive control), or 0.9% saline as the negative control till 22 days. Compared with saline group, the tumor volumes of the I-2 group and I-6 group were respectively reduced by 52% and 67% with no obvious alteration in mouse weight (Fig. 9). Simultaneously，I-6 possessed stronger inhibitory effects on tumor volume compared with Tamoxifen (67% versus 36%). Thus, I-6 could inhibit breast tumor growth via inducing ERα degradation.
3. Conclusion
In conclusion, we have successfully developed a peptide PROTAC by recruiting ubiquitin E3 ligase, and applied it in the degradation of intracellular ERα (Table 1). Compound I-6, a peptide-based PROTAC, exhibited strong antiproliferative activity, high cellular uptake and pro-apoptosis effects on ERα-positive cancer cells with negligible cytotoxicity toward ERα-negative cells (Fig. 3, Fig. 5, Fig. 6 and Fig. 7). Meanwhile, I-6 could degrade intracellular ERα by activating the ubiquitin proteasome pathway as to inhibit ERα-dependent tumor cell proliferation (Fig. 4 and Fig. 8). In addition, in vivo experiments indicated that I-6 performed much stronger potency in inhibition of tumors growth than tamoxifen (Fig. 9). Thus, the present work signifies that the compound I-6 may be a promising candidate which extends the breast cancer therapy. In summary, PROTACs possess dual-function chemical characteristics structurally. However, a molecule with multiple functions at the same time whose microstructure is bound to be large and complex, and the macroscopic property is bringing bottlenecks in pharmacokinetics and biopharmaceutics. Nevertheless, medicinal chemistry does not have to be constrained by Lipinski's regulation of drug 5 (RO5)[31]. Rational strategies and applicability to a wide range of protein targets have been widely recognized by the pharmaceutical field and have broad prospects for development.
4. Experimental section
4.1 Materials and Animals
All reagents were purchased as reagent grade and used without further purification. Nα-Protected amino acids were purchased from Nanjing Peptide Biotech Ltd. Rink amide MBHA resin (loading 0.58 mmol g_1) and N-Hydroxybenzotriazole (HOBt) were purchased from GL Biochem Ltd. Trifluoroacetic acid (TFA) and N, N-diisopropylethyl amine (DIPEA) and were purchased from Aladdin. N， N-diisopropylcarbodiimide (DIC) was purchased from Ark Pharm. Fluorescein isothiocyanate isomer (FITC) was purchased from Bide Pharmatech. All other reagents, unless otherwise accounts, were obtained from Sigma-Aldrich Co. (Saint Louis, MO) and used as received. Hoechst 33342, RIPA buffer and BCA Protein Assay Kit were purchased from Beyotime Biotechnology. Annexin V–FITC/PI Detection Kit were purchased from Jiangsu KeyGEN BioTECH Ltd. Rabbit polyclonal antibody (ESR1 polyclonal antibody, GAPDH polyclonal antibody, UBB polyclonal antibody, PR polyclonal antibody, VDR polyclonal antibody and Alexa Fluor 647 antirabbit IgG antibody) were purchased from ABclonal. Microwave procedures were performed in the Microwave Peptide Synthesizer (CEM, Matthews, NC, USA).
4.2 Synthesis and purification of the compounds
Peptides were synthesis by standard solid-phase peptide synthesis (SPPS) methodology on Fmoc-Rink Amide MBHA resin using a microwave synthesizer, as already described in previous research[32]. The synthesis route of compounds was shown in Fig.S1. Crude peptides were purified by Shimadzu LC-10 preparative reverse-phase high-performance liquid chromatography with C18 column (5 µm, 340 × 28 mm). Purity analysis and characterization were analyzed by UPLC/MS (Waters UPLC with the ACQUITY TQD; Waters Corporation, Milford, MA, USA) with a Waters ACQUITY UPLCBEH C18 column (1.7 × 50 mm,Waters). The purities of the compounds were above 95%.
4.3 Circular Dichroism Spectroscopy
CD spectra were performed using a Jasco J-810 spectropolarimeter and with obtained by wavelengths from 250 to 190 nm with a 1 mm cell at a scan speed of 20 nm/s at 20℃. Compounds were dissolved in ddH2O at concentrations of about 100 µM. Two scans per sample were averaged, and the averaged spectrum was smoothed. Percent helicity was calculated based on the equation reported by Arora[33]. Helicity % = [θ]222/[θ]max. [θ] = (−44000 + 250T)(1 − k/n) (k = 4 and n = number of amino acid residues in the peptide, T = 20 °C)
4.4 Cell Culture
Human breast MCF-7 and MDA-MB-231 cancer cell lines, human ovarian A2780 cancer cell lines, human umbilical vein endothelial cells HUVEC and mouse breast cancer cells 4T1, were obtained from KeyGEN BioTECH (Nanjing, China). MCF-7 and 4T1 cells were cultured in RPMI 1640 supplemented with 10% FBS （Hyclone Laboratories). A2780 and MDA-MB-231 cells were grown in high glucose DMEM supplied with 10% FBS. All media were supplemented with 1% antibiotics (penicillin and streptomycin). All the cells were incubated in an atmosphere of 5% CO2 at 37 °C.
4.5 Cytotoxicity Assay
Cell viability was determined by MTT method with a minor modification[34]. cells during logarithmic growth phase were seeded in 96-well plates at a density of 7 × 103 cells and gowned 12 h. The compounds were added to cells with different final concentrations (ranging from 5 µM to 80 µM) and incubated in an atmosphere of 5% CO2 at 37 °C. for 48 h. MTT dye (10 µL of 2.5 mg/mL in PBS) was added to each well 4 h prior to experiment termination. Then, the plates were centrifuged at 1200 rpm for 20 min and the supernatant was removed without disturbing the formazan crystals and cells in the wells. 150 µL of DMSO was added to dissolve formazan crystals and the plates agitated on a plate shaker for 5 min. The absorbance at 490 nm was read on a microplate reader (Thermo, USA). The IC50 values of compounds were calculated by GraphPad Prism 7.0 software (San Diego, CA, USA) according to the dose−response curves. Experiments were performed three times.
4.6 Flow Cytometry Analysis Cellular uptake
A total of 6 × 104 adherent MCF-7 cells was seeded in 12-well plates and cultured for 48 h in complete growth medium. Then, cells were treated with FITC labeled compounds in medium supplemented with 5% FBS for 12 h. The cells were washed twice with ice-cold PBS, followed by digested with 0.25% trypsin for 2 min. After that, cells were collected in flow pipe and analyzed by flow cytometry (Accuri C6). A minimum of 10 000 gated events were acquired and the data was analyzed using Flow Jo V10 software. Experiments were performed three times.
4.7 Flow Cytometry Analysis of Apoptosis
The reported procedures which was employed for detecting the percentage of apoptotic cells was modified slightly[34]. In brief, apoptotic cells were quantitated by flow cytometry with annexin V–FITC/PI Detection Kit. MCF-7 cells (4 × 105 cells/well) were seeded overnight in 6-well plates with 2 mL complete growth medium. Cells were then treated with various concentrations of FITC labeled compounds in FBS-free medium for 12 h at 37 °C. The cells were washed three with ice-cold PBS, followed by digested with 0.25% trypsin without EDTA and collected in tubes. Subsequently, later process was carried out according to the manufacturer’s instructions, and then, analyzed using the flow cytometer (Accuri C6).
4.8 Western Blot Analysis
The western blot procedure was conducted on the basis of the protocol reported previously[35,36]. MCF-7 cells were seeded in 6-well plates (4 × 105 cells/well). The cells were then treated with different concentrations of compounds for 12 h. The mediums containing compounds was discarded, and the cells were washed with PBS buffer three times. Cellular proteins were obtained in the RIPA lysis buffer (Beyotime) containing PMSF and denatured by boiling bath for 10 min, and protein concentration was quantified by BCA method following the manufacturer’s instructions of the kit. 40 ug of cell lysate was resolved on gradient SDS−PAGE gel and then transferred to PVDF membranes, which were then blocked in 5% bovine serum albumin (BSA) in TBST buffer (10 mM Tris-HCl, pH 7.6, 150 mM NaCl, and 0.1% Tween 20) to block nonspecific binding for 1 h. Subsequently, the membranes were incubated with primary antibodies (ESR1 antibody, PR antibody, VDR antibody, UBB antibody and GAPDH antibody) at 4 °C overnight. After washing the PVDF membrane with TBST buffer three times, the PVDF membrane was incubated with HRP-conjugated secondary antibody at room temperature for 2 h. The protein bands were imaged with Tanon High sig ECL western blotting and a luminescent image analyzer (Tanon 5500). Band intensity was analyzed by Image J software. GAPDH was used for confirming equal loading in each lane in the protein samples.
4.9 Immunofluorescence assay
MCF-7 cells during logarithmic growth phase were seeded in 6-well plates and grown overnight in RPMI 1640 supplemented with 10% FBS. Then, cells were treated with FITC labeled compounds in medium supplemented with 5% FBS for 12 h. The cells were washed twice with ice-cold PBS, followed by fixed with a 4% paraformaldehyde (PFA) at 37 ° C for 15 min. For immunofluorescence, after fixation, the cells were treated with 0.25% Triton X-100 for 5 min, blocked with 5% BSA in PBST for 30 min followed by washing with PBST for 3 × 10 min. The cells were incubated with ESR1 polyclonal antibody at a dilution rate of 1:50 in 3% BSA for 1 h. After washing with PBST, the cells were incubated with Alexa Fluor 647 antirabbit IgG antibody at a dilution rate of 1:100 for 40 min at room temperature. The plate was added 1 mL of PBS buffer per well, wrapped the plate with tin foil and placed on ice box. Fluorescence images were obtained with fluorescence microscopy (Nikon Ts2R).
4.10 Animal Model
4T1 tumor-bearing BALB/c mice (female, 18-22 g, Certificate number: NO. 20170005004344) were purchased from Keygen Biotech (Jiangsu, China). All the animals involved were treated in accordance with protocols approved by the ethical committee of China Pharmaceutical University. All animal experimental protocols adhered to the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH publication 85-23, revised 1986). 24 mice were divided into four groups (six mice per group) randomly and administrated with 0.9% saline solution, I-2 (10 µmol/kg), I-6 (10 µmol/kg), Tamoxifen (10 µmol/kg) once a day via peritumoral injection. Tumor size was measured by vernier caliper during a period of 21 days. Tumor volume was calculated using the formula: tumor volume = 1/2ab2 (where a is the largest length and b is the smallest width). After the mouse were sacrificed, necropsies were performed and the tumors were removed, weighed.
4.11 Statistical analysis
Comparisons among groups were statistically analyzed by one-way ANOVA in GraphPad Prism 7.0. A value of p < 0.05 was considered significant. Data were presented as mean ± SD for three independent tests. Acknowledgements This work was supported by the National Natural Science Foundation of China (No. 81872733 & No. 81673299). Conflict of interest The authors have no conflicts of interest to declare.
PANNIFER A D, PICKFORD A R, PRIOR S H, READ C M, SCOTT A, BROWN D G, XU B,
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Highlights
Synthesis of cell-permeable peptide-based PROTACs targeting estrogen receptor α. I-6 possessed strong effect on ERα positive cells MCF-7 toxicity (IC50 ~9.7µM). I-6 exhibited membrane penetrability and pro-apoptosis activity and tumor growth inhibitory activities in vivo. I-6 selectively recruited ERα to the VHL E3 ligase complex, leading to the degradation of ERα in a proteasome-dependent manner. All results indicated PROTAC I-6 may be a promising candidate which extends the breast cancer therapy. Declaration of interests ☑The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:
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