- Email: [email protected]
Development of stapled short helical peptides capable of inhibiting vitamin D receptor (VDR)–coactivator interactions
Bioorganic & Medicinal Chemistry Letters 23 (2013) 4292–4296
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
Development of stapled short helical peptides capable of inhibiting vitamin D receptor (VDR)–coactivator interactions Yosuke Demizu a,⇑, Saori Nagoya a, Manami Shirakawa a, Megumi Kawamura a,b, Nanako Yamagata a, Yukiko Sato a, Mitsunobu Doi c, Masaaki Kurihara a,b,⇑ a b c
Division of Organic Chemistry, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya, Tokyo 158-8501, Japan Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama 226-8501, Japan Osaka University of Pharmaceutical Sciences, Osaka 569-1094, Japan
a r t i c l e
i n f o
a b s t r a c t
Article history: Received 29 April 2013 Revised 27 May 2013 Accepted 1 June 2013 Available online 10 June 2013
We synthesized stapled helical leucine-based peptides (DPI-01-07) containing 2-aminoisobutyric acid and a covalent cross-linked unit as inhibitors of vitamin D receptor (VDR)–coactivator interactions. The effects of these peptides on the human VDR were examined in an inhibition assay based on the receptor cofactor assay system, and one of them, DPI-07, exhibited potent inhibitory activity (IC50: 3.2 lM). Ó 2013 Elsevier Ltd. All rights reserved.
Keywords: Vitamin D receptor VDR–coactivator interaction inhibitor Stapled helical peptide Protein–protein interaction
The vitamin D receptor (VDR), a nuclear receptor (NR), regulates various biological actions such as calcium uptake, cell proliferation and differentiation, and immune modulation. 1a,25-Dihydroxyvitamin D3 [1a,25(OH)2D3] is an endogenous VDR ligand and has also been demonstrated to regulate these biological events via the VDR.1,2 Therefore, agonistic 1a,25(OH)2D3 derivatives (secosteroi-
dal VDR ligands) and non-secosteroidal VDR ligands are considered to be therapeutic candidates for the treatment of osteoporosis, various types of rickets, secondary hyperparathyroidism, psoriasis, autoimmune diseases, and cancer.3–10 On the other hand, antagonistic VDR ligands are considered to be useful for the treatment of conditions involving hypersensitivity to 1a,25(OH)2D3, such as
(a)
(b)
O Boc
N H
O
H N
N * H 3
O
L
X
O O
n H N
N H
O
X
O
H N O
L
n N * CO2Me H 7
L
A
R3,7R-2
Figure 1. (a) Chemical structure of the stapled heptapeptides. (b) X-ray structure of the stapled heptapeptide R3,7R-2. The covalent side chain is shown in green. The nomenclature R3,7R refers to a peptide with R configurations at its 3rd and 7th positions.
⇑ Corresponding authors. Tel.: +81 3 3700 1141; fax: +81 3 3707 6950. E-mail addresses: [email protected] (Y. Demizu), [email protected] (M. Kurihara). 0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.bmcl.2013.06.002
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coactivator LXXLL
inhibition
coactivator LXXLL target gene
target gene
stapled short helical peptide containing the LXXLL motif
VDR
VDR
Figure 2. Illustration of the mechanism by which the stapled short helical peptides inhibited VDR–coactivator interactions.
Paget’s disease of bone,11 which results in an abnormal bone architecture. To date, several secosteroidal VDR antagonists have been developed.10 In addition, small molecules12–14 and peptides15–17 containing the consensus sequence LXXLL [L: leucine (Leu), X: any amino acid residue] have been demonstrated to inhibit VDR– coactivator interactions. Therefore, they are also considered to be drug candidates for reducing VDR-mediated transactivation. In order to bind to the VDR, small molecules/peptides must contain three Leu residues or Leu mimics.15–17 Furthermore, small peptides have to have an a-helical structure to efficiently interact with the VDR. It is sometimes difficult to form stable a-helices in short peptides. However, a,a-disubstituted a-amino acids (dAAs)18–20 and a covalent cross-linking system21–24 have been demonstrated to be useful for stabilizing the helical structures of such peptides. Especially, 2-aminoisobutyric acid (Aib) has been used as a strong helical promoter.25–28 The covalent cross linker at the i and i+3
O Boc
N H
O
H N
N * H
O
Table 1 IC50 values of peptides DPI-01-07 against human VDR–cofactor interactions according to a receptor cofactor assay systema Entry
Peptide
IC50 (lM)
1 2 3 4 5 6 7b
H-S3,7S-2-OH (DPI-01) H-S3,7R-2-OH (DPI-02) H-R3,7S-2-OH (DPI-03) H-R3,7R-2-OH (DPI-04) H-hS3,7S-2-OH (DPI-05) H-hS3,7hS-2-OH (DPI-06) H-hS3,7hSdOH-2-OH (DPI-07)
610 430 610 600 520 220 3.2
a b
The EC50 value of the positive control, 1a,25(OH)2D3, was 7.7 nM. An isomeric mixture of DPI-07 was examined in the assay.
positions on peptides has been found to be also useful for the helix-stabilization strategy, as reported by Grubbs and Verdine.22,29Recently, we reported that the combined use of 2-
O
n H N
O N H
O
H N
O
O 1) 1M NaOH aq., MeOH, rt, 12h
n
N * CO 2Me H 2) 6M HCl aq., THF, rt, 6h
O
O
H N
H 2N
N * H
O
S 3, 7S-2 S 3, 7R-2 R3, 7S-2 R3, 7R-2 hS3, 7S-2 hS3, 7hS-2
O O
n H N
N H
O
O
H N O
H-S 3,7S-2-OH (DPI-01) H-S 3,7R-2-OH (DPI-02) H-R3, 7S-2-OH (DPI-03) H-R3, 7R-2-OH (DPI-04) H-hS3, 7S-2-OH (DPI-05) H-hS3, 7hS-2-OH (DPI-06)
HO
O
Boc
N H
O
H N
N H
O
O H N
O N H
O
H N
N H
O
CO2Me
2) OsO 4, NMO, H 2O/acetone, rt, 24h
2) 6M HCl aq., THF, rt, 6h
O
N H
H N O
N H
O H N
O
O N H
H N O
OH
O H N
Boc
O
hS3, 7hSdOH-2 HO
H 2N
OH
O 1) 2nd Grubbs cat., CH 2Cl 2, rt, 24h
O
hS3, 7hS-1
1) 1M NaOH aq., MeOH, rt, 12h
n N * CO2H H
O N H
O H N
O
O N H
H N O
O N H
CO2H
H-hS3, 7hSdOH-2-OH (DPI-07)
Scheme 1. Synthesis of the stapled heptapeptides. The nomenclature hS indicates a L-homoserine (L-Hse) derivative.
O N H
CO2Me
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Figure 3. CD spectrum (190–260 nm region) of hS3,7hS-2dOH in TFE solution. Peptide concentration: 0.5 mM.
aminoisobutyric acid (Aib) and a cross-linked unit stabilized the helical structures of short Leu-based peptides with structure A (Fig. 1)as reported by Grubbs and Verdine.30 These peptides were able to efficiently catalyze the enantioselective epoxidation of a,b-unsaturated ketones.31As peptides A contain the LXXLL motif, we considered that any short peptide based on this structure
(a)
(b) HO
OH
O O N H
should bind to the VDR and inhibit VDR–coactivator interactions (Fig. 2). Here, we synthesized several stapled heptapeptides containing the LXXLL motif and evaluated their ability to inhibit VDR–coactivator interactions. The stapled peptides S3,7S-2, S3,7R-2, R3,7S-2, R3,7R-2, hS3,7S-2, and hS3,7hS-2 were prepared according to the previously reported methods.31 Then, their C- and N-termini were deprotected to afford C- and N-terminal-free peptides; that is, H-S3,7S-2-OH (DPI-01), H-S3,7R-2-OH (DPI-02), H-R3,7S-2-OH (DPI-03), H-R3,7 R-2-OH (DPI-04), H-hS3,7S-2-OH (DPI-05), and H-hS3,7hS-2-OH (DPI-06), respectively. The stapled peptide hS3,7hS-2dOH, which possessed two hydroxyl group side chains, was synthesized from the linear peptide hS3,7hS-131 via a ruthenium-catalyzed intramolecular ring-closing metathesis reaction followed by osmium catalyzed dihydroxylation.32 Finally, the C- and N-terminal protecting groups of hS3,7hS-2dOH were removed to obtain H-hS3,7hS-2dOH-OH (DPI-07) (Scheme 1).33 The isomeric mixture of DPI-07 was not separated. The inhibitory activity of the synthetic peptides DPI-01-07 against VDR-coactivator interactions was evaluated using the EnBio receptor cofactor assay system (RCAS) for the VDR kit (Fujikura Kasei Co., Ltd) according to the manufacturer’s instructions.34 The results are summarized in Table 1. Most of the peptides exhibited weak activity against VDR–coactivator interactions (IC50: 220– 610 lM, entries 1–6); however, DPI-07, which possesses the side-chain hydroxyl groups, displayed 70-fold greater activity than the other peptides (IC50: 3.2 lM, entry 7). The dominant conformations of the N- and C-terminal protected peptides S3,7S-2, S3,7R-2, R3,7S-2, R3,7R-2, hS3,7S-2, hS3,7hS2, and hS3,7hS-2dOH were analyzed using the CD spectra in 2,2,2-tri-
H N 2 O
O N H
O H N
O
O N 5 H
H N 6 O
O N H
H N O
DPI-07 derivative B
(c)
Leu630 Leu633
Leu634
Figure 4. (a) Chemical structure of the modeled DPI-07 derivative (B). (b) Modeled structure of B bound to the VDR. The VDR surface is shown in gray. (c) Overlay of the X-ray structure of the coactivator fragment (yellow, PDB ID 3AUN) bound to the VDR and the calculated structure of B bound to the VDR.
Y. Demizu et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4292–4296
fluoroethanol (TFE) solution. The CD spectrum ofhS3,7hS-2dOH showed negative maxima at around 206 and 222 nm, which indicates that hS3,7hS-2dOH was folded into a right-handed helical structure as the preferred secondary structure (Fig. 3).35 The other peptides S3,7S-2, S3,7R-2, R3,7S-2, R3,7R-2, hS3,7S-2, and hS3,7hS-2 had the similar CD patterns and intensities to hS3,7hS-2dOH in TFE solution, respectively.30,31,36 Next, we used the X-ray co-crystal structure (PDB: 3AUN) of VDR bound to the LXXLL motif found in its coactivators to perform docking studies.37 Accordingly, a docking model of a DPI-07 derivative (B) (Fig. 4a)38 bound to the VDR was constructed based on a conformational search in MacroModel (ver. 9.1). AMBER⁄ was used as the force field, and more than 10,000 conformers for B were optimized. The most stable conformation is shown in Figure 4b. In the modeled structure of B, the three Leu residues at the 2nd, 5th, and 6th positions are oriented around the hydrophobic region (Ile234, Ile238, Leu259, Ala263, and Val417) of the VDR. The positions of these three Leu residues in B displayed good agreement with those of the leucine residues in the LXXLL coactivator motif (Leu630, Leu633, and Leu634) during its binding to the VDR (Fig. 4c).37 Furthermore, when B binds to the VDR its hydroxyl group side chains are located on either side of the Leu-containing motif. These hydroxyl groups could increase the affinity of DPI07 for water molecules surrounding the protein, and hence, DPI07 strongly binds to the protein. Although all synthetic peptides had the similar helicity, the peptides without any hydroxyl groups did not show strong inhibitory activity (IC50: 220–610 lM). These results also mean that the side-chain hydroxyl groups of the stapled peptide play an important role to develop the strong affinity. In summary, we developed stapled short helical peptides (DPI01-07) containing an LXXLL motif. The ability of these peptides to inhibit VDR–coactivator interactions was evaluated in an inhibition assay. Peptides DPI-01-06, which do not contain hydroxyl groups in their covalent side chains, exhibited weak inhibitory activity (IC50: 220–610 lM), whereas DPI-07, which possesses the side-chain hydroxyl groups, displayed strong inhibitory activity (IC50: 3.2 lM). Stapled short helical peptides like DPI-07 might be useful for treating conditions involving hypersensitivity to 1a,25(OH)2D3. Transcriptional inhibition assays, the derivatization of further peptides, and studies of their effects on other nuclear receptors are currently underway. Acknowledgments This study was supported, in part, by Grants-in-Aid for Scientific Research (M.K.) from the Japan Society for the Promotion of Science, and a grant from the Kaneka Award for Synthetic Organic Chemistry, Japan (Y.D.). References and notes 1. Feldman, D.; Glorieux, F. H.; Pike, J. W. Vitamin D, 2nd ed.; Elsevier Academic Press: New York, 2005. 2. DeLuca, H. F. FASEB J. 1998, 2, 224. 3. Asano, L.; Ito, I.; Kuwabara, N.; Waku, T.; Yanagisawa, J.; Miyachi, H.; Shimizu, T. FEBS Lett. 2013, 587, 957. 4. Kashiwagi, H.; Ono, Y.; Ohta, M.; Itoh, S.; Ichikawa, F.; Harada, S.; Takeda, S.; Sekiguchi, N.; Ishigai, M.; Takahashi, T. Bioorg. Med. Chem. 2013, 1823, 21. 5. Ciesielski, F.; Sato, Y.; Chebaro, Y.; Moras, D.; Dejaegere, A.; Rochel, N. J. Med. Chem. 2012, 55, 8440. 6. Abe, J.; Nagai, Y.; Higashikuni, R.; Iida, K.; Hirokawa, T.; Nagai, H.; Kominato, K.; Tsuchida, T.; Hirata, M.; Inada, M.; Miyaura, C.; Nagasawa, K. Org. Biomol. Chem. 2012, 10, 7826. 7. Sawada, D.; Tsukuda, Y.; Saito, H.; Kakuda, S.; Takimoto-Kamimura, M.; Ochiai, E.; Takenouchi, K.; Kittaka, A. J. Am. Chem. Soc. 2011, 133, 7215. 8. Fujii, S.; Masuno, H.; Taoda, Y.; Kano, A.; Wongmayura, A.; Nakabayashi, M.; Ito, N.; Shimizu, M.; Kawachi, E.; Hirano, T.; Endo, Y.; Tanatani, A.; Kagechika, H. J. Am. Chem. Soc. 2011, 133, 20933. 9. Demizu, Y.; Nakatsu, A.; Honzawa, S.; Yamashita, A.; Sugiura, T.; Kittaka, A.; Kato, S.; Okuda, H.; Kurihara, M. Lett. Org. Chem. 2011, 8, 43.
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10. Other seco- and nonsecosteroidal agonistic and antagonistic VDR ligands were shown in a recent review: Carlberg, C.; Molnár, F.; Mouriño, A. Expert Opin. Ther. Patents 2012, 22, 417. 11. Roodman, G. D.; Windle, J. J. J. Clin. Invest. 2005, 115, 200. 12. Mita, Y.; Dodo, K.; Noguchi-Yachide, T.; Hashimoto, Y.; Ishikawa, M. Bioorg. Med. Chem. 2013, 21, 993. 13. Nandhikonda, P.; Lynt, W. Z.; McCallum, M. M.; Ara, T.; Baranowski, A. M.; Yuan, N. Y.; Pearson, D.; Bikle, D. D.; Guy, R. K.; Arnold, L. A. J. Med. Chem. 2012, 55, 4640. 14. Mita, Y.; Dodo, K.; Noguchi-Yachide, T.; Miyachi, H.; Makishima, M.; Hashimoto, Y.; Ishikawa, M. Bioorg. Med. Chem. Lett. 2010, 20, 1712. 15. Zella, L. A.; Chang, C.-Y.; McDonnell, D. P.; Pike, J. W. Arch. Biochem. Biophys. 2007, 460, 206. 16. Pike, J. W.; Pathrose, P.; Barmina, O.; Chang, C.-Y.; McDonnell, D. P.; Yamamoto, H.; Shevde, N. K. J. Cell. Biochem. 2003, 88, 252. 17. Pathrose, P.; Barmina, O.; Chang, C.-Y.; McDonnell, D. P.; Shevde, N. K.; Pike, J. W. J. Bone Miner. Res. 2002, 17, 2196. 18. Royo, S.; Borggraeve, W. M. D.; Peggion, C.; Formaggio, F.; Crisma, M.; Jiménez, A. I.; Cativiela, C.; Toniolo, C. J. Am. Chem. Soc. 2005, 127, 2036. 19. Demizu, Y.; Doi, M.; Kurihara, M.; Maruyama, T.; Suemune, H.; Tanaka, M. Chem. Eur. J. 2012, 18, 2430. 20. Demizu, Y.; Doi, M.; Sato, Y.; Tanaka, M.; Okuda, H.; Kurihara, M. Chem. Eur. J. 2011, 17, 11107. 21. Azzarito, V.; Long, K.; Murphy, N. S.; Wilson, A. J. Nat. Chem. 2013, 5, 161. 22. Kim, Y.-W.; Grossmann, T. N.; Verdine, G. L. Nat. Protoc. 2011, 6, 761. 23. Ousaka, N.; Inai, Y.; Kuroda, R. J. Am. Chem. Soc. 2008, 130, 12266. 24. Wang, D.; Liao, W.; Arora, P. S. Angew. Chem., Int. Ed. 2005, 44, 6525. 25. Toniolo, C.; Crisma, M.; Formaggio, F.; Peggion, C.; Broxterman, Q. B.; Kaptein, B. Biopolymers (Pept. Sci.) 2004, 76, 162. 26. Karle, I. L. Biopolymers (Pept. Sci.) 2001, 60, 351. 27. Venkatraman, J.; Shankaramma, S. C.; Balaram, P. Chem. Rev. 2001, 101, 3131– 3152. 28. Oba, M.; Demizu, Y.; Yamagata, N.; Sato, Y.; Doi, M.; Tanaka, M.; Suemune, H.; Okuda, H.; Kurihara, M. Tetrahedron 2010, 66, 2293. 29. Blackwell, H. E.; Sadowsky, J. D.; Howard, R. J.; SaMpson, J. N.; Chao, J. A.; Steinmetz, W. E.; O’Leary, D. J.; Grubbs, R. H. J. Org. Chem. 2001, 66, 5291. 30. Yamagata, N.; Demizu, Y.; Sato, Y.; Doi, M.; Tanaka, M.; Nagasawa, K.; Okuda, H.; Kurihara, M. Tetrahedron Lett. 2011, 52, 798. 31. Demizu, Y.; Yamagata, N.; Nagoya, S.; Sato, Y.; Doi, M.; Tanaka, M.; Nagasawa, K.; Okuda, H.; Kurihara, M. Tetrahedron 2011, 67, 6155. 32. In an inert atmosphere, a solution of hS3,7hS-1 (71 mg, 0.075 mmol) and Grubbs catalyst 2nd generation (32 mg, 0.04 mmol) in CH2Cl2 (20 mL) was stirred at room temperature for 12 h. The solution was then poured into water and extracted with CH2Cl2. The combined organic layer was dried over Na2SO4, and the solvent was removed under reduced pressure to afford the stapled peptide hS3,7hS-2 (61 mg, 88%), which was used for the subsequent reaction without further purification. A solution of the above peptide, Nmethylmorpholine-N-oxide (NMO, 20 mg, 0.17 mmol), and 4% aqueous osmium tetroxide (25 lL) was dissolved in acetone/t-BuOH/water (7 mL, 3/ 3/1) and then stirred at room temperature for 24 h. Next, saturated aqueous sodium thiosulfate (30 mL) was added to the solution and stirred at room temperature for 30 min. The resultant solution was diluted with AcOEt, washed with 1% aqueous HCl and brine, and dried over Na2SO4. After the solvent had been removed, the residue was purified by column chromatography on silica gel (AcOEt/MeOH = 20:1) to give hS3,7hSdOH-2 (39 mg, 62%). Spectroscopic data for hS3,7hSdOH-2: Foam; ½a24 D 33.9 (c 0.50, CHCl3); IR (in CHCl3): m = 3326, 2959, 1665, 1529, 1251, 1159 cm1; 1H NMR (400 MHz, CDCl3) 7.96 (d, J = 6.4 Hz, 1H), 7.56 (d, J = 4.8 Hz, 1H), 7.26 (m, 2H), 7.16 (d, J = 10.0 Hz, 1H), 6.98 (d, J = 4.8 Hz, 1H), 6.56 (m, 1H), 4.99–5.12 (m, 2H), 4.50–4.78 (m, 2H), 4.25 (m, 1H), 4.13 (m, 1H), 3.94–4.02 (m, 3H), 3.73 (s, 3H), 3.54–3.68 (m, 6H), 3.22–3.46 (m, 3H), 2.22 (m, 1H), 1.81–1.94 (m, 4H), 1.61–1.71 (m, 10H), 1.43–1.48 (m, 14H), 1.00–1.30 (m, 2H), 0.89–0.99 (m, 24H); [HR-ESI(+)]: m/z calcd for C46H83N7O14Na [M+Na]+: 980.5890; actual: 980.5883. 33. A solution of hS3,7hSdOH-2 (30 mg, 0.03 mmol) and 1 M aqueous NaOH (0.06 mL) in MeOH (1 mL) was stirred at room temperature for 12 h. Then, the solution was neutralized with 1 M aqueous HCl, extracted with AcOEt, and dried over Na2SO4. After the solvent had been removed, the residue was purified by column chromatography on silica gel (AcOEt/MeOH = 5:1) to give hS3,7hSdOH-2-carboxylic acid. Next, 6 M aqueous HCl (0.05 mL) was added to a solution of the above acid in tetrahydrofuran (1 mL) at 0 °C, before being stirred at room temperature for 5 h. The removal of the solvent afforded the Nand C-terminal-free heptapeptide H-hS3,7hSdOH-2-OH (DPI-07) (20 mg, 81%). 1 Spectroscopic data for DPI-07: Foam; ½a24 D 16.1 (c 1.00, CHCl3); H NMR (400 MHz, CD3OD) d 4.68 (m, 1H), 4.20–4.48 (m, 4H), 3.96 (m, 1H), 3.48–3.81 (m, 10H), 1.47–2.39 (m, 22H), 0.95–1.06 (m, 24H); [HR-ESI(+)]: m/z calcd for C40H74N7O12 [M+H]+: 844.5395; actual: 844.5350. 34. Synthesized peptides were dissolved in DMSO to final concentrations of 2.5 106, 2.5 105, 2.5 104, and 2.5 103 M. The relative activity of the positive control 1a,25(OH)2D3 was calculated the formula B/Bmax = (C B)/ (A B)%, where A is the OD450 value of the positive control in an SRC(+) well minus the OD450 value of the positive control in an SRC() well; B is the OD450 value of the negative control in an SRC(+) well minus the OD450 value of the positive control in an SRC() well; C is the OD450 value of the peptide sample in an SRC(+) well minus the OD450 value of the peptide sample in an SRC() well. All peptides were precipitated in 2.5 103 M.
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35. Demizu, Y.; Tanaka, M.; Nagano, M.; Kurihara, M.; Doi, M.; Maruyama, T.; Suemune, H. Chem. Pharm. Bull. 2007, 55, 840. 36. The helicity of stapled peptides was higher than that of the corresponding linear peptides. Furthermore, the inhibitory activity of stapled peptides showed stronger than that of the linear peptides (for instance, 220 lM for the stapled peptide H-hS3,7hS-2-OH, 500 lM for the corresponding linear peptide H-hS3,7hS-1-OH).
37. Demizu, Y.; Takahashi, T.; Kaneko, F.; Sato, Y.; Okuda, H.; Ochiai, E.; Horie, K.; Takagi, K.; Kakuda, S.; Takimoto-Kamimura, M.; Kurihara, M. Bioorg. Med. Chem. Lett. 2011, 21, 6104. 38. The N- and C-termini of DPI-07 are capped with N-acetyl and methylamide groups, respectively. It was determined that DIP-07 exists as a mixture of two stereoisomers.
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
Development of stapled short helical peptides capable of inhibiting vitamin D receptor (VDR)–coactivator interactions Yosuke Demizu a,⇑, Saori Nagoya a, Manami Shirakawa a, Megumi Kawamura a,b, Nanako Yamagata a, Yukiko Sato a, Mitsunobu Doi c, Masaaki Kurihara a,b,⇑ a b c
Division of Organic Chemistry, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya, Tokyo 158-8501, Japan Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama 226-8501, Japan Osaka University of Pharmaceutical Sciences, Osaka 569-1094, Japan
a r t i c l e
i n f o
a b s t r a c t
Article history: Received 29 April 2013 Revised 27 May 2013 Accepted 1 June 2013 Available online 10 June 2013
We synthesized stapled helical leucine-based peptides (DPI-01-07) containing 2-aminoisobutyric acid and a covalent cross-linked unit as inhibitors of vitamin D receptor (VDR)–coactivator interactions. The effects of these peptides on the human VDR were examined in an inhibition assay based on the receptor cofactor assay system, and one of them, DPI-07, exhibited potent inhibitory activity (IC50: 3.2 lM). Ó 2013 Elsevier Ltd. All rights reserved.
Keywords: Vitamin D receptor VDR–coactivator interaction inhibitor Stapled helical peptide Protein–protein interaction
The vitamin D receptor (VDR), a nuclear receptor (NR), regulates various biological actions such as calcium uptake, cell proliferation and differentiation, and immune modulation. 1a,25-Dihydroxyvitamin D3 [1a,25(OH)2D3] is an endogenous VDR ligand and has also been demonstrated to regulate these biological events via the VDR.1,2 Therefore, agonistic 1a,25(OH)2D3 derivatives (secosteroi-
dal VDR ligands) and non-secosteroidal VDR ligands are considered to be therapeutic candidates for the treatment of osteoporosis, various types of rickets, secondary hyperparathyroidism, psoriasis, autoimmune diseases, and cancer.3–10 On the other hand, antagonistic VDR ligands are considered to be useful for the treatment of conditions involving hypersensitivity to 1a,25(OH)2D3, such as
(a)
(b)
O Boc
N H
O
H N
N * H 3
O
L
X
O O
n H N
N H
O
X
O
H N O
L
n N * CO2Me H 7
L
A
R3,7R-2
Figure 1. (a) Chemical structure of the stapled heptapeptides. (b) X-ray structure of the stapled heptapeptide R3,7R-2. The covalent side chain is shown in green. The nomenclature R3,7R refers to a peptide with R configurations at its 3rd and 7th positions.
⇑ Corresponding authors. Tel.: +81 3 3700 1141; fax: +81 3 3707 6950. E-mail addresses: [email protected] (Y. Demizu), [email protected] (M. Kurihara). 0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.bmcl.2013.06.002
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Y. Demizu et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4292–4296
coactivator LXXLL
inhibition
coactivator LXXLL target gene
target gene
stapled short helical peptide containing the LXXLL motif
VDR
VDR
Figure 2. Illustration of the mechanism by which the stapled short helical peptides inhibited VDR–coactivator interactions.
Paget’s disease of bone,11 which results in an abnormal bone architecture. To date, several secosteroidal VDR antagonists have been developed.10 In addition, small molecules12–14 and peptides15–17 containing the consensus sequence LXXLL [L: leucine (Leu), X: any amino acid residue] have been demonstrated to inhibit VDR– coactivator interactions. Therefore, they are also considered to be drug candidates for reducing VDR-mediated transactivation. In order to bind to the VDR, small molecules/peptides must contain three Leu residues or Leu mimics.15–17 Furthermore, small peptides have to have an a-helical structure to efficiently interact with the VDR. It is sometimes difficult to form stable a-helices in short peptides. However, a,a-disubstituted a-amino acids (dAAs)18–20 and a covalent cross-linking system21–24 have been demonstrated to be useful for stabilizing the helical structures of such peptides. Especially, 2-aminoisobutyric acid (Aib) has been used as a strong helical promoter.25–28 The covalent cross linker at the i and i+3
O Boc
N H
O
H N
N * H
O
Table 1 IC50 values of peptides DPI-01-07 against human VDR–cofactor interactions according to a receptor cofactor assay systema Entry
Peptide
IC50 (lM)
1 2 3 4 5 6 7b
H-S3,7S-2-OH (DPI-01) H-S3,7R-2-OH (DPI-02) H-R3,7S-2-OH (DPI-03) H-R3,7R-2-OH (DPI-04) H-hS3,7S-2-OH (DPI-05) H-hS3,7hS-2-OH (DPI-06) H-hS3,7hSdOH-2-OH (DPI-07)
610 430 610 600 520 220 3.2
a b
The EC50 value of the positive control, 1a,25(OH)2D3, was 7.7 nM. An isomeric mixture of DPI-07 was examined in the assay.
positions on peptides has been found to be also useful for the helix-stabilization strategy, as reported by Grubbs and Verdine.22,29Recently, we reported that the combined use of 2-
O
n H N
O N H
O
H N
O
O 1) 1M NaOH aq., MeOH, rt, 12h
n
N * CO 2Me H 2) 6M HCl aq., THF, rt, 6h
O
O
H N
H 2N
N * H
O
S 3, 7S-2 S 3, 7R-2 R3, 7S-2 R3, 7R-2 hS3, 7S-2 hS3, 7hS-2
O O
n H N
N H
O
O
H N O
H-S 3,7S-2-OH (DPI-01) H-S 3,7R-2-OH (DPI-02) H-R3, 7S-2-OH (DPI-03) H-R3, 7R-2-OH (DPI-04) H-hS3, 7S-2-OH (DPI-05) H-hS3, 7hS-2-OH (DPI-06)
HO
O
Boc
N H
O
H N
N H
O
O H N
O N H
O
H N
N H
O
CO2Me
2) OsO 4, NMO, H 2O/acetone, rt, 24h
2) 6M HCl aq., THF, rt, 6h
O
N H
H N O
N H
O H N
O
O N H
H N O
OH
O H N
Boc
O
hS3, 7hSdOH-2 HO
H 2N
OH
O 1) 2nd Grubbs cat., CH 2Cl 2, rt, 24h
O
hS3, 7hS-1
1) 1M NaOH aq., MeOH, rt, 12h
n N * CO2H H
O N H
O H N
O
O N H
H N O
O N H
CO2H
H-hS3, 7hSdOH-2-OH (DPI-07)
Scheme 1. Synthesis of the stapled heptapeptides. The nomenclature hS indicates a L-homoserine (L-Hse) derivative.
O N H
CO2Me
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Figure 3. CD spectrum (190–260 nm region) of hS3,7hS-2dOH in TFE solution. Peptide concentration: 0.5 mM.
aminoisobutyric acid (Aib) and a cross-linked unit stabilized the helical structures of short Leu-based peptides with structure A (Fig. 1)as reported by Grubbs and Verdine.30 These peptides were able to efficiently catalyze the enantioselective epoxidation of a,b-unsaturated ketones.31As peptides A contain the LXXLL motif, we considered that any short peptide based on this structure
(a)
(b) HO
OH
O O N H
should bind to the VDR and inhibit VDR–coactivator interactions (Fig. 2). Here, we synthesized several stapled heptapeptides containing the LXXLL motif and evaluated their ability to inhibit VDR–coactivator interactions. The stapled peptides S3,7S-2, S3,7R-2, R3,7S-2, R3,7R-2, hS3,7S-2, and hS3,7hS-2 were prepared according to the previously reported methods.31 Then, their C- and N-termini were deprotected to afford C- and N-terminal-free peptides; that is, H-S3,7S-2-OH (DPI-01), H-S3,7R-2-OH (DPI-02), H-R3,7S-2-OH (DPI-03), H-R3,7 R-2-OH (DPI-04), H-hS3,7S-2-OH (DPI-05), and H-hS3,7hS-2-OH (DPI-06), respectively. The stapled peptide hS3,7hS-2dOH, which possessed two hydroxyl group side chains, was synthesized from the linear peptide hS3,7hS-131 via a ruthenium-catalyzed intramolecular ring-closing metathesis reaction followed by osmium catalyzed dihydroxylation.32 Finally, the C- and N-terminal protecting groups of hS3,7hS-2dOH were removed to obtain H-hS3,7hS-2dOH-OH (DPI-07) (Scheme 1).33 The isomeric mixture of DPI-07 was not separated. The inhibitory activity of the synthetic peptides DPI-01-07 against VDR-coactivator interactions was evaluated using the EnBio receptor cofactor assay system (RCAS) for the VDR kit (Fujikura Kasei Co., Ltd) according to the manufacturer’s instructions.34 The results are summarized in Table 1. Most of the peptides exhibited weak activity against VDR–coactivator interactions (IC50: 220– 610 lM, entries 1–6); however, DPI-07, which possesses the side-chain hydroxyl groups, displayed 70-fold greater activity than the other peptides (IC50: 3.2 lM, entry 7). The dominant conformations of the N- and C-terminal protected peptides S3,7S-2, S3,7R-2, R3,7S-2, R3,7R-2, hS3,7S-2, hS3,7hS2, and hS3,7hS-2dOH were analyzed using the CD spectra in 2,2,2-tri-
H N 2 O
O N H
O H N
O
O N 5 H
H N 6 O
O N H
H N O
DPI-07 derivative B
(c)
Leu630 Leu633
Leu634
Figure 4. (a) Chemical structure of the modeled DPI-07 derivative (B). (b) Modeled structure of B bound to the VDR. The VDR surface is shown in gray. (c) Overlay of the X-ray structure of the coactivator fragment (yellow, PDB ID 3AUN) bound to the VDR and the calculated structure of B bound to the VDR.
Y. Demizu et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4292–4296
fluoroethanol (TFE) solution. The CD spectrum ofhS3,7hS-2dOH showed negative maxima at around 206 and 222 nm, which indicates that hS3,7hS-2dOH was folded into a right-handed helical structure as the preferred secondary structure (Fig. 3).35 The other peptides S3,7S-2, S3,7R-2, R3,7S-2, R3,7R-2, hS3,7S-2, and hS3,7hS-2 had the similar CD patterns and intensities to hS3,7hS-2dOH in TFE solution, respectively.30,31,36 Next, we used the X-ray co-crystal structure (PDB: 3AUN) of VDR bound to the LXXLL motif found in its coactivators to perform docking studies.37 Accordingly, a docking model of a DPI-07 derivative (B) (Fig. 4a)38 bound to the VDR was constructed based on a conformational search in MacroModel (ver. 9.1). AMBER⁄ was used as the force field, and more than 10,000 conformers for B were optimized. The most stable conformation is shown in Figure 4b. In the modeled structure of B, the three Leu residues at the 2nd, 5th, and 6th positions are oriented around the hydrophobic region (Ile234, Ile238, Leu259, Ala263, and Val417) of the VDR. The positions of these three Leu residues in B displayed good agreement with those of the leucine residues in the LXXLL coactivator motif (Leu630, Leu633, and Leu634) during its binding to the VDR (Fig. 4c).37 Furthermore, when B binds to the VDR its hydroxyl group side chains are located on either side of the Leu-containing motif. These hydroxyl groups could increase the affinity of DPI07 for water molecules surrounding the protein, and hence, DPI07 strongly binds to the protein. Although all synthetic peptides had the similar helicity, the peptides without any hydroxyl groups did not show strong inhibitory activity (IC50: 220–610 lM). These results also mean that the side-chain hydroxyl groups of the stapled peptide play an important role to develop the strong affinity. In summary, we developed stapled short helical peptides (DPI01-07) containing an LXXLL motif. The ability of these peptides to inhibit VDR–coactivator interactions was evaluated in an inhibition assay. Peptides DPI-01-06, which do not contain hydroxyl groups in their covalent side chains, exhibited weak inhibitory activity (IC50: 220–610 lM), whereas DPI-07, which possesses the side-chain hydroxyl groups, displayed strong inhibitory activity (IC50: 3.2 lM). Stapled short helical peptides like DPI-07 might be useful for treating conditions involving hypersensitivity to 1a,25(OH)2D3. Transcriptional inhibition assays, the derivatization of further peptides, and studies of their effects on other nuclear receptors are currently underway. Acknowledgments This study was supported, in part, by Grants-in-Aid for Scientific Research (M.K.) from the Japan Society for the Promotion of Science, and a grant from the Kaneka Award for Synthetic Organic Chemistry, Japan (Y.D.). References and notes 1. Feldman, D.; Glorieux, F. H.; Pike, J. W. Vitamin D, 2nd ed.; Elsevier Academic Press: New York, 2005. 2. DeLuca, H. F. FASEB J. 1998, 2, 224. 3. Asano, L.; Ito, I.; Kuwabara, N.; Waku, T.; Yanagisawa, J.; Miyachi, H.; Shimizu, T. FEBS Lett. 2013, 587, 957. 4. Kashiwagi, H.; Ono, Y.; Ohta, M.; Itoh, S.; Ichikawa, F.; Harada, S.; Takeda, S.; Sekiguchi, N.; Ishigai, M.; Takahashi, T. Bioorg. Med. Chem. 2013, 1823, 21. 5. Ciesielski, F.; Sato, Y.; Chebaro, Y.; Moras, D.; Dejaegere, A.; Rochel, N. J. Med. Chem. 2012, 55, 8440. 6. Abe, J.; Nagai, Y.; Higashikuni, R.; Iida, K.; Hirokawa, T.; Nagai, H.; Kominato, K.; Tsuchida, T.; Hirata, M.; Inada, M.; Miyaura, C.; Nagasawa, K. Org. Biomol. Chem. 2012, 10, 7826. 7. Sawada, D.; Tsukuda, Y.; Saito, H.; Kakuda, S.; Takimoto-Kamimura, M.; Ochiai, E.; Takenouchi, K.; Kittaka, A. J. Am. Chem. Soc. 2011, 133, 7215. 8. Fujii, S.; Masuno, H.; Taoda, Y.; Kano, A.; Wongmayura, A.; Nakabayashi, M.; Ito, N.; Shimizu, M.; Kawachi, E.; Hirano, T.; Endo, Y.; Tanatani, A.; Kagechika, H. J. Am. Chem. Soc. 2011, 133, 20933. 9. Demizu, Y.; Nakatsu, A.; Honzawa, S.; Yamashita, A.; Sugiura, T.; Kittaka, A.; Kato, S.; Okuda, H.; Kurihara, M. Lett. Org. Chem. 2011, 8, 43.
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10. Other seco- and nonsecosteroidal agonistic and antagonistic VDR ligands were shown in a recent review: Carlberg, C.; Molnár, F.; Mouriño, A. Expert Opin. Ther. Patents 2012, 22, 417. 11. Roodman, G. D.; Windle, J. J. J. Clin. Invest. 2005, 115, 200. 12. Mita, Y.; Dodo, K.; Noguchi-Yachide, T.; Hashimoto, Y.; Ishikawa, M. Bioorg. Med. Chem. 2013, 21, 993. 13. Nandhikonda, P.; Lynt, W. Z.; McCallum, M. M.; Ara, T.; Baranowski, A. M.; Yuan, N. Y.; Pearson, D.; Bikle, D. D.; Guy, R. K.; Arnold, L. A. J. Med. Chem. 2012, 55, 4640. 14. Mita, Y.; Dodo, K.; Noguchi-Yachide, T.; Miyachi, H.; Makishima, M.; Hashimoto, Y.; Ishikawa, M. Bioorg. Med. Chem. Lett. 2010, 20, 1712. 15. Zella, L. A.; Chang, C.-Y.; McDonnell, D. P.; Pike, J. W. Arch. Biochem. Biophys. 2007, 460, 206. 16. Pike, J. W.; Pathrose, P.; Barmina, O.; Chang, C.-Y.; McDonnell, D. P.; Yamamoto, H.; Shevde, N. K. J. Cell. Biochem. 2003, 88, 252. 17. Pathrose, P.; Barmina, O.; Chang, C.-Y.; McDonnell, D. P.; Shevde, N. K.; Pike, J. W. J. Bone Miner. Res. 2002, 17, 2196. 18. Royo, S.; Borggraeve, W. M. D.; Peggion, C.; Formaggio, F.; Crisma, M.; Jiménez, A. I.; Cativiela, C.; Toniolo, C. J. Am. Chem. Soc. 2005, 127, 2036. 19. Demizu, Y.; Doi, M.; Kurihara, M.; Maruyama, T.; Suemune, H.; Tanaka, M. Chem. Eur. J. 2012, 18, 2430. 20. Demizu, Y.; Doi, M.; Sato, Y.; Tanaka, M.; Okuda, H.; Kurihara, M. Chem. Eur. J. 2011, 17, 11107. 21. Azzarito, V.; Long, K.; Murphy, N. S.; Wilson, A. J. Nat. Chem. 2013, 5, 161. 22. Kim, Y.-W.; Grossmann, T. N.; Verdine, G. L. Nat. Protoc. 2011, 6, 761. 23. Ousaka, N.; Inai, Y.; Kuroda, R. J. Am. Chem. Soc. 2008, 130, 12266. 24. Wang, D.; Liao, W.; Arora, P. S. Angew. Chem., Int. Ed. 2005, 44, 6525. 25. Toniolo, C.; Crisma, M.; Formaggio, F.; Peggion, C.; Broxterman, Q. B.; Kaptein, B. Biopolymers (Pept. Sci.) 2004, 76, 162. 26. Karle, I. L. Biopolymers (Pept. Sci.) 2001, 60, 351. 27. Venkatraman, J.; Shankaramma, S. C.; Balaram, P. Chem. Rev. 2001, 101, 3131– 3152. 28. Oba, M.; Demizu, Y.; Yamagata, N.; Sato, Y.; Doi, M.; Tanaka, M.; Suemune, H.; Okuda, H.; Kurihara, M. Tetrahedron 2010, 66, 2293. 29. Blackwell, H. E.; Sadowsky, J. D.; Howard, R. J.; SaMpson, J. N.; Chao, J. A.; Steinmetz, W. E.; O’Leary, D. J.; Grubbs, R. H. J. Org. Chem. 2001, 66, 5291. 30. Yamagata, N.; Demizu, Y.; Sato, Y.; Doi, M.; Tanaka, M.; Nagasawa, K.; Okuda, H.; Kurihara, M. Tetrahedron Lett. 2011, 52, 798. 31. Demizu, Y.; Yamagata, N.; Nagoya, S.; Sato, Y.; Doi, M.; Tanaka, M.; Nagasawa, K.; Okuda, H.; Kurihara, M. Tetrahedron 2011, 67, 6155. 32. In an inert atmosphere, a solution of hS3,7hS-1 (71 mg, 0.075 mmol) and Grubbs catalyst 2nd generation (32 mg, 0.04 mmol) in CH2Cl2 (20 mL) was stirred at room temperature for 12 h. The solution was then poured into water and extracted with CH2Cl2. The combined organic layer was dried over Na2SO4, and the solvent was removed under reduced pressure to afford the stapled peptide hS3,7hS-2 (61 mg, 88%), which was used for the subsequent reaction without further purification. A solution of the above peptide, Nmethylmorpholine-N-oxide (NMO, 20 mg, 0.17 mmol), and 4% aqueous osmium tetroxide (25 lL) was dissolved in acetone/t-BuOH/water (7 mL, 3/ 3/1) and then stirred at room temperature for 24 h. Next, saturated aqueous sodium thiosulfate (30 mL) was added to the solution and stirred at room temperature for 30 min. The resultant solution was diluted with AcOEt, washed with 1% aqueous HCl and brine, and dried over Na2SO4. After the solvent had been removed, the residue was purified by column chromatography on silica gel (AcOEt/MeOH = 20:1) to give hS3,7hSdOH-2 (39 mg, 62%). Spectroscopic data for hS3,7hSdOH-2: Foam; ½a24 D 33.9 (c 0.50, CHCl3); IR (in CHCl3): m = 3326, 2959, 1665, 1529, 1251, 1159 cm1; 1H NMR (400 MHz, CDCl3) 7.96 (d, J = 6.4 Hz, 1H), 7.56 (d, J = 4.8 Hz, 1H), 7.26 (m, 2H), 7.16 (d, J = 10.0 Hz, 1H), 6.98 (d, J = 4.8 Hz, 1H), 6.56 (m, 1H), 4.99–5.12 (m, 2H), 4.50–4.78 (m, 2H), 4.25 (m, 1H), 4.13 (m, 1H), 3.94–4.02 (m, 3H), 3.73 (s, 3H), 3.54–3.68 (m, 6H), 3.22–3.46 (m, 3H), 2.22 (m, 1H), 1.81–1.94 (m, 4H), 1.61–1.71 (m, 10H), 1.43–1.48 (m, 14H), 1.00–1.30 (m, 2H), 0.89–0.99 (m, 24H); [HR-ESI(+)]: m/z calcd for C46H83N7O14Na [M+Na]+: 980.5890; actual: 980.5883. 33. A solution of hS3,7hSdOH-2 (30 mg, 0.03 mmol) and 1 M aqueous NaOH (0.06 mL) in MeOH (1 mL) was stirred at room temperature for 12 h. Then, the solution was neutralized with 1 M aqueous HCl, extracted with AcOEt, and dried over Na2SO4. After the solvent had been removed, the residue was purified by column chromatography on silica gel (AcOEt/MeOH = 5:1) to give hS3,7hSdOH-2-carboxylic acid. Next, 6 M aqueous HCl (0.05 mL) was added to a solution of the above acid in tetrahydrofuran (1 mL) at 0 °C, before being stirred at room temperature for 5 h. The removal of the solvent afforded the Nand C-terminal-free heptapeptide H-hS3,7hSdOH-2-OH (DPI-07) (20 mg, 81%). 1 Spectroscopic data for DPI-07: Foam; ½a24 D 16.1 (c 1.00, CHCl3); H NMR (400 MHz, CD3OD) d 4.68 (m, 1H), 4.20–4.48 (m, 4H), 3.96 (m, 1H), 3.48–3.81 (m, 10H), 1.47–2.39 (m, 22H), 0.95–1.06 (m, 24H); [HR-ESI(+)]: m/z calcd for C40H74N7O12 [M+H]+: 844.5395; actual: 844.5350. 34. Synthesized peptides were dissolved in DMSO to final concentrations of 2.5 106, 2.5 105, 2.5 104, and 2.5 103 M. The relative activity of the positive control 1a,25(OH)2D3 was calculated the formula B/Bmax = (C B)/ (A B)%, where A is the OD450 value of the positive control in an SRC(+) well minus the OD450 value of the positive control in an SRC() well; B is the OD450 value of the negative control in an SRC(+) well minus the OD450 value of the positive control in an SRC() well; C is the OD450 value of the peptide sample in an SRC(+) well minus the OD450 value of the peptide sample in an SRC() well. All peptides were precipitated in 2.5 103 M.
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35. Demizu, Y.; Tanaka, M.; Nagano, M.; Kurihara, M.; Doi, M.; Maruyama, T.; Suemune, H. Chem. Pharm. Bull. 2007, 55, 840. 36. The helicity of stapled peptides was higher than that of the corresponding linear peptides. Furthermore, the inhibitory activity of stapled peptides showed stronger than that of the linear peptides (for instance, 220 lM for the stapled peptide H-hS3,7hS-2-OH, 500 lM for the corresponding linear peptide H-hS3,7hS-1-OH).
37. Demizu, Y.; Takahashi, T.; Kaneko, F.; Sato, Y.; Okuda, H.; Ochiai, E.; Horie, K.; Takagi, K.; Kakuda, S.; Takimoto-Kamimura, M.; Kurihara, M. Bioorg. Med. Chem. Lett. 2011, 21, 6104. 38. The N- and C-termini of DPI-07 are capped with N-acetyl and methylamide groups, respectively. It was determined that DIP-07 exists as a mixture of two stereoisomers.