Bioorganic & Medicinal Chemistry Letters 25 (2015) 1925–1928
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Histone H3 peptide based LSD1-selective inhibitors Taeko Kakizawa a, Yosuke Ota b, Yukihiro Itoh b, Hiroki Tsumoto c, Takayoshi Suzuki b,d,⇑ a
Department of Chemistry and Biochemistry, School of Advanced Science and Engineering, Waseda University, Shinjuku, Tokyo 169-8555, Japan Graduate School of Medical Science, Kyoto Prefectural University of Medicine, 1-5 Shimogamohangi-cho, Sakyo-ku, Kyoto 606-0823, Japan c Research Team for Mechanism of Aging, Tokyo Metropolitan Institute of Gerontology, 35-2 Sakae-cho, Itabashi-ku, Tokyo 173-0015, Japan d CREST, Japan Science and Technology Agency (JST), 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan b
a r t i c l e
i n f o
Article history: Received 21 February 2015 Revised 10 March 2015 Accepted 12 March 2015 Available online 20 March 2015 Keywords: Histone H3 Peptide Lysine-specific demethylase 1 (LSD1) Inhibitor
a b s t r a c t A series of candidates for the histone H3 peptide based LSD1-selective inhibitor were designed and synthesized. Among peptides 1a–c and 2a–c, peptide 1a, which has a phenylcyclopropylamine (PCPA) moiety at Lys-4 of the 21 amino acid residues of histone H3, was the most potent LSD1-selective inhibitor. Truncation studies of peptide 1a revealed the significance of the peptide sequence length. These findings will be useful for the further development of histone H3 peptide based LSD1-selective inhibitors. Ó 2015 Elsevier Ltd. All rights reserved.
Chromatin contains DNA and the full complement of DNAbound proteins, including non-histone proteins in addition to the core histones. The chromatin architecture is a key determinant in the regulation of epigenetic gene expression, and is strongly influenced by the posttranslational modification of histones, such as acetylation, methylation, and phosphorylation.1 Histone methylation is a dynamic process regulated by the addition of methyl groups by histone methyltransferases and the removal of methyl groups from mono- and di-methylated lysines by lysine-specific demethylase 1 (LSD1), and from mono-, di- and tri-methylated lysines by Jumonji C-domain containing demethylases.1–3 LSD1 is a histone lysine demethylase that demethylates mono- and di-methylated Lys4 of histone H3 (H3K4me1/2) and H3K9me1/ 2.2,4,5 LSD1 can also demethylate methylated lysines on nonhistone proteins, such as p53, E2F1, DNA methyltransferase 1, and STAT3.6–9 LSD1 has a fair degree of structural similarity and homology to amine oxidases, including monoamine oxidases (MAOs), all of which are flavin-dependent amine oxidases that catalyze the oxidation of nitrogen–hydrogen bonds and/or nitrogen–carbon bonds.2 In addition, it has been suggested that LSD1 is associated with certain disease states, including cancer, herpes simplex infection, and globin disorders.5,10–12 Therefore, LSD1 is an interesting target for the development of new drugs to treat cancer and other diseases. To date, a number of LSD1 inhibitors have been identified, and some of them are peptide⇑ Corresponding author. Tel./fax: +81 75 703 4937. E-mail address: [email protected] (T. Suzuki). http://dx.doi.org/10.1016/j.bmcl.2015.03.030 0960-894X/Ó 2015 Elsevier Ltd. All rights reserved.
based inhibitors.13 We recently reported histone H3 peptide– phenylcyclopropylamine (PCPA) conjugate 1a (Fig. 1), an LSD1 inhibitor.14 Following that finding, we performed further investigations of histone H3 based peptides in an effort to find novel LSD1selective inhibitors. We describe herein the design, synthesis, and enzyme (LSD1 and MAOs) inhibitory activity of a series of histone H3 based peptides. PCPA-Lys-4 H3-21 (1a) (Fig. 1) is a potent LSD1 inhibitor bearing a PCPA moiety at Lys-4 in the 21 amino acid residues of histone H3 (H3-21).14 Peptide 1a displayed strong and time-dependent inactivation of LSD1. The proposed mechanism of the LSD1 inactivation by 1a is shown in Figure 1. It is suggested that peptide 1a is efficiently recognized by LSD1, and the PCPA moiety inactivates LSD1 by single-electron transfer, radical opening of the cyclopropyl ring, and covalent bond formation with FAD in the active site of LSD1. We initially designed histone H3 peptide based LSD1 inhibitor candidates 1b and 1c (Fig. 2) in which the PCPA moiety is replaced with 2,5-dihydro-1H-pyrrole (DHP) (1b) and 1,2,3,6-tetrahydropyridine (THP) (1c). Figure 3a shows the catalytic mechanism of the demethylation of methylated lysine substrates. First, the methylated lysine substrate is converted into an iminium cation through oxidation of the amine by FAD. Next, the addition of a water molecule to the iminium cation and the subsequent release of formaldehyde afford the demethylated lysine product. As shown in Figure 3b, cyclic allyl amines 1b and 1c were expected to be oxidized to the iminium cation, as in the case of the methylated lysine substrate. Then, the formyl moiety would be generated by the
1926
T. Kakizawa et al. / Bioorg. Med. Chem. Lett. 25 (2015) 1925–1928 R N
H3 C H3 C
N
O NH
N
R N
H 3C H 3C
N
N
R N
H 3C
NH
O Ph
FAD
O
H 3C
N
N
O Ph
R
O
HN
HN
O NH H3 C H3 C
ART
N H
NH
N H
NH 2
O
N
H2 O
Ph
HN
R
N
H3 C
N
H3 C
N
O HO
Ph
O
N
O NH
O Ph
FAD-PCPA adduct
QTARKSTGGKAPRKQLA
N H
O
N H
O
O N H
PCPA-Lys-4 H3-21 (1a)
O H3-21
Figure 1. Proposed mechanism of LSD1 inhibition by PCPA-Lys-4 H3-21 (1a).
n
N
OMs
N
n
a ART
QTARKSTGGKAPRKQLA
N H
ART
N H
O
DHP-Lys-4 H3-21 (1b): n = 1 THP-Lys-4 H3-21 (1c): n = 2
ART
QTARKSTGGKAPRKQLA
N H
O
O 1b: n = 1 1c: n = 2
Mesyl-Lys-4 H3-21
Figure 2. Structures of THP-Lys-4 H3-21 (1b) and DHP-Lys-4 H3-21 (1c).
QTARKSTGGKAPRKQLA
Scheme 1. Reagents and conditions: (a) DHP or THP, Et3N, H2O, CH3CN, rt, 7.2% for 1b, 31% for 1c.
addition of a water molecule. However, in this case, the iminium cation could be re-formed by the intramolecular condensation between the formyl group and the amine. Finally, the iminium cation would be nucleophilically attacked by N5 of the reduced FAD, thereby leading to LSD1 inactivation.15 Peptides 1b and 1c were synthesized from their precursor peptide mesyl-Lys-4 H3-2116–18 by the displacement of the mesyl group with the corresponding amine (Scheme 1).14 Specifically,
DHP and THP were treated with mesyl-Lys-4 H3-21 to give desired peptide derivatives DHP-Lys-4 H3-21 (1b) and THP-Lys-4 H3-21 (1c). Peptides 1a, 1b, and 1c were subjected to an assay to determine their inhibitory activity toward recombinant LSD1 using an H2O2 detector, and the results are summarized in Table 1. Peptide 1b showed slightly lower potency against LSD1 (IC50 = 0.223 lM) than peptide 1a (IC50 = 0.148 lM), whereas peptide 1c displayed
a FADH-
FAD
HN CH 3 N CH 3 methylated lysine
O
HN CH3
O
LSD1
HN
HN
H 2O
CH 3
O
N
O
N CH2
OH
CH 3 NH
HCHO
demethylated lysine
b R H3 C H3 C
N
N
NH
N FAD
H 3C
O
H 3C FADH -
O
R N
H N
O NH
N O
R N
H3 C HN
HN O N n
LSD1
HN
O N
1b: n = 1 1c: n = 2
n
O NH
N O
O
N n 1b/1c-FAD adduct
H2 O HN
HN O
H3 C
H N
O N n HO
NH n H O
Figure 3. Expected mechanism of LSD1 inhibition by THP-Lys-4 H3-21 (1b) and DHP-Lys-4 H3-21 (1c).
T. Kakizawa et al. / Bioorg. Med. Chem. Lett. 25 (2015) 1925–1928 Table 1 LSD1 and MAO inhibitory activities of PCPA and peptides 1 and 2 IC50a (lM)
Compound
PCPA PCPA-Lys-4 H3-21 (1a) DHP-Lys-4 H3-21 (1b) THP-Lys-4 H3-21 (1c) PCPA-Lys-9 H3-21 (2a) DHP-Lys-9 H3-21 (2b) THP-Lys-9 H3-21 (2c) a
LSD1
MAO A
MAO B
>100 0.148 ± 0.325 0.223 ± 0.006 11.0 ± 0.3 0.283 ± 0.032 >100 88.0 ± 2.8
7.87 ± 1.95 >100 >100 >100 >100 >100 >100
4.90 ± 0.24 >100 >100 >100 >100 >100 >100
Values are means ± S.D. of at least three experiments.
Table 2 LSD1 and MAOs inhibitory activities of truncated peptides 3–6 Compound
PCPA-Lys-4 H3-21 (1a) PCPA-Lys-4 H3-17 (3) PCPA-Lys-4 H3-13 (4) PCPA-Lys-4 H3-9 (5) PCPA-Lys-4 H3-5 (6) a b
Sequencea
IC50b (lM) LSD1
MAO A
MAO B
ARTXQTARKSTGGKAPRKQLA
0.148 ± 0.325
>100
>100
ARTXQTARKSTGGKAPR
0.158 ± 0.251
>100
>100
ARTXQTARKSTGG
0.356 ± 0.004
>100
>100
ARTXQTARK
0.443 ± 0.006
>100
>100
ARTXQ
>10
>100
>100
Amino acid X is a PCPA containing lysine residue. Values are means ± S.D. of at least three experiments.
weak LSD1 inhibitory activity (IC50 = 11.0 lM). Peptides 1a, 1b, and 1c were also subjected to an assay to determine their inhibitory activity toward monoamine oxidases MAO A and MAO B, to examine their selectivity for LSD1 rather than MAOs, because LSD1 is structurally homologous with MAOs.2 As shown in Table 1, peptides 1a, 1b, and 1c exhibited low inhibitory activity toward MAO A and MAO B (IC50 = >100 lM), suggesting their high selectivity for LSD1 rather than MAOs. As LSD1 is known to remove methyl groups from H3K9me1/2 as well as H3K4me1/2,4,5 we prepared three Lys-9 H3-21 peptides, namely, PCPA-Lys-9 H3-21 (2a), DHP-Lys-9 H3-21 (2b), and
1927
THP-Lys-9 H3-21 (2c) in a manner analogous to the synthesis of the corresponding Lys-4 peptides. Although peptide 2a showed relatively high LSD1 inhibitory activity (IC50 = 0.283 lM), the Lys-9 peptides tended to display low potency against LSD1 (IC50 = 0.283 lM (2a), >100 lM (2b), and 88.0 lM (2c)) compared with the corresponding Lys-4 peptides (IC50 = 0.148 lM (1a), 0.223 lM (1b), and 11.0 lM (1c)). This is probably due to the substrate preference of LSD1 to H3K4me1/2 over H3K9me1/2 in the enzyme assay conditions.2 As for the inhibitory activity toward MAOs, peptides 2a, 2b, and 2c inhibited neither MAO A nor MAO B (Table 1). To develop the downsized LSD1 inhibitors, we next performed truncation studies of the histone peptide. Because peptide 1a showed the highest potency against LSD1 among peptides 1a–c and 2a–c, we selected peptide 1a as the template sequence and removed amino acids in the C-terminal region. Several sequence lengths of the truncated peptides (Table 2) having a lysine residue containing PCPA (designated as amino acid X in the table) at the fourth position were synthesized using a procedure similar to the preparation of peptide 1a,14 and the inhibitory activities of the resultant peptides toward LSD and MAOs were evaluated. As shown in Table 2, the removal of the amino acids led to a gradual loss of the inhibitory activity toward LSD1, and they were inactive toward MAO A and MAO B. Peptide PCPA-Lys-4 H3-17 (3), which has four amino acid residues less than peptide 1a, showed mild loss of potency with IC50 = 0.156 lM. Shorter peptides PCPALys-4 H3-13 (4) and PCPA-Lys-4 H3-9 (5) maintained moderate inactivation efficiency with IC50 values of 0.356 lM and 0.443 lM, respectively. Pentapeptide PCPA-Lys-4 H3-5 (6) showed marked loss of potency, its IC50 being >10 lM. The loss of LSD1 inhibitory activity of the shorter peptides is considered to be due to the lack of the three consecutive c-turn structures that could be formed in the Lys-4 H3-21 peptides (Fig. 4).19 In summary, H3-21 peptides having several types of modified Lys-4 and Lys-9 residues were newly designed and synthesized, and their inhibitory activities toward LSD1 and MAOs were evaluated. Among peptides 1a–c and 2a–c, peptide 1a was the most potent and selective LSD1 inhibitor. To downsize peptide 1a, we synthesized histone H3 peptides having several sequence lengths and bearing the PCPA moiety in the side chain of Lys-4. The LSD1 inhibition efficiency was gradually decreased as the peptides were downsized. LSD1-selective inhibitors have considerable potential both for the development of novel therapeutic agents and as tools for biological research. Detailed studies of the histone H3 peptide based LSD1-selective inhibitors and their analogues are under way.
Figure 4. Secondary structure of H3 peptide at LSD1 catalytic site (PDB: 2UXN). View of conformation of H3 peptide (stick representation) (left). Schematic representation of H3 peptide (right). Three consecutive c-turn structures are indicated in bold.
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T. Kakizawa et al. / Bioorg. Med. Chem. Lett. 25 (2015) 1925–1928
Acknowledgements We thank Professor Takaki Koide and Dr. Miki Suzuki for technical support. This work is supported in part by a Grant-in-Aid for Scientific Research (T.K.; C, 26460159) from MEXT (The Ministry of Education, Culture, Sports, Science and Technology), Waseda University Grant for Special Research Projects (T.K.; project number 2014K-6139), JST CREST Program (T.S.), Takeda Science Foundation (T.S.), Terumo Life Science Foundation (T.S.), and Mochida Memorial Foundation for Medical and Pharmaceutical Research (T.S.). Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bmcl.2015.03. 030. References and notes 1. Itoh, Y.; Suzuki, T.; Miyata, N. Mol. Biosyst. 2013, 9, 873. 2. Shi, Y.; Lan, F.; Matson, C.; Mulligan, P.; Whetstine, J. R.; Cole, P. A.; Casero, R. A.; Shi, Y. Cell 2004, 119, 941. 3. Tsukada, Y.; Fang, J.; Erdjument-Bromage, H.; Warren, M. E.; Borchers, C. H.; Tempst, P.; Zhang, Y. Nature 2006, 439, 811. 4. Metzger, E.; Wissmann, M.; Yin, N.; Müller, J. M.; Schneider, R.; Peters, A. H.; Günther, T.; Buettner, R.; Schüle, R. Nature 2005, 437, 436.
5. Liang, Y.; Vogel, J. L.; Narayanan, A.; Peng, H.; Kristie, T. M. Nat. Med. 2009, 15, 1312. 6. Jing, H.; Sengupta, R.; Espejo, A. B.; Min Gyu, L.; Dorsey, J. A.; Richter, M.; Opravil, S.; Shiekhattar, R.; Bedford, M. T.; Jenuwein, T.; Berger, S. L. Nature 2007, 449, 105. 7. Kontaki, H.; Taliannidis, I. Mol. Cell 2010, 39, 152. 8. Jing, W.; Hevi, S.; Kurash, J. K.; Hong, L.; Gay, F.; Bajko, J.; Hui, S.; Weitao, S.; Hua, C.; Guoliang, X.; Gaudet, F.; En, L.; Taiping, C. Nat. Genet. 2009, 41, 125. 9. Yang, J.; Huang, J.; Dasgupta, M.; Sears, N.; Miyagi, M.; Wang, B.; Chance, M. R.; Chen, X.; Du, Y.; Wang, Y.; An, L.; Wang, Q.; Lu, T.; Zhang, X.; Wang, Z.; Stark, G. R. Proc. Natl. Acad. Sci. U.S.A. 2010, 107, 21499. 10. Schulte, J. H.; Lim, S.; Schramm, A.; Friedrichs, N.; Koster, J.; Versteeg, R.; Ora, I.; Pajtler, K.; Klein-Hitpass, L.; Kuhfittig-Kulle, S.; Metzger, E.; Schule, R.; Eggert, A.; Buettner, R.; Kirfel, J. Cancer Res. 2009, 69, 2065. 11. Schenk, T.; Chen, W. C.; Göllner, S.; Howell, L.; Jin, L.; Hebestreit, K.; Klein, H.U.; Popescu, A. C.; Burnett, A.; Mills, K.; Casero, R. A., Jr.; Marton, L.; Woster, P.; Minden, M. D.; Dugas, M.; Wang, J. C. Y.; Dick, J. E.; Müller-Tidow, C.; Petrie, K.; Zelent, A. Nat. Med. 2012, 18, 605. 12. Shi, L.; Cui, S.; Engel, J. D.; Tanabe, O. Nat. Med. 2013, 19, 291. 13. Suzuki, T.; Miyata, N. J. Med. Chem. 2011, 54, 8236. 14. Ogasawara, D.; Itoh, Y.; Tsumoto, H.; Kakizawa, T.; Mino, K.; Fukuhara, K.; Nakagawa, H.; Hasegawa, M.; Sasaki, R.; Mizukami, T.; Miyata, N.; Suzuki, T. Angew. Chem., Int. Ed. 2013, 52, 8620. 15. Wichitnithad, W.; O’Callaghan, J. P.; Miller, D. B.; Train, B. C.; Callery, P. S. Bioorg. Med. Chem. 2011, 19, 7482. 16. Culhane, J. C.; Wang, D.; Yen, P. M.; Cole, P. A. J. Am. Chem. Soc. 2010, 132, 3164. 17. Szewczuk, L. M.; Culhane, J. C.; Yang, M.; Majumdar, A.; Yu, H.; Cole, P. A. Biochemistry 2007, 46, 6892. 18. Culhane, J. C.; Szewczuk, L. M.; Liu, X.; Da, G.; Marmorstein, R.; Cole, P. A. J. Am. Chem. Soc. 2006, 128, 4536. 19. Yang, M.; Culhane, J. C.; Szewczuk, L. M.; Gocke, C. B.; Brautigam, C. A.; Tomchick, D. R.; Machius, M.; Cole, P. A.; Yu, H. Nat. Struct. Mol. Biol. 2007, 14, 535.
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Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
Histone H3 peptide based LSD1-selective inhibitors Taeko Kakizawa a, Yosuke Ota b, Yukihiro Itoh b, Hiroki Tsumoto c, Takayoshi Suzuki b,d,⇑ a
Department of Chemistry and Biochemistry, School of Advanced Science and Engineering, Waseda University, Shinjuku, Tokyo 169-8555, Japan Graduate School of Medical Science, Kyoto Prefectural University of Medicine, 1-5 Shimogamohangi-cho, Sakyo-ku, Kyoto 606-0823, Japan c Research Team for Mechanism of Aging, Tokyo Metropolitan Institute of Gerontology, 35-2 Sakae-cho, Itabashi-ku, Tokyo 173-0015, Japan d CREST, Japan Science and Technology Agency (JST), 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan b
a r t i c l e
i n f o
Article history: Received 21 February 2015 Revised 10 March 2015 Accepted 12 March 2015 Available online 20 March 2015 Keywords: Histone H3 Peptide Lysine-specific demethylase 1 (LSD1) Inhibitor
a b s t r a c t A series of candidates for the histone H3 peptide based LSD1-selective inhibitor were designed and synthesized. Among peptides 1a–c and 2a–c, peptide 1a, which has a phenylcyclopropylamine (PCPA) moiety at Lys-4 of the 21 amino acid residues of histone H3, was the most potent LSD1-selective inhibitor. Truncation studies of peptide 1a revealed the significance of the peptide sequence length. These findings will be useful for the further development of histone H3 peptide based LSD1-selective inhibitors. Ó 2015 Elsevier Ltd. All rights reserved.
Chromatin contains DNA and the full complement of DNAbound proteins, including non-histone proteins in addition to the core histones. The chromatin architecture is a key determinant in the regulation of epigenetic gene expression, and is strongly influenced by the posttranslational modification of histones, such as acetylation, methylation, and phosphorylation.1 Histone methylation is a dynamic process regulated by the addition of methyl groups by histone methyltransferases and the removal of methyl groups from mono- and di-methylated lysines by lysine-specific demethylase 1 (LSD1), and from mono-, di- and tri-methylated lysines by Jumonji C-domain containing demethylases.1–3 LSD1 is a histone lysine demethylase that demethylates mono- and di-methylated Lys4 of histone H3 (H3K4me1/2) and H3K9me1/ 2.2,4,5 LSD1 can also demethylate methylated lysines on nonhistone proteins, such as p53, E2F1, DNA methyltransferase 1, and STAT3.6–9 LSD1 has a fair degree of structural similarity and homology to amine oxidases, including monoamine oxidases (MAOs), all of which are flavin-dependent amine oxidases that catalyze the oxidation of nitrogen–hydrogen bonds and/or nitrogen–carbon bonds.2 In addition, it has been suggested that LSD1 is associated with certain disease states, including cancer, herpes simplex infection, and globin disorders.5,10–12 Therefore, LSD1 is an interesting target for the development of new drugs to treat cancer and other diseases. To date, a number of LSD1 inhibitors have been identified, and some of them are peptide⇑ Corresponding author. Tel./fax: +81 75 703 4937. E-mail address: [email protected] (T. Suzuki). http://dx.doi.org/10.1016/j.bmcl.2015.03.030 0960-894X/Ó 2015 Elsevier Ltd. All rights reserved.
based inhibitors.13 We recently reported histone H3 peptide– phenylcyclopropylamine (PCPA) conjugate 1a (Fig. 1), an LSD1 inhibitor.14 Following that finding, we performed further investigations of histone H3 based peptides in an effort to find novel LSD1selective inhibitors. We describe herein the design, synthesis, and enzyme (LSD1 and MAOs) inhibitory activity of a series of histone H3 based peptides. PCPA-Lys-4 H3-21 (1a) (Fig. 1) is a potent LSD1 inhibitor bearing a PCPA moiety at Lys-4 in the 21 amino acid residues of histone H3 (H3-21).14 Peptide 1a displayed strong and time-dependent inactivation of LSD1. The proposed mechanism of the LSD1 inactivation by 1a is shown in Figure 1. It is suggested that peptide 1a is efficiently recognized by LSD1, and the PCPA moiety inactivates LSD1 by single-electron transfer, radical opening of the cyclopropyl ring, and covalent bond formation with FAD in the active site of LSD1. We initially designed histone H3 peptide based LSD1 inhibitor candidates 1b and 1c (Fig. 2) in which the PCPA moiety is replaced with 2,5-dihydro-1H-pyrrole (DHP) (1b) and 1,2,3,6-tetrahydropyridine (THP) (1c). Figure 3a shows the catalytic mechanism of the demethylation of methylated lysine substrates. First, the methylated lysine substrate is converted into an iminium cation through oxidation of the amine by FAD. Next, the addition of a water molecule to the iminium cation and the subsequent release of formaldehyde afford the demethylated lysine product. As shown in Figure 3b, cyclic allyl amines 1b and 1c were expected to be oxidized to the iminium cation, as in the case of the methylated lysine substrate. Then, the formyl moiety would be generated by the
1926
T. Kakizawa et al. / Bioorg. Med. Chem. Lett. 25 (2015) 1925–1928 R N
H3 C H3 C
N
O NH
N
R N
H 3C H 3C
N
N
R N
H 3C
NH
O Ph
FAD
O
H 3C
N
N
O Ph
R
O
HN
HN
O NH H3 C H3 C
ART
N H
NH
N H
NH 2
O
N
H2 O
Ph
HN
R
N
H3 C
N
H3 C
N
O HO
Ph
O
N
O NH
O Ph
FAD-PCPA adduct
QTARKSTGGKAPRKQLA
N H
O
N H
O
O N H
PCPA-Lys-4 H3-21 (1a)
O H3-21
Figure 1. Proposed mechanism of LSD1 inhibition by PCPA-Lys-4 H3-21 (1a).
n
N
OMs
N
n
a ART
QTARKSTGGKAPRKQLA
N H
ART
N H
O
DHP-Lys-4 H3-21 (1b): n = 1 THP-Lys-4 H3-21 (1c): n = 2
ART
QTARKSTGGKAPRKQLA
N H
O
O 1b: n = 1 1c: n = 2
Mesyl-Lys-4 H3-21
Figure 2. Structures of THP-Lys-4 H3-21 (1b) and DHP-Lys-4 H3-21 (1c).
QTARKSTGGKAPRKQLA
Scheme 1. Reagents and conditions: (a) DHP or THP, Et3N, H2O, CH3CN, rt, 7.2% for 1b, 31% for 1c.
addition of a water molecule. However, in this case, the iminium cation could be re-formed by the intramolecular condensation between the formyl group and the amine. Finally, the iminium cation would be nucleophilically attacked by N5 of the reduced FAD, thereby leading to LSD1 inactivation.15 Peptides 1b and 1c were synthesized from their precursor peptide mesyl-Lys-4 H3-2116–18 by the displacement of the mesyl group with the corresponding amine (Scheme 1).14 Specifically,
DHP and THP were treated with mesyl-Lys-4 H3-21 to give desired peptide derivatives DHP-Lys-4 H3-21 (1b) and THP-Lys-4 H3-21 (1c). Peptides 1a, 1b, and 1c were subjected to an assay to determine their inhibitory activity toward recombinant LSD1 using an H2O2 detector, and the results are summarized in Table 1. Peptide 1b showed slightly lower potency against LSD1 (IC50 = 0.223 lM) than peptide 1a (IC50 = 0.148 lM), whereas peptide 1c displayed
a FADH-
FAD
HN CH 3 N CH 3 methylated lysine
O
HN CH3
O
LSD1
HN
HN
H 2O
CH 3
O
N
O
N CH2
OH
CH 3 NH
HCHO
demethylated lysine
b R H3 C H3 C
N
N
NH
N FAD
H 3C
O
H 3C FADH -
O
R N
H N
O NH
N O
R N
H3 C HN
HN O N n
LSD1
HN
O N
1b: n = 1 1c: n = 2
n
O NH
N O
O
N n 1b/1c-FAD adduct
H2 O HN
HN O
H3 C
H N
O N n HO
NH n H O
Figure 3. Expected mechanism of LSD1 inhibition by THP-Lys-4 H3-21 (1b) and DHP-Lys-4 H3-21 (1c).
T. Kakizawa et al. / Bioorg. Med. Chem. Lett. 25 (2015) 1925–1928 Table 1 LSD1 and MAO inhibitory activities of PCPA and peptides 1 and 2 IC50a (lM)
Compound
PCPA PCPA-Lys-4 H3-21 (1a) DHP-Lys-4 H3-21 (1b) THP-Lys-4 H3-21 (1c) PCPA-Lys-9 H3-21 (2a) DHP-Lys-9 H3-21 (2b) THP-Lys-9 H3-21 (2c) a
LSD1
MAO A
MAO B
>100 0.148 ± 0.325 0.223 ± 0.006 11.0 ± 0.3 0.283 ± 0.032 >100 88.0 ± 2.8
7.87 ± 1.95 >100 >100 >100 >100 >100 >100
4.90 ± 0.24 >100 >100 >100 >100 >100 >100
Values are means ± S.D. of at least three experiments.
Table 2 LSD1 and MAOs inhibitory activities of truncated peptides 3–6 Compound
PCPA-Lys-4 H3-21 (1a) PCPA-Lys-4 H3-17 (3) PCPA-Lys-4 H3-13 (4) PCPA-Lys-4 H3-9 (5) PCPA-Lys-4 H3-5 (6) a b
Sequencea
IC50b (lM) LSD1
MAO A
MAO B
ARTXQTARKSTGGKAPRKQLA
0.148 ± 0.325
>100
>100
ARTXQTARKSTGGKAPR
0.158 ± 0.251
>100
>100
ARTXQTARKSTGG
0.356 ± 0.004
>100
>100
ARTXQTARK
0.443 ± 0.006
>100
>100
ARTXQ
>10
>100
>100
Amino acid X is a PCPA containing lysine residue. Values are means ± S.D. of at least three experiments.
weak LSD1 inhibitory activity (IC50 = 11.0 lM). Peptides 1a, 1b, and 1c were also subjected to an assay to determine their inhibitory activity toward monoamine oxidases MAO A and MAO B, to examine their selectivity for LSD1 rather than MAOs, because LSD1 is structurally homologous with MAOs.2 As shown in Table 1, peptides 1a, 1b, and 1c exhibited low inhibitory activity toward MAO A and MAO B (IC50 = >100 lM), suggesting their high selectivity for LSD1 rather than MAOs. As LSD1 is known to remove methyl groups from H3K9me1/2 as well as H3K4me1/2,4,5 we prepared three Lys-9 H3-21 peptides, namely, PCPA-Lys-9 H3-21 (2a), DHP-Lys-9 H3-21 (2b), and
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THP-Lys-9 H3-21 (2c) in a manner analogous to the synthesis of the corresponding Lys-4 peptides. Although peptide 2a showed relatively high LSD1 inhibitory activity (IC50 = 0.283 lM), the Lys-9 peptides tended to display low potency against LSD1 (IC50 = 0.283 lM (2a), >100 lM (2b), and 88.0 lM (2c)) compared with the corresponding Lys-4 peptides (IC50 = 0.148 lM (1a), 0.223 lM (1b), and 11.0 lM (1c)). This is probably due to the substrate preference of LSD1 to H3K4me1/2 over H3K9me1/2 in the enzyme assay conditions.2 As for the inhibitory activity toward MAOs, peptides 2a, 2b, and 2c inhibited neither MAO A nor MAO B (Table 1). To develop the downsized LSD1 inhibitors, we next performed truncation studies of the histone peptide. Because peptide 1a showed the highest potency against LSD1 among peptides 1a–c and 2a–c, we selected peptide 1a as the template sequence and removed amino acids in the C-terminal region. Several sequence lengths of the truncated peptides (Table 2) having a lysine residue containing PCPA (designated as amino acid X in the table) at the fourth position were synthesized using a procedure similar to the preparation of peptide 1a,14 and the inhibitory activities of the resultant peptides toward LSD and MAOs were evaluated. As shown in Table 2, the removal of the amino acids led to a gradual loss of the inhibitory activity toward LSD1, and they were inactive toward MAO A and MAO B. Peptide PCPA-Lys-4 H3-17 (3), which has four amino acid residues less than peptide 1a, showed mild loss of potency with IC50 = 0.156 lM. Shorter peptides PCPALys-4 H3-13 (4) and PCPA-Lys-4 H3-9 (5) maintained moderate inactivation efficiency with IC50 values of 0.356 lM and 0.443 lM, respectively. Pentapeptide PCPA-Lys-4 H3-5 (6) showed marked loss of potency, its IC50 being >10 lM. The loss of LSD1 inhibitory activity of the shorter peptides is considered to be due to the lack of the three consecutive c-turn structures that could be formed in the Lys-4 H3-21 peptides (Fig. 4).19 In summary, H3-21 peptides having several types of modified Lys-4 and Lys-9 residues were newly designed and synthesized, and their inhibitory activities toward LSD1 and MAOs were evaluated. Among peptides 1a–c and 2a–c, peptide 1a was the most potent and selective LSD1 inhibitor. To downsize peptide 1a, we synthesized histone H3 peptides having several sequence lengths and bearing the PCPA moiety in the side chain of Lys-4. The LSD1 inhibition efficiency was gradually decreased as the peptides were downsized. LSD1-selective inhibitors have considerable potential both for the development of novel therapeutic agents and as tools for biological research. Detailed studies of the histone H3 peptide based LSD1-selective inhibitors and their analogues are under way.
Figure 4. Secondary structure of H3 peptide at LSD1 catalytic site (PDB: 2UXN). View of conformation of H3 peptide (stick representation) (left). Schematic representation of H3 peptide (right). Three consecutive c-turn structures are indicated in bold.
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Acknowledgements We thank Professor Takaki Koide and Dr. Miki Suzuki for technical support. This work is supported in part by a Grant-in-Aid for Scientific Research (T.K.; C, 26460159) from MEXT (The Ministry of Education, Culture, Sports, Science and Technology), Waseda University Grant for Special Research Projects (T.K.; project number 2014K-6139), JST CREST Program (T.S.), Takeda Science Foundation (T.S.), Terumo Life Science Foundation (T.S.), and Mochida Memorial Foundation for Medical and Pharmaceutical Research (T.S.). Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bmcl.2015.03. 030. References and notes 1. Itoh, Y.; Suzuki, T.; Miyata, N. Mol. Biosyst. 2013, 9, 873. 2. Shi, Y.; Lan, F.; Matson, C.; Mulligan, P.; Whetstine, J. R.; Cole, P. A.; Casero, R. A.; Shi, Y. Cell 2004, 119, 941. 3. Tsukada, Y.; Fang, J.; Erdjument-Bromage, H.; Warren, M. E.; Borchers, C. H.; Tempst, P.; Zhang, Y. Nature 2006, 439, 811. 4. Metzger, E.; Wissmann, M.; Yin, N.; Müller, J. M.; Schneider, R.; Peters, A. H.; Günther, T.; Buettner, R.; Schüle, R. Nature 2005, 437, 436.
5. Liang, Y.; Vogel, J. L.; Narayanan, A.; Peng, H.; Kristie, T. M. Nat. Med. 2009, 15, 1312. 6. Jing, H.; Sengupta, R.; Espejo, A. B.; Min Gyu, L.; Dorsey, J. A.; Richter, M.; Opravil, S.; Shiekhattar, R.; Bedford, M. T.; Jenuwein, T.; Berger, S. L. Nature 2007, 449, 105. 7. Kontaki, H.; Taliannidis, I. Mol. Cell 2010, 39, 152. 8. Jing, W.; Hevi, S.; Kurash, J. K.; Hong, L.; Gay, F.; Bajko, J.; Hui, S.; Weitao, S.; Hua, C.; Guoliang, X.; Gaudet, F.; En, L.; Taiping, C. Nat. Genet. 2009, 41, 125. 9. Yang, J.; Huang, J.; Dasgupta, M.; Sears, N.; Miyagi, M.; Wang, B.; Chance, M. R.; Chen, X.; Du, Y.; Wang, Y.; An, L.; Wang, Q.; Lu, T.; Zhang, X.; Wang, Z.; Stark, G. R. Proc. Natl. Acad. Sci. U.S.A. 2010, 107, 21499. 10. Schulte, J. H.; Lim, S.; Schramm, A.; Friedrichs, N.; Koster, J.; Versteeg, R.; Ora, I.; Pajtler, K.; Klein-Hitpass, L.; Kuhfittig-Kulle, S.; Metzger, E.; Schule, R.; Eggert, A.; Buettner, R.; Kirfel, J. Cancer Res. 2009, 69, 2065. 11. Schenk, T.; Chen, W. C.; Göllner, S.; Howell, L.; Jin, L.; Hebestreit, K.; Klein, H.U.; Popescu, A. C.; Burnett, A.; Mills, K.; Casero, R. A., Jr.; Marton, L.; Woster, P.; Minden, M. D.; Dugas, M.; Wang, J. C. Y.; Dick, J. E.; Müller-Tidow, C.; Petrie, K.; Zelent, A. Nat. Med. 2012, 18, 605. 12. Shi, L.; Cui, S.; Engel, J. D.; Tanabe, O. Nat. Med. 2013, 19, 291. 13. Suzuki, T.; Miyata, N. J. Med. Chem. 2011, 54, 8236. 14. Ogasawara, D.; Itoh, Y.; Tsumoto, H.; Kakizawa, T.; Mino, K.; Fukuhara, K.; Nakagawa, H.; Hasegawa, M.; Sasaki, R.; Mizukami, T.; Miyata, N.; Suzuki, T. Angew. Chem., Int. Ed. 2013, 52, 8620. 15. Wichitnithad, W.; O’Callaghan, J. P.; Miller, D. B.; Train, B. C.; Callery, P. S. Bioorg. Med. Chem. 2011, 19, 7482. 16. Culhane, J. C.; Wang, D.; Yen, P. M.; Cole, P. A. J. Am. Chem. Soc. 2010, 132, 3164. 17. Szewczuk, L. M.; Culhane, J. C.; Yang, M.; Majumdar, A.; Yu, H.; Cole, P. A. Biochemistry 2007, 46, 6892. 18. Culhane, J. C.; Szewczuk, L. M.; Liu, X.; Da, G.; Marmorstein, R.; Cole, P. A. J. Am. Chem. Soc. 2006, 128, 4536. 19. Yang, M.; Culhane, J. C.; Szewczuk, L. M.; Gocke, C. B.; Brautigam, C. A.; Tomchick, D. R.; Machius, M.; Cole, P. A.; Yu, H. Nat. Struct. Mol. Biol. 2007, 14, 535.
