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Letter
Structural basis for the effective myostatin inhibition of the mouse myostatin prodomain–derived minimum peptide Tomo Asari, Kentaro Takayama, Akari Nakamura, Takahiro Shimada, Akihiro Taguchi, and Yoshio Hayashi ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.6b00420 • Publication Date (Web): 23 Nov 2016 Downloaded from http://pubs.acs.org on November 29, 2016
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ACS Medicinal Chemistry Letters
Structural basis for the effective myostatin inhibition of the mouse myostatin prodomain–derived minimum peptide Tomo Asari, Kentaro Takayama*, Akari Nakamura, Takahiro Shimada, Akihiro Taguchi and Yoshio Hayashi*. Department of Medicinal Chemistry, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan. KEYWORDS: α-helix, inhibitor, myostatin, peptide, structure-activity relationship. ABSTRACT: Myostatin inhibition is one of the promising strategies for treating muscle atrophic disorders, including muscular dystrophy. It is well known that the myostatin prodomain derived from the myostatin precursor acts as an inhibitor of mature myostatin. In our previous study, myostatin inhibitory minimum peptide 1 (WRQNTRYSRIEAIKIQILSKLRL-amide) was discovered from the mouse myostatin prodomain. In the present study, alanine scanning of 1 demonstrated that the key amino acid residues for the effective inhibitory activity are rodent-specific Tyr and C-terminal aliphatic residues, in addition to N-terminal Trp residue. Subsequently, we designed five Pro-substituted peptides and examined the relationship between secondary structure and inhibitory activity. As a result, we found that Pro-substitutions of Ala or Gln residues around the center of 1 significantly decreased both αhelicity and inhibitory activity. These results suggested that an α-helical structure possessing hydrophobic faces formed around the C-terminus is important for inhibitory activity.
Myostatin, a transforming growth factor β (TGF-β) superfamily protein, is a promising target for treating muscle atrophic disorders such as muscular dystrophy, cancer cachexia, and sarcopenia. As previously reported, the lack of myostatin leads to a significant increase in muscular mass, whereas its overexpression induces cachexia.1,2 Based on these findings, several strategies to induce muscular growth have been investigated. In particular, much attention has been focused on myostatin inhibitory molecules, including neutralizing antibodies,3 prodomain proteins,4,5 soluble decoys of active type II receptors (ActRII),6 interacting proteins (growth and differentiation factor–associated serum protein, GASP),7 follistatin8 and follistatin-related protein5,9 are focused. Treatment with neutralizing antibodies increased muscle mass and strength in Duchenne muscular dystrophy model mdx mice,3 while decoy receptors prevented muscle wasting and cancer-induced cardiac atrophy in tumor-bearing mice.6 It is well known that the prodomain of myostatin interacts with mature myostatin to form an inactive complex in the extracellular matrix and in serum.2,5,10 In a previous structural study of TGF-β1, the N-terminal α-helical region of its prodomain was found to be buried in the type I receptor–binding pocket of the mature domain.11 Indeed, Walton et al. reported that aliphatic (Ile and Leu) residues in the α-helical region of the prodomain plays a key role in TGF-β1 inactivation.12 This α-helical region, which includes aliphatic residues, is highly conserved throughout the TGF-β superfamily. In 2004, Jiang et al. reported that the human myostatin prodomain–derived fragment, which consists of 74 residues (positions: 19–92 in the prodomain), shows the significant myostatin inhibitory activity as a glutathione S–transferase fusion protein.4 As shown in Figure 1, our group recently identified a mouse myo-
statin prodomain–derived minimum peptide 1 (23 residues, including a conserved α-helical region spanning positions 21– 43)13 through the earlier discovery of core inhibitory fragment (p29) consisting of 29 residues (position: 19–47).14 Intramuscular injection of these prodomain–derived peptides, including p29, significantly increased muscle mass in muscular dystrophy model mdx mice. Additionally, we determined the importance of Trp residue (position 21) and the mouse-derived Arg-Tyr sequence (positions 26 and 27) in the synthetic peptide inhibitor 1 using a cell-based luciferase reporter assay. Next, we carried out a structure activity relationship (SAR) study of 1 in order to develop a more potent derivative inhibitor. Our first SAR study focused on the N-terminal Trp of minimum peptide 1 yielded the derivative 2, which is three times as potent as 1, registering an IC50 value of 1.2 µM on the reporter assay (Figure 1).15 However, the structural basis for effective myostatin inhibition by the mouse-derived minimum peptide 1 is still unclear. Here, we performed Ala scanning of 1 to identify other key residues and investigated the relationship between secondary structure and inhibitory activity by synthesizing Pro-substituted peptides. 19
p29 peptide 1 peptide 2
27
43
47
-amide -amide
X = 2-naphthoxyacetyl group
Figure 1. Structures of myostatin inhibitory peptides p29, 1 and 2, derived from the prodomain of mouse myostatin.13-15 Numbers added to the top of amino acid indicate the position in the prodomain sequence of mouse myostatin.
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In this study, all peptides were synthesized by the 9fluorenylmethoxycarbonyl (Fmoc)-based solid-phase peptide synthesis (Fmoc-SPPS) method as previously reported.13 Prepared resins bearing protected peptides were treated with trifluoroacetic acid (TFA)-m-cresol-thioanisole-1,2-ethanedithiol (EDT) (4.0 mL, 40:1:1:1) for 150 min at room temperature. The resulting crude peptides were purified by preparative reversed–phase high–performance liquid chromatography (RPHPLC). All peptides were characterized by electrospray ionization time-of-flight mass spectrometry (ESI-TOF MS) (see Supporting Information). Each peptide was at least 95% pure, as determined by RP-HPLC analysis at 230 nm. The myostatin inhibitory activities of the peptides were measured by a luciferase reporter assay as previously reported (see Supporting Information).15 Briefly, pre-mixed 8 ng/mL (0.32 nM) recombinant human myostatin, with or without synthetic peptides, was added to human embryonic kidney 293 (HEK293) cells for 4 h. These cells were transiently transfected with myostatin-responsive (SBE)4-luc reporter beforehand. The myostatin-dependent reporter activity was measured by luminometer. Recombinant protein of mouse myostatin prodomain at 10 nM was used as a positive control. The results for each peptide are presented as a relative value compared to the reporter activity of myostatin alone (=100). Secondary structure analysis of peptides was performed by measuring circular dichroic (CD) spectra as previously reported (see Supporting Information).13 Briefly, peptides were dissolved to yield a final concentration of 5 µM in 20 mM sodium phosphate buffer (pH 7.4) containing 10% 2,2,2trifluoroethanol (TFE). CD spectra of the synthetic peptides were measured at 25 °C. To identify the key residues for the effective myostatin in-
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hibition of 1, we synthesized twenty-two different Alasubstituted peptides (Figure 2A) and evaluated their inhibitory activities (Figure 2B). Peptide 1 at 10 µM blocked the myostatin activity completely, performing similarly to the prodomain protein. Similarly, R22A, Q23A, N24A, T25A, R26A, S28A, R29A, E31A, K34A, Q36A, S39A, K40A and R42A also suppressed the myostatin-dependent reporter activity, suggesting that side chain structures of these residues are less relevant to the inhibitory activity. On the other hand, the inhibitory activity of W21A was not observed as expected from our previous report.13 Focusing on the mouse-derived Arg-Tyr sequence at positions 26 and 27 of 1, Ala substitution of Tyr at position 27 (Y27A) only resulted in a significant decrease in myostatin inhibitory activity (Figure 2B). This decrease suggests that the mouse-derived Tyr enhanced the inhibitory activity compared with human-derived Ser. Interestingly, sequence alignment focused on peptide 1 showed that rat and rabbit, in addition to mouse among several species (Figure S1), only possess Tyr at position 27, implying that searching in the rodent sequence with this specific Tyr led us to the successful identification of synthetic peptide inhibitor 1 in our previous study.13 In the C-terminal region, the importance of all aliphatic residues (Ile and Leu) for the effective myostatin inhibition of 1 was demonstrated by the results of I30A, I33A, I35A, I37A, L38A, L41A and L43A (Figure 2B). Walton et al. had already reported that the five hydrophobic residues corresponding to positions 30, 37, 38, 41 and 43 in 1 contribute to TGF-β1 inactivation.12 In the present study, we discovered the necessity for other residues, namely Ile at positions 33 and 35. The inhibitory activities of I33A and I35A were quite weak at 10 µM, suggesting that Ile residues at positions 33 and 35 are indispensable for the myostatin inhibition by peptide 1.
Figure 2. (A) Structures of Ala-substituted peptides. The numbers above each amino acid indicates its position in the prodomain sequence of mouse myostatin. (B) Luciferase reporter assay to determine the ability of Ala-substituted peptides to inhibit myostatin relative to peptide 1. Cell line: HEK293; peptide concentration: 10 µM; positive control (prodomain: mouse recombinant) concentration: 10 nM; myostatin concentration: 8 ng/mL (0.32 nM); incubation: 4 h. Values represent means ± SD (n= 3).
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Next, we focused on the rodent-specific Tyr at position 27 and synthesized a series of peptide derivatives to perform an SAR study (Figure 3A). As shown in Figure 3B, Y27F. Y27y (small letter: D-form) and Y27W at 10 µM were able to significantly inhibit myostatin activity, whereas Y27H, Y27Q, Y27R and Y27E showed weak inhibition. Moreover, the potency of the former three peptide derivatives is almost identical since no significant difference was observed among them at lower concentrations (3 µM) (data not shown). These results suggested that the position 27 can accommodate aromatic hydrophobic amino acids bearing phenyl or indolyl group at side chain, i.e., Tyr, Phe and Trp,16 without any restriction on either L- or D-isomers. To investigate the relationship between secondary structure and inhibitory activity, we measured the CD spectra of these peptide derivatives. We previously reported that peptide 1 tends to form an α-helix structure in the presence of TFE.13 Similarly, a series of peptide derivatives also displayed the characteristic absorptions at 208 and 222 nm in the presence of 10% TFE (Figure S2), indicating that the secondary structure of 1 is not greatly affected by the amino acid substitution at position 27. Previously, we also reported that the N-terminal fragment of 1 forms a random coiled structure.13 This finding and our present results, including Y27y, suggest that the myostatin inhibitory activity of peptide is dependent on the side chain structure at position 27. A)
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peptide 1
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Y27F Y27y Y27W Y27H Y27Q Y27R Y27E
-amide -amide -amide
-amide -amide -amide
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-amide -amide -amide -amide -amide
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myostatin myostatin alone prodomain peptide 1 N24P S28P A32P Q36P K40P
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Figure 4. (A) Structures of Pro-substituted peptides. The numbers above each amino acid indicates its position in the prodomain sequence of mouse myostatin. (B) Luciferase reporter assay to determine the ability of of Pro-substituted peptides to inhibit myostatin relative to peptide 1. Cell line: HEK293; peptide concentration: 10 µM; positive control (prodomain: mouse recombinant) concentration: 10 nM; myostatin concentration: 8 ng/mL (0.32 nM); incubation: 4 h. Values represent means ± SD (n= 3). (C) CD spectra of Pro-substituted peptides in the presence of 10% TFE solution prepared in 20 mM sodium phosphate buffer (pH 7.4); peptide concentration, 5 µM.
Y27W
Y27H
Y27Q
Y27R
Y27E
Figure 3. (A) Structures of Tyr-substituted peptides. The numbers above each amino acid indicates its position in the prodomain sequence of mouse myostatin. (B) Luciferase reporter assay to determine the ability of of Tyr-substituted peptides to inhibit myostatin relative to peptide 1. Cell line: HEK293; peptide concentration: 10 µM; positive control (prodomain: mouse recombinant) concentration: 10 nM; myostatin concentration: 8 ng/mL (0.32 nM); incubation: 4 h. Values represent means ± SD (n= 3).
Moreover, to examine the relationship between the secondary structure and the inhibitory activity, we synthesized five different peptide derivatives, N24P, S28P, A32P, Q36P and K40P, each with a potentially α-helix-breaking Pro substitution targeting every fourth residue in peptide 1 from position 24 to 40. These five residues were selected from among Alasubstitutable amino acids (Figure 4A).17 As shown in Figures 4B and 4C, the myostatin inhibitory activities and secondary structures of these peptides were evaluated similarly using the reporter assay and CD spectra measurement, respectively. In the N-terminal random coiled region of 1, Pro substitutions at positions 24 and 28 did not significantly decrease their αhelicity, whereas the latter led to a moderate loss of inhibitory activity. These results implicate that the side chain orientation of Tyr at position 27 in S28P may be improperly fixed by the introduction of Pro at the neighboring position 28. In the C-
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terminal α-helical region, Pro substitutions at positions 32, 36 and 40 broke the α-helix structure of 1 (Figure 4C). On the other hand, a significant decrease in inhibitory activity was observed in A32P and Q36P although the influence of broken secondary structure on the inhibitory activity was limited in K40P (Figure 4B). These results suggested that the α-helix structure formed around position 32–36 of peptide 1 plays an especially important role in effective myostatin inhibition. A) Ile30 Ile33 Ile37 Lys40
Ile35
30, 33 and 37 and that the spatial restriction of Leu residues at positions 41 and 43 is not so strong. Furthermore, the necessity for Ile at position 35 of 1 was first determined in this study since it corresponds to Gly in TGF-β1. Hence, it raises the possibility that the second (minor) hydrophobic phase is formed by Leu at position 38 as shown in Figure 5. In conclusion, Ala scanning of a mouse-derived myostatin inhibitory minimum peptide 1 afforded the key residues at positions 21, 27, 30, 33, 35, 37, 38, 41 and 43 for the effective inhibition. In the N-terminal random coiled region, the rodentspecific Tyr at position 27 could be substituted with other aromatic hydrophobic residues such as Phe and Trp and the Dform of Tyr without affecting the secondary structure. In the C-terminal region, the α-helix formed around positions 32–36 was especially important for effective myostatin inhibition by 1. These findings would be valuable for future SAR studies to discover and develop more potent myostatin inhibitors.
Leu41 Leu38
ASSOCIATED CONTENT Leu43
Supporting Information Materials, Experimental procedure, Analytical data for all peptide derivatives, analytical HPLC chromatograms and Figures S1-2. The Supporting Information is available free of charge on the ACS Publications website.
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Corresponding Author
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* To whom correspondence should be addressed: Department of Medicinal Chemistry, Tokyo University of Pharmacy and Life Sciences, Tokyo 192-0392, Japan. Tel: +81 42 676 3275. Fax: +81 42 676 3279. E-mail address:
[email protected] (Y. H.) and
[email protected] (K.T.).
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34 29
K
R 41
40
L 30
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33 26
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ACKNOWLEDGMENT
I
Figure 5. (A) Predicted 3D helical model and (B) 2D helical wheel focused on positions 26–43 in peptide 1. The hydrophobic face formed by aliphatic residues 30, 33, 37, and 41 is highlighted in blue; another hydrophobic face based on residues 35 and 38 is highlighted in red. Leu at position 43 is highlighted in purple.
Based on these findings, we finally drew a predicted αhelical 3D model (MOE, Molecular Operating Environment System) and 2D wheel focused on positions 26–43 of 1 similarly to a previous report concerning TGF-β1.12 Additionally, we highlighted the aliphatic residues that form the major and minor hydrophobic faces in blue and red, respectively (Figure 5). Interestingly, Leu at position 43 (highlighted in purple) was not included in both hydrophobic faces although it was determined as one of key residues in Ala scanning as shown in Figure 2. Hence, this residue may relate to other interaction with myostatin. Additionally, although Leu at position 41 seems to participate in forming the major hydrophobic face, the myostatin inhibition by K40P only slightly decreased compared with 1 without the structural influence of breaks in its α-helical nature as mentioned above (Figure 4). These considerations imply that a real major hydrophobic face in an active form of 1 is composed of three Ile residues at positions
This research was supported by the Japan Society for the Promotion of Sciences (JSPS) KAKENHI, including Grants-in-Aid for Scientific Research (B) 15H04658 (K.T. and Y.H.), Platform for Drug Discovery, Informatics and Structural Life Science funded by the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan (Y.H.), MEXT-supported Program for the Strategic Research Foundation at Private Universities, and an Intramural Research Grant (26-8) for Neurological and Psychiatric Disorders on NCNP (Y.H.). The authors thank Mr. Shota Takayama, Mr. Yusuke Saga and Ms. Yuko Sohma for peptide synthesis, CD spectra measurement and cell-based assay.
ABBREVIATIONS ActRII, activin type II receptor; CD, circular dichroism; DIPCI, N,N'-diisopropylcarbodiimide; DMEM, Dulbecco’s Modified Eagle’s Medium; DMF, N,N-dimethylformamide; DMSO, dimethylsulfoxide; EDT, 1,2-ethanedithiol; ES, electrospray; FBS, fetal bovine serum; Fmoc, 9-fluorenylmethoxycarbonyl; GASP, growth and differentiation factor-associated serum protein; HEK293, human embryonic kidney 293; HOBt, 1hydroxybenzotriazole; MOE, Molecular Operating Environment; MS, mass spectrometry; RP-HPLC, reverse-phase highperformance liquid chromatography; SAR, structure activity relationship; SBE, Smad binding element; SPPS, solid-phase peptide synthesis; TFA, trifluoroacetic acid; TFE, 2,2,2-trifluoroethanol; TGF-β, transforming growth factor-β; TOF, time-of-flight.
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REFERENCES (1) McPherron, A. C.; Lawler, A. M.; Lee, S.-J. Regulation of skeletal muscle in mice by a new TGF-β superfamily member. Nature 1997, 387, 83-90. (2) Zimmers, T. A.; Davies, M. V.; Koniaris, L. G.; Haynes, P.; Esquela, A. F.; Tomkinson, K. N.; McPherron, A. C.; Wolfman, N. M.; Lee, S.-J. Induction of cachexia in mice by systemically administrated myostatin. Science 2002, 296, 1486-1488. (3) Bogdanovich, S.; Krag, T. O.; Barton, E. R.; Morris, L. D.; Whittemore L. A.; Ahima, R. S.; Khurans, T. S. Functional improvement of dystrophic muscle by myostatin blockade. Nature 2002, 420, 418-421. (4) Jiang, M.-S.; Liang, L.; Wang, S.; Ratovitski, T.; Holmstrom, J.; Barker, C.; Stotish, R. Characterization and identification of the inhibitory domain of GDF-8 propeptide. Biochem. Biophys. Res. Commun. 2004, 315, 525-531. (5) Hill, J. J.; Davies, M. V.; Pearson, A. A.; Wang, J. W.; Hewick, R. M.; Wolfman, N. M.; Qiu, Y. The myostatin propeptide and the follistatin-related gene are inhibitory binding proteins of myostatin in normal serum. J. Biol. Chem. 2002, 277, 40735-40741. (6) Zhou, X.; Wang, J. L.; Lu, J.; Song, Y.; Kwak, K. S.; Jiao, Q.; Rosenfeld, R.; Chen, Q.; Boone, T.; Scott Simonet, W.; Lacey, D. L.; Goldberg, A. L.; Han, H. Q. Reversal of cancer cachexia and muscle wasting by ActRIIB antagonism leads to prolonged survival. Cell 2010, 142, 531-543. (7) Lee, S.-J.; Lee, Y.-S. Regulation of GDF-11 and myostatin by GASP-1 and GASP-2. Proc. Natl. Acad. Sci. U. S. A. 2013, 110, E3713-3722. (8) Lee, Y.-S.; Lee, S.-J.; Zimmers, T. A.; Soleimani, A.; Matzuk, M. M.; Tsuchida, K.; Cohn, R. D.; Barton, E. R. Regulation of Muscle Mass by Follistatin and Activins, Mol. Endocrinol. 2010, 24, 1998-2008. (9) Nakatani, M.; Takehara, Y.; Sugino, H.; Matsumoto, M.; Hashimoto, O.; Hasegawa, Y.; Murakami, T.; Uezumi, A.; Takeda, S.; Noji, S.; Sunada, Y.; Tsuchida, K. Transgenic expression of a myostatin inhibitor derived from follistatin increases skeletal muscle mass
and ameliorates dystrophic pathology in mdx mice. FASEB J. 2008, 22, 477-487. (10) Anderson, S. B.; Goldberg, A. L.; Whitman, M. Identification of a novel pool of extracellular pro-myostatin in skeletal muscle. J. Biol. Chem. 2008, 283, 7027-7035. (11) Shi, M.; Zhu, J.; Wang, R.; Chen, X.; Mi, L.; Walz, T.; Springer, T. A. Latent TGF-β structure and activation. Nature 2011, 474, 343-349. (12) Walton, K. L.; Makanji, Y.; Chen, J.; Wilce, M. C.; Chan, K. L.; Robertson, D. M.; Harrison, C. A. Two distinct region of latencyassociated peptide coordinate stability of the latent transforming growth factor-β1 complex. J. Biol. Chem. 2010, 285, 17029-17037. (13) Takayama, K.; Noguchi, Y.; Aoki, S.; Takayama, S.; Yoshida, M.; Asari, T.; Yakushiji, F.; Nishimatsu, S.; Ohsawa, Y.; Itoh, F.; Negishi, Y.; Sunada, Y.; Hayashi, Y. Identification of the minimum peptide from mouse myostatin prodomain for human myostatin inhibition. J. Med. Chem. 2015, 58, 1544-1549. (14) Ohsawa, Y.; Takayama, K.; Nishimatsu, S.; Okada, T.; Fujino, M.; Fukai, Y.; Murakami, T.; Hagiwara, H.; Itoh, F.; Tsuchida, K.; Hayashi, Y.; Sunada, Y. The Inhibitory Core of the Myostatin Prodomain: Its Interaction with Both Type I and II Membrane Receptors, and Potential to Treat Muscle Atrophy. PLoS One. 2015, 10, e0133713. (15) Takayama, K.; Nakamura, A.; Rentier, C.; Mino, Y.; Asari, T.; Saga, Y.; Taguchi, A.; Yakushiji, F.; Hayashi, Y. Effect of NTerminal Acylation on the Activity of Myostatin Inhibitory Peptides. ChemMedChem. 2016, 11, 845-849. (16) Wimley, W. C.; White, S. H. Experimentally determined hydrophobicity scale for proteins at membrane interfaces. Nat. Struct. Biol. 1996, 3, 842-848. (17) Nilsson, I.; Sääf, A.; Whitley, P.; Gafvelin, G.; Waller, C.; von Heijne, G. Proline-induced disruption of a transmembrane alpha-helix in its natural environment. J. Mol. Biol. 1998, 284, 1165-1175.
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Graphic entry for the Table of Contents (TOC) Ile30 Ile33
Tyr27
Ile37
Aromatic hydrophobic structure
Lys40
Ile35
Leu41 Leu38
Minor hydrophobic face
Leu43
Myostatin inhibitory peptide 1
Ile30
Ile3
Tyr27 Aromatic hydrophobic structure
Ile35 Leu38
Minor hydrophobic face
Myostatin inhibitory p
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p29 1 peptide 1 2 3 peptide 2 4 5
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CAWRQNTRYSRIEAIKIQILSKLRLETAP
WRQNTRYSRIEAIKIQILSKLRL-amide
ACS Paragon Plus Environment XRQNTRYSRIEAIKIQILSKLRL -amide
(X = 2-naphthoxyacetyl group)
A) peptide 1 1 2W21A
3 4R22A
5Q23A
6 7N24A
8 9T25A
10 R26A
11 12 Y27A
13 14 S28A
15 16 R29A
17 I30A
18 19 E31A
20 21 I33A
22 23 K34A
24 I35A
25 26 Q36A
27 28 I37A
29 30 L38A
31 S39A
32 33 K40A
34 35 L41A
36 37 R42A
38 39 L43A
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myostatin (-) ARQNTRYSRIEAIKIQILSKLRL-amide
myostatin alone WAQNTRYSRIEAIKIQILSKLRL-amide
prodomain WRANTRYSRIEAIKIQILSKLRL-amide
peptide 1 W21A
WRQATRYSRIEAIKIQILSKLRL-amide
R22A
WRQNARYSRIEAIKIQILSKLRL-amide
Q23A
WRQNTAYSRIEAIKIQILSKLRL-amide
N24A
WRQNTRASRIEAIKIQILSKLRL-amide
T25A
WRQNTRYARIEAIKIQILSKLRL-amide
R26A
WRQNTRYSAIEAIKIQILSKLRL-amide
Y27A
S28A
WRQNTRYSRAEAIKIQILSKLRL-amide
R29A
WRQNTRYSRIAAIKIQILSKLRL-amide
I30A
WRQNTRYSRIEAAKIQILSKLRL-amide
E31A
WRQNTRYSRIEAIAIQILSKLRL-amide
I33A
WRQNTRYSRIEAIKAQILSKLRL-amide
K34A
I35A
WRQNTRYSRIEAIKIAILSKLRL-amide
Q36A
WRQNTRYSRIEAIKIQALSKLRL-amide
I37A
WRQNTRYSRIEAIKIQIASKLRL-amide
L38A
WRQNTRYSRIEAIKIQILAKLRL-amide
S39A
WRQNTRYSRIEAIKIQILSALRL-amide
K40A
WRQNTRYSRIEAIKIQILSKARL-amide
L41A
ACS-amide Paragon WRQNTRYSRIEAIKIQILSKLAL
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L43A
WRQNTRYSRIEAIKIQILSKLRA-amide
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W21A
R22A
Q23A
N24A
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R26A
Y27A
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R29A
I30A
E31A
I33A
K34A
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Q36A
I37A
L38A
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K40A
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WRQNTRFSRIEAIKIQILSKLRL-amide
WRQNTRySRIEAIKIQILSKLRL-amide
WRQNTRWSRIEAIKIQILSKLRL-amide
WRQNTRHSRIEAIKIQILSKLRL-amide
WRQNTRQSRIEAIKIQILSKLRL-amide
WRQNTRRSRIEAIKIQILSKLRL-amide
WRQNTRESRIEAIKIQILSKLRL-amide
1 2Y27F 3 Y27y 4 5Y27W 6 7Y27H 8 9Y27Q 10 Y27R 11 12 Y27E 13 14 15 relative luciferase activity 16 17 0 20 40 60 80 100 18 19 myostatin (-) 20 myostatin alone 21 prodomain 22 23 peptide 1 24 Y27F
25 26 Y27y
27 Y27W
28 29 Y27H
30 Y27Q
31 32 Y27R
ACS Paragon Plus Environment 33 Y27E
34 35
B)
myostatin (-)
myostatin alone
prodomain
peptide 1
Y27F
Y27y
Y27W
Y27H
Y27Q
Y27R
Y27E
A) peptide 1
ACS Medicinal 27 Chemistry 21 32 Letters 36
Page 10 of 12 43
WRQNTRYSRIEAIKIQILSKLRL-amide
WRQPTRYSRIEAIKIQILSKLRL-amide
WRQNTRYPRIEAIKIQILSKLRL-amide
WRQNTRYSRIEPIKIQILSKLRL-amide
WRQNTRYSRIEAIKIPILSKLRL-amide
WRQNTRYSRIEAIKIQILSPLRL-amide
1 2N24P 3 S28P 4 5A32P 6 7Q36P 8 9K40P 10 11 relative luciferase activity 12 0 20 40 60 80 100 13 14 15 myostatin (-) 16 myostatin alone 17 prodomain 18 19 peptide 1 20 N24P
21 22 S28P
23 A32P
24 25 Q36P
26 K40P
27 28 29 30 30000 31 peptide wild type1 32 33 N24P N24P 34 20000 35 S28P S28P 36 A32P A32P 37 38 Q36P 10000 Q36P 39 40 K40P K40P 41 42 0 43 44 45 46 47 -10000 48 49 50 51 -20000 ACS Paragon Plus Environment 52 190 200 210 220 230 240 250 53 54 wavelength (nm)
B)
myostatin (-)
myostatin alone
prodomain
peptide 1
N24P
S28P
A32P
Q36P
K40P
[θ] deg cm2/dmol
C)
Page 11 of 12 A) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50
ACS Medicinal Chemistry Letters
Ile30 Ile33 Ile37 Lys40
Ile35
Leu41 Leu38 Leu43
B) 35 42 31
R
I
28
S
39
S
E
32
A
38
43
L
L
27
Y
36
Q
34 29
K
R 41
40
L 30
33
I
37 Plus26 I ACS Paragon Environment
I
R
K
ACS MedicinalIleChemistry Letters Page 12 of 12 30
Ile33
Tyr27
1 2 3 4
Ile37
Aromatic hydrophobic structure
Lys40
Ile35
Leu41
ACS ParagonLeuPlus Environment Minor hydrophobic face
38
Leu43
Myostatin inhibitory peptide 1
