Development of Myostatin Inhibitory d-Peptides to Enhance the Potency, Increasing Skeletal Muscle Mass in Mice

Abstract

Myostatin is a key negative regulator of skeletal muscle growth, and myostatin inhibitors are attractive tools for the treatment of muscular atrophy. Previously, we reported a series of 14-29-mer peptide myostatin inhibitors, including a potent derivative, MIPE-1686, a 16-mer N-terminal-free l-peptide with three unnatural amino acids and a propensity to form β-sheets. However, the in vivo biological stability of MIPE-1686 is a concern for its development as a drug. In the present study, to develop a more stable myostatin inhibitory d-peptide (MID), we synthesized various retro-inverso versions of a 16-mer peptide. Among these, an arginine-containing derivative, MID-35, shows a potent and equivalent in vitro myostatin inhibitory activity equivalent to that of MIPE-1686 and considerable stability against biodegradation. The in vivo potency of MID-35 to increase the tibialis anterior muscle mass in mice is significantly enhanced over that of MIPE-1686, and MID-35 can serve as a new entity for the prolonged inactivation of myostatin in skeletal muscle.

Figure 1

Sequences of previously reported 16-mer peptides MIPE-1686 and 7c(24) and newly synthesized peptides 7c-ri, MID-35, MID-36, and MID-39 using an Fmoc-based solid-phase peptide synthesis (SPPS) method. The numbers above each amino acid indicate the position relative to the N-terminus. The lowercase letter indicates the d-amino acid. The underline indicates the substituted amino acid from peptide 7c or 7c-ri. The hyphen means deletion of the corresponding amino acid.

Figure 2

Results of a luciferase reporter assay of the activities of myostatin inhibitory d-peptides. (A) 7c-ri, (B) MID-35, and (C) MID-36 and MID-39. Cells, HEK293-cell-transfected Smad-responsive reporter (pGL4.48[luc2P/SBE/Hygro]) and control (pGL4.74[hRluc/TK]) plasmids; peptide concentration, 0.3 μM; positive control (prodomain, mouse recombinant) concentration, 10 nM; myostatin concentration, 8 ng/mL (0.32 nM); starvation with serum-free DMEM, 8 h; incubation with or without peptides, 4 h. Values represent means ± SD (N = 3).

Figure 3

Luciferase reporter assays to evaluate the dose-dependent inhibition by MIPE-1686 and MID-35 against myostatin (A), GDF-11 (B), activin A (C), and TGF-β1 (D). Cells, HEK293-cell-transfected Smad-responsive reporter (pGL4.48[luc2P/SBE/Hygro]) and control (pGL4.74[hRluc/TK]) plasmids; peptide concentration, 0.025–2 μM (A), 0.08–6 μM (B), 0.025–18 μM (C), and 0.025–18 μM for MID-35 or 0.074–54 μM for MIPE-1686 (D); positive control (SB431542) concentration, 5 μM; myostatin, GDF-11, and activin A concentrations, 8 ng/mL (0.32 nM); TGF-β1 concentration, 2.5 ng/mL (0.1 nM); starvation with serum-free DMEM, 8 h; incubation with or without peptides, 4 h. Values represent means ± SD (N = 3). Curve fitting was performed using KaleidaGraph 4.5. The sigmoidal curve is normalized with the minimum values in each assay. The vertical dotted line (gray) represents a concentration of 1 μM.

Figure 4

(A,B) Representative analytical RP-HPLC chromatograms of (A) MIPE-1686 and (B) MID-35 incubated in a solution of trypsin from bovine pancreas (1 μg/mL) on 50 mM Tris buffer (pH 7.5) containing 150 mM NaCl, 10 mM CaCl₂, and 0.05% Brij-35 at 37 °C for 400 min. Column, COSMOSIL 5C18-AR-II (4.6 × 150 mm); binary solvent system, a linear gradient of CH₃CN (25–40%, 30 min) in 0.1% aqueous TFA; flow, 1.0 mL/min; detection, UV 220 nm. Asterisks identify unknown peaks. (C) Sequence and mass spectrometric data of metabolite tm1 derived from MIPE-1686.

Figure 5

(A) Weight of tibialis anterior (TA) muscles in 8-week-old male C57BL/6J mice treated with MIPE-1686 (left TA; 0.75 mM in saline, 40 μL) or saline (right TA; 40 μL). The TA weight is normalized with the body weight (BW). Results are presented as mean values ± SD (N = 4). (B) Weight of TA muscles in 8-week-old male C57BL/6J mice treated with MID-35 (left TA; 0.75 mM in saline, 40 μL) or saline (right TA; 40 μL). The TA weight is normalized with the body weight (BW). Results are presented as mean values ± SD (N = 5). (C) Increase (%) in TA weight upon treatment with peptides. The saline-injected TA weight in each mouse is 100%. The values are presented as mean values ± SD (MID-35, N = 5; MIPE-1686, N = 4). **p < 0.01.