2022 Volume 70 Issue 6 Pages 413-419
The enhancement of basic research based on biomolecule-derived peptides has the potential to elucidate their biological function and lead to the development of new drugs. In this review, two biomolecules, namely “neuromedin U (NMU)” and “myostatin,” are discussed. NMU, a neuropeptide first isolated from the porcine spinal cord, non-selectively activates two types of receptors (NMUR1 and NMUR2) and displays a variety of physiological actions, including appetite suppression. The development of receptor-selective regulators helps elucidate each receptor’s detailed biological roles. A structure–activity relationship (SAR) study was conducted to achieve this purpose using the amidated C-terminal core structure of NMU for receptor activation. Through obtaining receptor-selective hexapeptide agonists, molecular functions of the core structure were clarified. Myostatin is a negative regulator of skeletal muscle growth and has attracted attention as a target for treating atrophic muscle disorders. Although the protein inhibitors, such as antibodies and receptor-decoys have been developed, the inhibition by smaller molecules, including peptides, is less advanced. Focusing on the inactivation mechanism by prodomain proteins derived from myostatin-precursor, a first mid-sized α-helical myostatin-inhibitory peptide (23-mer) was identified from the mouse sequence. The detailed SAR study based on this peptide afforded the structural requirements for effective inhibition. The subsequent computer simulation proposed the docking mode at the activin type I receptor binding site of myostatin. The resulting development of potent inhibitors suggested the existence of a more appropriate binding mode linked to their β-sheet forming properties, suggesting that further investigations might be needed.
A mid-sized peptide1) having high affinity and specificity to the target molecule is an attractive tool for understanding biological phenomena and accelerating drug development. Functional elucidation of peptide hormones such as insulin, natriuretic peptides, calcitonin, gonadotropin-releasing hormone, and glucagon-like peptide-1 (GLP-1) has contributed to today’s healthcare. It facilitated the generation of respective analogs used in clinical practice.2) Basic research on GLP-1 identified dipeptidyl peptidase-4 inhibitors as a peptidomimetic, expanding the options for diabetes treatment. Similar to these hormones, a peptide recognizing a broad region due to the molecular size can regulate protein–protein interactions (PPI) formed between relatively shallow parts of the molecular surface.3) The enhanced biological activity of peptide molecules is often afforded by introducing unnatural amino acids, cyclization, and side-chain stapling, inducing conformational fixation.4,5) Hence, molecular function-directed studies using biomolecules are meaningful in pharmaceutical science. This review discusses two biomolecules, neuromedin U (NMU) and myostatin, to illustrate this topic.
NMU is a neuropeptide initially isolated in 1985 from the porcine spinal cord.6) As shown in Fig. 1, human NMU (hNMU) consists of 25 amino acid residues. Its C-terminal amidated heptapeptide structure 1 (Phe19-Leu20-Phe21-Arg22-Pro23-Arg24-Asn25-amide) is common in mammals and is a core for activation of two types of NMU receptors (NMUR1, NMUR2).7) The physiological responses via these receptors have been investigated by the intracerebroventricular administration of NMU or by use of knockout (KO) mice. Hanada et al. reported that centrally-acting NMU induces the secretion of corticotropin-releasing hormone from hypothalamic paraventricular nucleus (PVN)8) where NMUR2 is highly expressed.9) This is related to decreased food intake and body weight,8,10,11) enhancement of catabolic function,11) induction of stress response,12) and suppression of gastric acid secretion.13) In addition, NMU has been reported to be involved in the regulation of cardiovascular function via sympathetic nerve activation,14) suppression of prolactin secretion,15) regulation of circadian oscillation,16) and glucose homeostasis.17,18) Moreover, NMU-mediated allergic inflammation, which is involved with activations of mast cells,19) eosinophils20) and type 2 innate lymphoid cells21–23) expressing NMUR1, has attracted attention over the years. Among them, anti-obesity effects, including appetite suppression, have received interest for drug development. Peripheral administration of high-molecular-weight human NMU (hNMU)-conjugates, bearing polyethylene glycol and human serum albumin, to respective NMU receptor-KO mice demonstrated that both NMUR1 and NMUR2 engage in the anorectic action.24,25) Benzon et al. suggested that NMUR2 plays an important role in the regulation of high-fat diet intake.26) The development of receptor-selective agonists is beneficial for accelerating receptor-directed studies regarding various NMU actions. Hence, we focused on the C-terminal typical structure 1 to obtain receptor-selective modulators and conducted a detailed structure–activity relationship (SAR) study (see Section 2, Fig. 2).
The numbers above each amino acid indicate the position in the sequence of human NMU.
Discovered by McPherron et al. in 1997, myostatin of the transforming growth factor-β (TGF-β) superfamily is an endogenous negative regulatory factor of skeletal muscle mass.27) In mice, systemic overexpression of myostatin induces the cachexia symptoms,28) whereas its genetic defect increases muscle mass up to 2-fold.27) Therefore, myostatin is a promising target for treating atrophic muscle disorders such as muscular dystrophy, cancer cachexia, sarcopenia, and disuse muscle atrophy. In 2002, Bogdanovich et al. reported that the neutralizing antibody increases muscle mass and strength in Duchenne-type dystrophic (DMD) model mdx mice.29) Subsequently, several protein-based myostatin inhibitors, including the soluble decoy of activin type II receptor30) and the humanized antibody31) progressed to clinical trials. However, their clinical use against muscle wasting has not been achieved yet.32,33) Therefore, there is a need for new therapeutic strategies and tools to overcome muscle atrophy.34) The active myostatin is produced by the final cleavage between prodomain and mature domain by furin-like proteases after the disulfide dimerization of the mature domain. It can form a ternary complex with activin type I and II receptors to activate the intracellular Smad2/3 signaling like other TGF-β superfamily members.35–38) Structural analysis reveals that myostatin, a dimer of the mature domain, is inactivated by the complex formation with two prodomain proteins (Fig. 3A) and stored on the extracellular matrix via latent TGF-β binding protein.28,39) In circulation, follistatin (Fst) and follistatin-like3 (Fstl3) glycoproteins also contribute to the inactivation of myostatin.40) We focused on the binding mode of these endogenous myostatin-inactivating proteins (prodomain, Fst) and tried to identify the myostatin inhibitory peptides (see Section 3).
The numbers above each amino acid indicate the position in the prodomain. The underlined residues play a key role in effective myostatin inhibition identified by Ala-scan.64) The bold style indicates rodent-specific residues. The table lists common interaction residues of myostatin in four proposed docking models (models 1 to 4).69)
Porcine NMU-8 (pNMU-8, YFLFRPRN-amide, Fig. 1), the endogenous short form of porcine NMU (25-mer), shows the full agonistic activity of hNMU. The mammalian common segment 1 (FLFRPRN-amide), the Tyr-deleted form of porcine NMU-8, is a partial agonist to both human NMU receptors.41) We attempted to develop smaller peptidic full agonists based on peptide 1 (Phe19-Leu20-Phe21-Arg22-Pro23-Arg24-Asn25-amide) and clarify its molecular function for modulating the agonistic activity of the respective human NMU receptors. We synthesized a series of the C-terminal core peptide derivatives of NMU (CPN) based on peptide 1 and evaluated their agonistic activities in Chinese hamster ovary (CHO) cells stably expressing each human NMU receptor. First, the acyl derivative N-terminal Phe19 was prepared, capping the N-terminal to make it resistant to digestion by exopeptidases. Then, the agonistic activities of 3-phenylpropionyl-hexapeptide 2 (Fig. 1), the N-terminal amino group-deleted form of peptide 1, were well maintained to both receptors and peptide 2 was used in the following detailed SAR study.41) Moreover, introducing 3-(3-pyridyl)propionyl and 3-cyclohexylpropinyl groups at this position afforded good selectivity toward NMUR1 and NMUR2, respectively.41) These results represented a breakthrough in the development of receptor-selective modulators (CPN-124 and CPN-116, respectively) as shown in Fig. 2. The derivatization of the Pro23-Arg24-Asn25-amide region decreased the agonistic activity of lead peptide 2, suggesting that the Pro23-Arg24-Asn25-amide structure is indispensable for the potent activation of NMU receptors.41) Therefore, we concentrated on derivatizations on other amino acid residues (Leu20, Phe21, Arg22) and the N-terminal acyl moiety.
The relatively bulky amino acid residue at positions 20 and 21, such as Trp and 2-naphthylalanine (2-Nal) are important for the selective activation of NMUR1.41–43) As a first NMUR1 agonist, CPN-124 bearing 2-Nal20 was reported in 2014 (Fig. 2); it showed a partial agonistic activity [EC50 = 2.2 nM, i.a., (intrinsic activity) = 79%].41) We found that introducing 2-thienylacetyl group at the N-terminus, Trp20 and Phe(4-F)21 (4-fluorophenylalanine), improves the efficacy and the agonistic activity through the synthesis of the most potent agonist CPN-170 (Fig. 2; EC50, NMUR1 = 0.083 nM, EC50, NMUR2 = 2.6 nM); however, its NMUR1 selectivity (approx. 30-fold) was lower than that of CPN-124 (approx. 50-fold).42) Therefore, the ongoing SAR study focused on position 21 of CPN-170. Consequently, α-methylated Trp, (α-Me)Trp21, displayed selective and full agonistic activities to NMUR1 (EC50 = 0.25 nM).43) This hexapeptide agonist, designated as CPN-267 (Fig. 2), is now commercially available.
In addition to the development of NMUR1 agonists, we analyzed the stability of peptide derivatives in serum. In rat and human serum, CPN-170, among our synthesized peptidic agonists, was rapidly degraded with the t1/2 value within 10 min, and the metabolite CPN-170-m1 cleaved at the amide bond between Arg24 and Asn25 was identified.42) Biodegradation at this site has also been reported by other groups.44,45) About 80% of the intact CPN-170 remained after incubation in citrated human plasma for 120 min.46) Further, we were interested in proteases activated in serum. Thrombin is one of such serine proteases, the factor IIa, involved in the center of the blood coagulation cascade. It recognizes the Pro-Arg sequence and cleaves the C-terminal amide bond of Arg.47–50) PPACK, a thrombin inhibitor, dramatically suppresses the rapid degradation of not only CPN-170 but also hNMU. However, other serine protease inhibitors such as PPACK-II (kallikrein inhibitor) and aprotinin were ineffective.46) Thus, we found that thrombin is a key enzyme that biodegrades NMU and its peptide derivatives in serum.
Surprisingly, CPN-267 with the same C-terminal sequence (Pro23-Arg24-Asn25-amide) has enhanced stability compared to CPN-170.43) The derivative bearing Trp at position 21 only exhibits slight stabilization43) indicating that α-methylation of Trp21 has multiple impacts not only on NMUR1 activation but also deterioration of thrombin recognition. We proposed derivatization at a position distant from the enzymatic cleavage site to stabilize peptidic molecules and maintain bioactivity. The P4 position of the substrate peptide interacts with the aryl-binding site (S4 pocket) of thrombin.47,51) We think that the side chain of Trp21 (P4 residue) is constrained by α-methylation to an unfavorable orientation. Furthermore, the success of alternative derivatization to increase the bulkiness of the P4 side-chain was confirmed by synthesizing CPN-223 (Fig. 2; EC50 = 3.2 nM, i.a., = 75%), a partial agonist improving NMUR1 selectivity (>300-fold).52) Instead of Phe21 in CPN-124, Bph21 (biphenylalanine) was introduced into P4 position to generate the steric hindrance against the S4 pocket of thrombin. The α-methylation of P4-Phe in CPN-124 causes the loss of the agonistic activity towards NMU receptors. This suggests that binding of derivatives bearing the 3-(3-pyridyl)propionyl group at the N-terminus (CPN-124 and CPN-223) to NMUR1 is different from that of 2-thienylacetyl hexapeptides (CPN-170 and CPN-267).
The introduction of an aliphatic amino acid (such as Leu21) at position 21 and a primary amine structure with a shorter side chain at position 22 is effective for the selective activation of NMUR2 in addition to the N-terminal 3-cyclohexylpropionyl group.41) CPN-116 bearing Dap22 (α,β-diaminopropionic acid) was reported in 2014 as the first NMUR2 selective agonist41) (Fig. 2); it is commercially available. However, the instability in phosphate buffer via the Nα-to-Nβ-acyl migration reaction was found later.53) Since the mechanism of phosphate-mediated acyl migration via five-membered ring intermediate was anticipated,54,55) chemical stability-enhanced CPN-219 bearing side-chain-extended Dab22 (α,γ-diaminobutanonic acid) was developed; it showed similar agonistic activity to CPN-116 (EC50 = 2.3 nM).53)
As the sequences of CPN-116 and CPN-219 are recognized by thrombin, they are degraded in serum.46) Recently, intranasal administration has attracted attention as a method for direct brain delivery (nose-to-brain delivery).56–58) Focusing on the activation of centrally expressed NMUR2, we demonstrated that administering CPN-116 and CPN-219 to the nasal cavity in male ddY mice markedly suppresses the body weight gain, which cannot be achieved via intraperitoneal injection.53,59) Nagai et al. reported that subcutaneous administration of high molecular weight conjugates selectively activated NMU receptors and decreased the bodyweight of obese-induced mice, resulting from the favorable pharmacokinetics in the circulation. However, the diarrhea score was transiently observed in the NMUR2-selective agonist.60) Therefore, intranasal administration might be a promising route for relieving digestive symptoms of NMUR2 receptor agonists.
We focused on the inactivation mechanism of myostatin by the prodomain protein as shown in Fig. 3A61); namely, the N-terminal α-helical region of prodomain embedded in the activin type I receptor binding site of myostatin was effective in identifying its minimum inhibitory segment. Jiang et al. previously reported that the glutathione S-transferase-fused 74-mer segment derived from the prodomain, including the corresponding α-helical region, can antagonize the myostatin activity.62) Synthesizing several segment peptides derived from the mouse sequence, we discovered the 23-mer inhibitory core peptide 3 spanning position 21–43 containing the corresponding α-helical region in the prodomain63) (Fig. 3B). Surprisingly, the human-derived version is not an effective inhibitor despite having the same amino acid sequence as peptide 3, except for positions 26 and 27 (Lys and Ser in human sequence, respectively).63) We carried out detailed SAR studies with peptide 3 to elucidate the molecular function and obtain a more potent derivative. The myostatin inhibitory activity and the secondary structure of peptide derivatives were analyzed by luciferase reporter assay and circular dichroic (CD) spectrum measurement.
We conducted an Ala scan to elucidate the contribution of each amino acid included in peptide 3. It suggested that nine amino acid residues, the N-terminal Trp, Tyr at position 27, and all branched-chain amino acids (BCAAs; Ile and Leu), are essential for effective inhibition64) (Fig. 3B). The importance of the corresponding Ile and Leu residues in the prodomain precursor was previously indicated in the similar inactivated complex formation of TGF-β1.65,66) At position 35, since Gly is coded in the TGF-β1 sequence, the role of Ile35 in the myostatin one is interesting. Here, the introduction of Pro, an α-helix breaker, into peptide 3 demonstrated that the α-helix structure formation ability is one of the requirements for exerting the inhibitory activity, and the helix core resides around position 32–3664) (Fig. 3B). Hence, it implies that Ile35 might be involved in forming the effective hydrophobic face and other BCAAs.
At position 27, Tyr is a rodent-specific residue along with Arg26 (Fig. 3B), while other species, including humans, have Ser as described above. Our SAR study showed that Tyr27 of peptide 3 could be substituted by Trp, Phe, or D-Tyr without decreasing the inhibitory activity, suggesting that hydrophobic aromatic amino acid at position 27 is necessary for effective inhibition of myostatin by synthetic peptides.64) This means that the exploration of mouse sequence is a key point for the discovery of peptide 3.
At the N-terminal position 21, Trp has a major contribution to the inhibitory activity of peptide 3. The deletion of Trp leads to a three-times lower affinity to myostatin and a reduced inhibition ability.63) In addition, the N-terminal heptapeptide (position 21–27)-deleted 16-mer peptide 4 did not show any inhibitory activity63,67) (Fig. 4). These results mean that the cooperative effect of not only seven BCAAs but also Trp21 and Tyr27 is necessary for the inhibition of peptide 3 with IC50 value of 3.56 µM.68)
The numbers above each amino acid indicate the position in the prodomain. The underlined residue is the moiety derivatized from original peptide 3. a The respective IC50 values are taken from refs. 68 (peptides 3 and 5) and 67 (peptides 4 and MIPE-1686). b The structural content values (%) are from refs. 72 (peptides 3 and 5) and 67 (peptides 4 and MIPE-1686).
We performed the docking simulation of peptide 3 to myostatin using a molecular operating environment (MOE) based on the above findings.69) Using a human pro-myostatin structure 5NTU (Protein Data Bank code) as a template,61) homology-modeled mouse pro-myostatin was prepared under the force field of Amber10:EHT. Then, myostatin and peptide fragments (position 21–43) were extracted. Next, 10 energy minimized stable structures of the peptide with the constrained main chain at position 30–41 were docked to myostatin with the induced-fit mode; consequently, four promising models were proposed.69) Among these models, the common residues of myostatin to interact with peptide are Leu20, Asn41, Leu52, Leu60, Met101, and Ser109 (Fig. 3B). Therefore, residues probably function as a key for binding the synthetic peptide inhibitor. Additionally, it also suggested that Cys108 and Asn88 of myostatin are involved in recognizing peptidic Trp21 and Tyr27, respectively. Therefore, forming the complementary interaction around the key residues might be important, implying that this simulation provides valuable information for designing a new inhibitor.
Using the medicinal chemistry approach, we attempted to improve the inhibition ability and shorten the peptide-chain length. The chain-shortening generally reduces antigenicity and manufacturing cost and may enhance biological stability by decreasing protease-recognition sites.70) For developing a potent derivative, a hydrophobic residue-directed SAR study focused on the N-terminal Trp21 and 7 BCAAs of peptide 3 were performed. The N-terminal Trp21 and Leu38 were structurally optimized to 2-naphthyloxyacetic acid and Ile.68,71) In conjunction with the substitution of Ala32 with Trp, a new 22-mer peptide 5 was developed68) (Fig. 4). It exhibited about 10-fold higher potency (IC50 = 0.32 µM) than peptide 3. Interestingly, the CD spectrum secondary structure analysis indicated that peptide 5 tended to form a β-sheet structure. Alternatively, the cyclization to induce the α-helix structure formation was applied to peptide 5. However, a series of cyclic peptide derivatives displayed similar inhibitory potency and β-sheet-forming property as that of peptide 5.72) It seems that the potent derivatives, including peptide 5, interact with myostatin by a different binding mode than peptide 3.
In the above SAR, we were interested in introducing Trp and Ile into positions 32 and 38 to afford the β-sheet-forming property as the N-terminal 2-naphthyloxyacetylation did not affect the α-helical nature of peptide.71) We substituted these two residues in the 16-mer inactive peptide 4 bearing an α-helix-forming property. As expected, the prepared 16-mer inhibitory peptide formed a β-sheet structure.67) The subsequent SAR study yielded the most potent 16-mer inhibitor MIPE-1686 with the IC50 value of 0.13 µM that also forms predominantly β-sheets67) (Fig. 4). In the next step, recombinant aminopeptidase N, chymotrypsin C, and trypsin 3 were used to examine the proteolytic stability of MIPE-1686, a N-terminal-free L-based peptide that includes three unnatural amino acids (two cyclohexylglycine and one D-Trp). Surprisingly, MIPE-1686 maintained its intact state over 270 min in each enzyme solution.73) This stabilization by the derivatization of position to be distant from the cleavage site seems related to the tendency to form a β-sheet structure.73) Namely, it suggests that the modulation of the secondary structure is valuable for improving the proteolytic stability. Finally, to investigate the in vivo applicability, MIPE-1686 was directly injected into the muscle of DMD model mdx mice. The muscle mass and the grip strength were remarkably increased at day 42 after the twice injection at days 0 and 14,67) suggesting a promising candidate for a muscle-building agent.
We developed N-terminal acylated hexapeptide agonists to NMUR1 and NMUR2 through derivatizing Phe19-Leu20-Phe21-Arg22 sequence in the C-terminal NMU. These are currently the minimum peptidic agents.44,45,74–78) The one amino acid-shorter derivative, the pentapeptide-type agonist, has a weaker NMUR1 agonistic activity without improved serum stability.52) Additionally, we have never found the prominent NMUR2 agonistic activity in pentapeptide derivatives. Thereby, it suggests that the molecular size of at least hexapeptide is indispensable for both the potent NMU receptor activation and the enhanced serum stability. In the future, we hope that receptor-selective agonists such as CPN-267 and CPN-219 greatly contribute to in vivo functional analyses of respective NMU receptors.
While examining myostatin inactivation, we first discovered the synthetic peptide inhibitor 3 derived from the prodomain sequence of the mouse myostatin-precursor. In other words, peptide 3 acts as the first synthetic PPI inhibitor acting on myostatin and its receptors. The detailed SAR study of amino acid substitution of peptide 3 with the α-helical nature proposed the molecular mechanism of inhibition and also afforded the chain-shortened 16-mer MIPE-1686 bearing a β-sheet-forming property, representing the most potent among our synthesized derivatives. Although the alternative 14-mer inhibitor DF-3 derived from the N-terminal α-helical domain of Fst was discovered like that of peptide 3,79) the following SAR study did not achieve the IC50 value of sub-micromolar-order to develop the derivative DF-100 further.80) Therefore, it seems that a peptide of around 16-mer length is needed for the potent inhibition of myostatin in the present state. If the binding mode of MIPE-1686 to myostatin is clarified in the future work, we may discover the molecular mechanism for improving its inhibitory activity by comparing it with peptide 3. This might lead to a breakthrough in the design and development of a novel smaller molecule inhibitor.
I would like to express my sincere gratitude to Prof. Yoshio Hayashi (Tokyo University of Pharmacy and Life Sciences) for his fruitful support and encouragement, which lead to the opportunity to describe this review. Also, I am grateful for all co-workers and collaborators who dedicated their great contributions to this study. The research described herein was supported by the Japan Society for the Promotion of Sciences (JSPS) KAKENHI, including Grants-in-Aid for Young Scientist (B) 17K15484.
The author declares no conflict of interest.
This review of the author’s work was written by the author upon receiving the 2021 Pharmaceutical Society of Japan Award for Young Scientists.
