Suppressive Effects of Neuromedin U Receptor 2‑Selective Peptide Agonists on Appetite and Prolactin Secretion in Mice

Neuromedin U receptor 2 (NMUR2), which is expressed in the central nervous system (CNS) including the hypothalamus, has been noted as a therapeutic target against obesity. We previously reported that intranasal administration of CPN-219, a NMUR2-selective hexapeptide agonist, suppresses body weight gain in mice; however, there is no detailed information regarding its CNS effects. Recently, in addition to appetite suppression, stress responses and regulation of prolactin (PRL) secretion have also attracted attention. NMUR2 expressed in the hypothalamic tuberoinfundibular dopaminergic neurons has emerged as an alternative target for treating hyperprolactinemia. Here, CPN-219 decreased food intake up to 24 h after administration at a dose of 200 nmol, resulting in body weight gain suppression, although grooming and anxiety-like behaviors were transiently induced. Interestingly, the restraint stress-induced increase in plasma PRL levels was significantly suppressed at a lower dose of 20 nmol, indicating the potential for drug development as an anti-PRL agent of NMUR2-selective agonists

Structural Basis for the Effective Myostatin Inhibition of the Mouse Myostatin Prodomain-Derived Minimum Peptide

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 <b>1</b> (WRQN­TRY­SRIE­AIK­IQIL­SKLRL-amide) was discovered from the mouse myostatin prodomain. In the present study, alanine scanning of <b>1</b> 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 <b>1</b> significantly decreased both α-helicity and inhibitory activity. These results suggested that an α-helical structure possessing hydrophobic faces formed around the C-terminus is important for inhibitory activity

Effect of the Attachment of a Penetration Accelerating Sequence and the Influence of Hydrophobicity on Octaarginine-Mediated Intracellular Delivery

Arginine-rich cell-penetrating peptides (CPPs), including oligoarginine peptides, have been widely used as a tool for intracellular delivery of various molecules with low membrane permeability. We previously reported the enhanced cytosolic entry of arginine-rich CPPs by the attachment of a short peptide segment, the penetration accelerating sequence (Pas). In this study, the importance of hydrophobic sequences, especially phenylalanine residues, in the Pas segment was demonstrated for this enhanced translocation through cell membranes. The advantage of using Pas for intracellular delivery was particularly marked for delivering cargoes with a relatively small molecular weight, such as bioactive peptides. In addition, the results of this study indicate the important roles that the total hydrophobicity of the PasR8 conjugates play in cytosolic translocation and the eventual bioactivity thus attained

Discovery of Potent Hexapeptide Agonists to Human Neuromedin U Receptor 1 and Identification of Their Serum Metabolites

Neuromedin U (NMU) and S (NMS) display various physiological activities, including an anorexigenic effect, and share a common C-terminal heptapeptide-amide sequence that is necessary to activate two NMU receptors (NMUR1 and NMUR2). On the basis of this knowledge, we recently developed hexapeptide agonists <b>2</b> and <b>3</b>, which are highly selective to human NMUR1 and NMUR2, respectively. However, the agonists are still less potent than the endogenous ligand, hNMU. Therefore, we performed an additional structure–activity relationship study, which led to the identification of the more potent hexapeptide <b>5d</b> that exhibits similar NMUR1-agonistic activity as compared to hNMU. Additionally, we studied the stability of synthesized agonists, including <b>5d</b>, in rat serum, and identified two major biodegradation sites: Phe²-Arg³ and Arg⁵-Asn⁶. The latter was more predominantly cleaved than the former. Moreover, substitution with 4-fluorophenylalanine, as in <b>5d</b>, enhanced the metabolic stability at Phe²-Arg³. These results provide important information to guide the development of practical hNMU agonists

Structure–Activity Relationship Study of Leucyl-3-<i>epi</i>-deoxynegamycin for Potent Premature Termination Codon Readthrough

(+)-Negamycin, isolated from <i>Streptomyces purpeofuscus</i>, shows antimicrobial activity against Gram-negative bacteria and readthrough activity against nonsense mutations. Previously, we reported that two natural negamycin analogues, 5-deoxy-3-<i>epi</i>-negamycin and its leucine adduct, have more potent readthrough activity in eukaryocytes (COS-7 cells) than negamycin but possess no antimicrobial activity and no in vitro readthrough activity in prokaryotic systems. In the present study, on leucyl-3-<i>epi</i>-deoxynegamycin, a structure–activity relationship study was performed to develop more potent readthrough agents. In a cell-based readthrough assay, the derivative <b>13b</b> with an <i>o</i>-bromobenzyl ester functions as a prodrug and exhibits a higher readthrough activity against TGA-type PTC than the aminoglycoside G418. This ester (<b>13b</b>) shows an in vivo readthrough activity with low toxicity, suggesting that it has the potential for treatment of hereditary diseases caused by nonsense mutations

The Inhibitory Core of the Myostatin Prodomain: Its Interaction with Both Type I and II Membrane Receptors, and Potential to Treat Muscle Atrophy

<div><p>Myostatin, a muscle-specific transforming growth factor-β (TGF-β), negatively regulates skeletal muscle mass. The N-terminal prodomain of myostatin noncovalently binds to and suppresses the C-terminal mature domain (ligand) as an inactive circulating complex. However, which region of the myostatin prodomain is required to inhibit the biological activity of myostatin has remained unknown. We identified a 29-amino acid region that inhibited myostatin-induced transcriptional activity by 79% compared with the full-length prodomain. This inhibitory core resides near the N-terminus of the prodomain and includes an α-helix that is evolutionarily conserved among other TGF-β family members, but suppresses activation of myostatin and growth and differentiation factor 11 (GDF11) that share identical membrane receptors. Interestingly, the inhibitory core co-localized and co-immunoprecipitated with not only the ligand, but also its type I and type II membrane receptors. Deletion of the inhibitory core in the full-length prodomain removed all capacity for suppression of myostatin. A synthetic peptide corresponding to the inhibitory core (p29) ameliorates impaired myoblast differentiation induced by myostatin and GDF11, but not activin or TGF-β1. Moreover, intramuscular injection of p29 alleviated muscle atrophy and decreased the absolute force in caveolin 3-deficient limb-girdle muscular dystrophy 1C model mice. The injection suppressed activation of myostatin signaling and restored the decreased numbers of muscle precursor cells caused by caveolin 3 deficiency. Our findings indicate a novel concept for this newly identified inhibitory core of the prodomain of myostatin: that it not only suppresses the ligand, but also prevents two distinct membrane receptors from binding to the ligand. This study provides a strong rationale for the use of p29 in the amelioration of skeletal muscle atrophy in various clinical settings.</p></div

Development of a New Benzophenone–Diketopiperazine-Type Potent Antimicrotubule Agent Possessing a 2‑Pyridine Structure

A new benzophenone–diketopiperazine-type potent antimicrotubule agent was developed by modifying the structure of the clinical candidate plinabulin (<b>1</b>). Although the right-hand imidazole ring with a branched alkyl chain at the 5-position in <b>1</b> was critical for the potency of the antimicrotubule activity, we successfully substituted this moiety with a simpler 2-pyridyl structure by converting the left-hand ring from a phenyl to a benzophenone structure without decreasing the potency. The resultant compound <b>6b</b> (KPU-300) exhibited a potent cytotoxicity, with an IC₅₀ value of 7.0 nM against HT-29 cells, by strongly binding to tubulin (<i>K</i>_d = 1.3 μM) and inducing microtubule depolymerization

p29 restores the reduced myotube formation resulting from LGMD1C-causing mutant caveolin 3 (CAV3^P104L).

<p>(<b>A</b>) Wright-Giemsa-stained C2C12 cells expressing LGMD1C-causing Pro104Leu mutant caveolin 3 (CAV3^P104L) at 7 days after differentiation with (+) or without (–)1 μM p29 (<b>left</b>). Scale bar, 100 μm. Fusion indices of these cells following addition of 1 μM of p29 were calculated in triplicate as the percentage of the total nuclei in myotubes/mm² (<b>right</b>). Values are the means ± SD (<i>n</i> = 5). *<i>P</i> < 0.05. (<b>B</b>) (<b>C</b>) Phase-contrast (<b>left</b>) and fluorescence (<b>right</b>) images of MyHC in C2C12 myoblasts expressing the empty vector (mock) or Pro104Leu mutant caveolin 3 at 7 days after differentiation with (+) or without (–) 1 μM p29. Scale bar, 100 μm. (<b>C</b>) Immunoblot analysis of MyHC and β-actin in C2C12 cells expressing the empty vector (mock) or Pro104Leu mutant caveolin 3 (CAV3^P104L) at 7 days after differentiation with (+) or without (–) 1 μM p29 (<b>left</b>). Densitometric analysis (<b>right</b>). Values are mean ± SD fold increases compared with untreated C2C12 cells expressing the empty vector (mock) (<i>n</i> = 5). *<i>P</i> < 0.05.</p

The identified inhibitory core of the myostatin prodomain specifically suppresses myostatin and its analog, GDF11, and includes an AH that is evolutionarily conserved among several other TGF-β family members.

<p>(<b>A</b>) The full-length myostatin prodomain (f-Pro) and its inhibitory core (Pro11) inhibited the transcriptional activities of myostatin and GDF11, but not of TGF-β1 or activin A, in HEK293 cells. (<b>B, C</b>) Sequence alignment of the prodomains of myostatin in nine species (<b>B</b>) and nine TGF-β family members (<b>C</b>). Red indicates the identified inhibitory core of the myostatin prodomain, consisting of 29 amino acids. The AH structure (blue) of the TGF-β1 prodomain has been shown to bind to both its ligand and TSP-1* [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133713#pone.0133713.ref006" target="_blank">6</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133713#pone.0133713.ref018" target="_blank">18</a>]. Crystallographic analyses of TGF-β1 and its receptors have predicted that the random coiled structure (RC, green) and the AH are located closely to its type I receptor, whereas the latency lasso structure (LL, brown) is located close to its type II receptor** [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133713#pone.0133713.ref019" target="_blank">19</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133713#pone.0133713.ref020" target="_blank">20</a>].</p

Identification of the inhibitory core of the myostatin prodomain.

<p>(<b>A</b>) Truncation and deletion constructs of human myostatin prodomain:human Fc fusion proteins (<b>left</b>). Percentage inhibitory effect of each construct on myostatin activity in comparison with the full-length prodomain (f-Pro, <b>right</b>). (<b>B</b>) Recombinant myostatin-induced transcriptional activity in HEK293 human embryonic kidney cells co-transfected with a pGL3-(CAGA)₁₂-luciferase reporter gene, pCMV-β-Gal, and various prodomain region:Fc fusion constructs. Values are the mean ± SD (<i>n</i> = 6). RLU, relative luminescence units.</p