A new class of pentapeptide KISS1 receptor agonists with hypothalamic-pituitary-gonadal axis activation.

The kisspeptin (Kp, Kp-54, metastin)/KISS1R system plays crucial roles in regulating the secretion of gonadotropin-releasing hormone. Continuous administration of nonapeptide Kp analogs caused plasma testosterone depletion, whereas bolus administration caused strong plasma testosterone elevation in male rats. To develop a new class of small peptide drugs, we focused on stepwise N-terminal truncation of Kp analogs and discovered potent pentapeptide analogs. Benzoyl-Phe-azaGly-Leu-Arg(Me)-Trp-NH2 (16) exhibited high agonist activity for KISS1R and excellent metabolic stability in rat serum. A single injection of a 4-pyridyl analog (19) at the N-terminus of 16 into male Sprague Dawley rats caused a robust increase in plasma luteinizing hormone levels, but unlike continuous administration of nonapeptide Kp analogs, continuous administration of 19 maintained moderate testosterone levels in rats. These results indicated that small peptide drugs can be successfully developed for treating sex hormone deficiency.

Glycosaminoglycan Conjugation for Improving the Duration of Therapeutic Action of Glucagon-Like Peptide-1.

Glucagon-like peptide-1 (GLP-1) is an incretin peptide that plays a crucial role in lowering blood glucose levels and holds promise for treating type II diabetes. In this study, we synthesized GLP-1 derivatives that were conjugated with glycosaminoglycans (GAGs), i.e., chondroitin (CH) or heparosan (HPN), to address the major limitation in their clinical use of GLP-1, which is its short half-life in the body. After exploring a variety of CHs with different molecular sizes and heterobifunctional linkers having different alkyl chains, we obtained CH-conjugated GLP-1 derivatives that stayed in blood circulation much longer (T1/2 elim > 25 h) than unconjugated GLP-1 and showed blood glucose-lowering efficacy up to 120 h after subcutaneous injection in mice. By using the same optimized linker design, we eventually obtained a HPN-conjugated GLP-1 derivative with efficacy lasting 144 h. These results demonstrate that conjugation with GAG is a promising strategy for improving the duration of peptide drugs.

Differential effects of selective agonists of neuromedin U1 and U2 receptors in obese and diabetic mice.

BACKGROUND AND PURPOSE: Neuromedin U (NmU) may be a novel target for obesity treatment owing to its anorectic and energy expenditure enhancing effects. Although two receptors, NMU1 and NMU2, are both responsible for the NmU-mediated anti-obesity effects, the receptor agonist with the most appropriate profiles for treating obesity and diabetes in terms of efficacy and safety is as yet unknown. Thus, we developed and evaluated novel NMU1/2 receptor-selective agonists. EXPERIMENTAL APPROACH: Efficacy and safety were assessed in mice with diet-induced obesity (DIO) and those with leptin-deficient diabetes (ob/ob) through repeated peripheral administration of selective agonists to NMU1 (NMU-6102) and NMU2 (NMU-2084), along with non-selective NMU1/2 agonists (NMU-0002 and NMU-6014). We also performed immunohistochemistry for c-Fos protein expression in the brain to probe their mechanisms of action. KEY RESULTS: Although both non-selective NMU1/2 agonists and the NMU2-selective agonist had high efficacy compared with the NMU1-selective agonist, only the NMU2-selective agonist led to relatively low adverse effects, such as diarrhoea, in DIO mice. However, the non-selective NMU1/2 agonist and the NMU1-selective agonist, but not the NMU2-selective agonist, were effective in diabetic ob/ob mice. Mechanistically, NMU2-selective agonists preferentially activate pro-opiomelanocortin neurons in the hypothalamic arcuate nucleus but not in the paraventricular nucleus. CONCLUSIONS AND IMPLICATIONS: These results suggest that an NMU2 receptor-selective agonist may be a well-balanced drug for the treatment of obesity and that an NMU1 receptor-selective agonist may also be beneficial for treating obesity and diabetes once its side effects are minimized.

A novel LRP1-binding peptide L57 that crosses the blood brain barrier.

The blood-brain barrier (BBB) is a major obstacle to drug delivery into the central nervous system (CNS), in particular for macromolecules such as peptides and proteins. However, certain macromolecules can reach the CNS via a receptor-mediated transcytosis (RMT) pathway, and low-density lipoprotein receptor-related protein 1 (LRP1) is one of the promising receptors for RMT. An LRP1 ligand peptide, Angiopep-2, was reported to pass through the BBB and deliver covalently conjugated drugs into the CNS. While conjugation of LRP1 ligands with drugs would be an effective approach for drug delivery to the CNS, no other reliable LRP1 ligands have been reported to date. In this study, we aimed to identify novel LRP1 ligands to further investigate LRP1-mediated RMT. Using phage display technology, we obtained a novel peptide, L57 (TWPKHFDKHTFYSILKLGKH-OH), with an EC50 value of 45 nM for binding to cluster 4 (Ser3332-Asp3779) of LRP1. L57 was stable in mouse plasma for up to 20 min. In situ brain perfusion assay in mice revealed the significantly high BBB permeability of L57. In conclusion, we discovered L57, the first artificial LRP1-binding peptide with BBB permeability. Our findings will contribute to the development of RMT-based drugs for the treatment of CNS diseases.

Highly potent antiobesity effect of a short-length peptide YY analog in mice.

Continuous administration of a 14-amino acid peptide YY (PYY) analog, Ac-[d-Pro24,Pya(4)26,Cha27,36,Aib28,31,Lys30]PYY(23-36) (4), which has a high binding affinity and agonist activity for the neuropeptide Y2 receptor (Y2R), has previously shown an antiobesity effect in a 2-week diet-induced obesity (DIO) study in mice. However, there remained a possibility to obtain more potent analogs by further improving its pharmacokinetic profile. A combination of the N-terminal 4-imidazolecarbonyl moiety and three amino acid substitutions, trans-4-hydroxy-d-proline (d-Hyp)24, isovaline (Iva)25, and γ-methylleucine (γMeLeu)28, not only improved the binding affinity of the peptide for Y2R but also increased its anorectic activity in lean mice. In a 2-week DIO study in mice, continuous administration of 4-imidazolecarbonyl-[d-Hyp24,Iva25,Pya(4)26,Cha27,36,γMeLeu28,Lys30,Aib31]PYY(23-36) (31, PYY-1119) at a dose of 0.03mg/kg/day showed a highly potent antiobesity effect, with more than 10% body weight reduction.

Potent antiobesity effect of a short-length peptide YY-analogue continuously administered in mice.

The gastrointestinal peptide, peptide YY3-36 (PYY3-36) and its shorter peptide analogues have been reported to reduce appetite by activating the neuropeptide Y2 receptor (Y2R), which is associated with obesity and other metabolic diseases. A 14-amino acid PYY analogue, Ac-[d-Pro24,Cha27,28,36,Aib31]PYY(23-36) (3), showed high binding affinity and agonist activity for the Y2R, similar to that of PYY3-36, but had weak anorectic activity upon continuous administration in lean mice. Three amino acid substitutions [Pya(4)26, Aib28, Lys30], which contributed to the decreased hydrophobicity of 3, efficiently increased its anorectic activity. The compound containing these three amino acids, Ac-[d-Pro24,Pya(4)26,Cha27,36,Aib28,31,Lys30]PYY(23-36) (22), exerted more potent and durable food intake suppression than that by PYY3-36 in lean mice, as well as excellent Y2R agonist activity (EC50: 0.20nM) and good subcutaneous bioavailability (66.6%). The 11-day continuous administration of 22 at 1mg/kg/day successfully produced antiobese and antidiabetic effects, with more than 20% body weight loss in obese and Type 2 diabetes ob/ob model mice.

Potent Body Weight-Lowering Effect of a Neuromedin U Receptor 2-selective PEGylated Peptide.

Neuromedin U (NMU) is a neuropeptide that mediates a variety of physiological functions via its receptors, NMUR1 and NMUR2. Recently, there has been an increased focus on NMU as a promising treatment option for diabetes and obesity. A short form of NMU (NMU-8) has potent agonist activity for both receptors but is metabolically unstable. Therefore, we designed and synthesized NMU-8 analogues modified by polyethylene glycol (PEG; molecular weight, 20 kDa; PEG20k) via a linker. 3-(2-Naphthyl)alanine substitution at position 19 increased NMUR2 selectivity of NMU-8 analogues with retention of high agonist activity. Compound 37, an NMUR2-selective PEG20k analogue containing piperazin-1-ylacetyl linker, exhibited a potent body weight-lowering effect with concomitant inhibition of food intake in a dose-dependent manner (body weight loss of 12.4% at 30 nmol/kg) by once-daily repeated dosing for 2 weeks in mice with diet-induced obesity.

Antiobesity Effect of a Short-Length Peptide YY Analogue after Continuous Administration in Mice.

Gastrointestinal peptides such as peptide YY (PYY) can regulate appetite, which is relevant to the study of obesity. The intraperitoneal bolus administration of PYY3-36 and a 12-amino acid PYY analogue, benzoyl-[Cha27,28,36,Aib31]PYY25-36 (1), showed similar anorectic activity by activating the Y2 receptor (Y2R). However, food intake inhibition and body weight loss were not observed upon continuous subcutaneous administration of 1 with osmotic pumps in diet-induced obese (DIO) mice. N-Terminal elongation of 1, together with amino acid substitution at position 24, led to a hydrophilic 14-amino acid peptide, Ac-[d-Hyp24,Cha27,28,36,Aib31]PYY23-36 (18), that showed higher affinity and more potent agonist activity for Y2R and a robust anorectic activity with potency similar to that of PYY3-36. In addition, the continuous subcutaneous administration of 18 at 0.3 mg/(kg·day) induced significant body weight loss in DIO mice. These results suggest that a short-length PYY analogue can be a lead compound for antiobesity therapy in a sustained-release formulation.

PEGylated neuromedin U-8 shows long-lasting anorectic activity and anti-obesity effect in mice by peripheral administration.

Neuromedin U (NMU) is a neuropeptide found in the brain and gastrointestinal tract. The NMU system has been shown to regulate energy homeostasis by both a central and a peripheral mechanism. Peripheral administration of human NMU-25 was recently shown to inhibit food intake in mice. We examined the possibility that other NMU-related peptides exert an anorectic activity by intraperitoneal (i.p.) administration. We found that rat NMU-23 and its structurally-related peptide rat neuromedin S (NMS) significantly reduced food intake in lean mice, whereas NMU-8, an active fragment of the octapeptide sequence conserved in porcine, human and mouse NMU, had no effect. When rat NMU-23, NMU-8, and rat NMS were covalently conjugated to polyethylene glycol (PEG) (PEGylation) at the N-terminus of these peptides, PEGylated NMU-8 showed the most long-lasting and robust anorectic activity. The exploration of the linker between NMU-8 and PEG using hetero-bifunctional chemical cross-linkers led to an identification of PEGylated NMU-8 analogs with higher affinity for NMU receptors and with more potent anorectic activity in lean mice. The PEGylated NMU-8 showed potent and robust anorectic activity and anti-obesity effect in diet-induced obesity (DIO) mice by once-daily subcutaneous (s.c.) administration. These results suggest that PEGylated NMU-8 has the therapeutic potential for treatment of obesity.

A PEGylated analog of short-length Neuromedin U with potent anorectic and anti-obesity effects.

Neuromedin U (NMU) is a neuropeptide known to regulate food intake and energy homeostasis that is widely distributed in the gastrointestinal tract, hypothalamus, and pituitary. A short form of NMU, porcine NMU-8 has potent agonist activity for the receptors NMUR1 and NMUR2; however, its short half-life precludes its effective use in vivo. To address this limitation, we designed and synthesized NMU-8 analogs modified by polyethylene glycol (PEG) with a molecular weight of 30kDa (PEG30k) via a variety of linkers (i.e., ω-amino- and ω-imino-carboxylic acid linker). Integrated evaluation of NMUR1 and NMUR2 binding affinities in vitro and anorectic activity in mice revealed that the introduction of a linker with a rigid ring group, e.g., 2-(piperazin-1-yl)acetic acid (PipAc), yielded a highly potent anorectic peptide, PEG30k-PipAc-NMU-8 (14), possessing improved receptor binding affinity. Subsequent optimization of the molecular weight of the PEG moiety led to the discovery of a PEG20k conjugate (15), which exhibited significant anti-obesity effect upon once-daily subcutaneous administration in diet-induced obese mice with 10% and 22% body weight loss at doses of 10 and 30nmol/kg, respectively. In addition, 15 reduced the weights of the liver and adipose tissue in a dose-dependent manner and improved the plasma biochemical parameters, e.g., insulin, glutamic pyruvic transaminase, glutamic oxaloacetic transaminase, and total cholesterol. Thus, our results suggest that 15 (NMU-0002), which showed potent and long-lasting biological profiles in vivo, represents a candidate peptide for investigating the central and peripheral actions of NMU and its potential for clinical use.

Generation of a novel artificial TrkB agonist, BM17d99, using T7 phage-displayed random peptide libraries.

Tropomyosin receptor kinase B (TrkB) is a known receptor of brain-derived neurotrophic factor (BDNF). Because it plays a critical role in the regulation of neuronal development, maturation, survival, etc., TrkB is a good target for drugs against central nervous system diseases. In this study, we aimed to generate peptidic TrkB agonists by applying random peptide phage display technology. After the phage panning against recombinant Fc-fused TrkB (TrkB-Fc), agonistic phages were directly screened against TrkB-expressing HEK293 cells. Through subsequent screening of the first-hit BM17 peptide-derived focus library, we successfully obtained the BM17d99 peptide, which had no sequence similarity with BDNF but had TrkB-binding capacity. We then synthesized a dimeric BM17d99 analog peptide that could phosphorylate or activate TrkB by facilitating receptor homodimerization. Treatment of TrkB-expressing HEK293 cells with the dimeric BM17d99 analog peptide significantly induced the phosphorylation of TrkB, suggesting that homodimerization of TrkB was enhanced by the dimeric peptide. This report demonstrates that our approach is useful for the generation of artificial peptidic agonists of cell surface receptors.

A Short-Length Peptide YY Analogue with Anorectic Effect in Mice.

Peripheral administration of PYY3-36, a fragment of peptide YY (PYY), has been reported to reduce food intake by activating the neuropeptide Y2 receptor (Y2R). An N-terminally truncated PYY analogue, benzoyl-[Ala26,Ile28,31]PYY(25-36) (1), showed a relatively potent agonist activity for Y2R but a weak anorectic activity by intraperitoneal administration (2000 nmol/kg) in lean mice because of its markedly poor biological stability in the mouse serum. Notably, two cyclohexylalanine (Cha) substitutions for Tyr residues at positions 27 and 36 (4) improved the stability in the mouse serum concomitant with enhanced anorectic activity. Further optimization at positions 27, 28, 30, and 31 revealed that 21, containing Cha28 and Aib31 residues, showed a more potent anorectic activity than PYY3-36 at a low dose of 300 nmol/kg. The minimum effective dose by intraperitoneal administration of 21 was 30 nmol/kg (ca. 52 µg/kg) in mice, suggesting the biologic potential of short-length PYY3-36 analogues with a potent anorectic effect.

Discovery of an artificial peptide agonist to the fibroblast growth factor receptor 1c/ßKlotho complex from random peptide T7 phage display.

Fibroblast growth factor receptor-1c (FGFR1c)/ßKlotho (KLB) complex is a receptor of fibroblast growth factor 21 (FGF21). Pharmacologically, FGF21 shows anti-obesity and anti-diabetic effects upon peripheral administration. Here, we report the development of an artificial peptide agonist to the FGFR1c/KLB heterodimer complex. The peptide, F91-8A07 (LPGRTCREYPDLWWVRCY), was discovered from random peptide T7 phage display and selectively bound to the FGFR1c/KLB complex, but not to FGFR1c and KLB individually. After subsequent peptide dimerization using a short polyethyleneglycol (PEG) linker, the dimeric F91-8A07 peptide showed higher potent agonist activity than that of FGF21 in cultured primary human adipocytes. Moreover, the dimeric peptide led to an expression of the early growth response protein-1 (Egr-1) mRNA in vivo, which is a target gene of FGFR1c. To the best of our knowledge, this is the first report of a FGFR1c/KLB complex-selective artificial peptide agonist.

Design and Synthesis of an Investigational Nonapeptide KISS1 Receptor (KISS1R) Agonist, Ac-d-Tyr-Hydroxyproline (Hyp)-Asn-Thr-Phe-azaGly-Leu-Arg(Me)-Trp-NH2 (TAK-448), with Highly Potent Testosterone-Suppressive Activity and Excellent Water Solubility.

Metastin/kisspeptin is an endogenous ligand of KISS1 Receptor (KISS1R). Metastin and KISS1R are suggested to play crucial roles in regulating the secretion of gonadotropin-releasing hormone (GnRH), and continuous administration of metastin derivatives attenuated the plasma testosterone levels in male rats. Our optimization studies of metastin derivatives led to the discovery of 1 (Ac-d-Tyr-d-Trp-Asn-Thr-Phe-azaGly-Leu-Arg(Me)-Trp-NH2, TAK-683), which suppressed plasma testosterone in rats at lower doses than those of leuprolide. Although 1 possessed extremely potent pharmacological activity, 20 mg/mL aqueous solution of 1 has a gel formation property. In order to improve this physicochemical property, we substituted d-Trp at position 47 with a variety of amino acids; we identified that substitution with cyclic amino acids, which could change peptide conformation, retained its potency. Especially, analogue 24 (TAK-448) with trans-4-hydroxyproline (Hyp) at position 47 showed not only superior pharmacological activity to 1 but also excellent water solubility. Furthermore, 20 mg/mL aqueous solution of 24 did not show gel formation up to 5 days.

Development of antibody-siRNA conjugate targeted to cardiac and skeletal muscles.

Despite considerable efforts to develop efficient carriers, the major target organ of short-interfering RNAs (siRNAs) remains limited to the liver. Expanding the application outside the liver is required to increase the value of siRNAs. Here we report on a novel platform targeted to muscular organs by conjugation of siRNAs with anti-CD71 Fab' fragment. This conjugate showed durable gene-silencing in the heart and skeletal muscle for one month after intravenous administration in normal mice. In particular, 1µg siRNA conjugate showed significant gene-silencing in the gastrocnemius when injected intramuscularly. In a mouse model of peripheral artery disease, the treatment with myostatin-targeting siRNA conjugate by intramuscular injection resulted in significant silencing of myostatin and hypertrophy of the gastrocnemius, which was translated into the recovery of running performance. These data demonstrate the utility of antibody conjugation for siRNA delivery and the therapeutic potential for muscular diseases.

Physicochemically and pharmacokinetically stable nonapeptide KISS1 receptor agonists with highly potent testosterone-suppressive activity.

Modifications of metastin(45-54) produced peptide analogues with higher metabolic stability than metastin(45-54). N-terminally truncated nonapeptide 4 ([D-Tyr46,D-Pya(4)47,azaGly51,Arg(Me)53]metastin(46-54)) is a representative compound with both potent agonistic activity and metabolic stability. Although 4 had more potent testosterone-suppressant activity than metastin, it possessed physicochemical instability at pH 7 and insufficient in vivo activity. Instability at pH 7 was dependent upon Asn48 and Ser49; substitution of Ser49 with Thr49 reduced this instability and maintained KISS1 receptor agonistic activity. Furthermore, [D-Tyr46,D-Trp47,Thr49,azaGly51,Arg(Me)53,Trp54]metastin(46-54) (14) showed 2-fold greater [Ca2+]i-mobilizing activity than metastin(45-54) and an apparent increase in physicochemical stability. N-terminal acetylation of 14 resulted in the most potent analogue, 22 (Ac-[D-Tyr46,D-Trp47,Thr49,azaGly51,Arg(Me)53,Trp54]metastin(46-54)). With continuous administration, 22 possessed 10-50-fold more potent testosterone-suppressive activity in rats than 4. These results suggested that a controlled release of short-length KISS1 receptor agonists can suppress the hypothalamic-pituitary-gonadal axis and reduce testosterone levels. Compound 22 was selected for further preclinical evaluation for hormone-dependent diseases.

Pharmacologic profiles of investigational kisspeptin/metastin analogues, TAK-448 and TAK-683, in adult male rats in comparison to the GnRH analogue leuprolide.

Kisspeptin/metastin, a hypothalamic peptide, plays a pivotal role in controlling gonadotropin-releasing hormone (GnRH) neurons, and we have shown that continuous subcutaneous administration of kisspeptin analogues suppresses plasma testosterone in male rats. This study examined pharmacologic profiles of investigational kisspeptin analogues, TAK-448 and TAK-683, in male rats. Both analogues showed high receptor-binding affinity and potent and full agonistic activity for rat KISS1R, which were comparable to natural peptide Kp-10. A daily subcutaneous injection of TAK-448 and TAK-683 (0.008-8µmol/kg) for consecutive 7 days initially induced an increase in plasma luteinizing hormone and testosterone levels; however, after day 7, plasma hormone levels and genital organ weights were reduced. Continuous subcutaneous administrations of TAK-448 (≥10pmol/h, ca. 0.7nmol/kg/day) and TAK-683 (≥30pmol/h, ca. 2.1nmol/kg/day) induced a transient increase in plasma testosterone, followed by abrupt reduction of plasma testosterone to castrate levels within 3-7 days. This profound testosterone-lowering effect was sustained throughout 4-week dosing periods. At those dose levels, the weights of the prostate and seminal vesicles were reduced to castrate levels. These suppressive effects of kisspeptin analogues were more rapid and profound than those induced by the GnRH agonist analogue leuprolide treatment. In addition, TAK-683 reduced plasma prostate specific antigen (PSA) in the JDCaP androgen-dependent prostate cancer rat model. Thus, chronic administration of kisspeptin analogues may hold promise as a novel therapeutic approach for suppressing reproductive functions and hormone-related diseases such as prostate cancer. Further studies are warranted to elucidate clinical significance of TAK-448 and TAK-683.

Design, synthesis, and biological evaluation of novel investigational nonapeptide KISS1R agonists with testosterone-suppressive activity.

Metastin/kisspeptin is a 54 amino acid peptide ligand of the KISS1R receptor and is a critical regulator of GnRH secretion. The N-terminally truncated peptide, metastin(45-54), possesses a 10-fold higher receptor-binding affinity than full-length metastin and agonistic KISS1R activity but is rapidly inactivated in rodent plasma. We have developed a decapeptide analog [D-Tyr(45),D-Trp(47),azaGly(51),Arg(Me)(53)]metastin(45-54) with improved serum stability compared with metastin(45-54) but with decreased KISS1R agonistic activity. Amino acid replacements at positions 45-47 led to an enhancement of KISS1R agonistic activity and metabolic stability. N-terminal truncation resulted in a stable nonapeptide, [D-Tyr(46),D-Pya(4)(47),azaGly(51),Arg(Me)(53)]metastin(46-54), compound 26, which displayed KISS1R binding affinities comparable to metastin(45-54) and had improved serum stability. Compound 26 reduced plasma testosterone in male rats and is the first short-length metastin analog to possess testosterone suppressive activities. Compound 26 has led to the elucidation of investigational analogs TAK-683 and TAK-448, both of which have undergone clinical evaluation for hormone-dependent diseases such as prostate cancer.

Chronic administration of the metastin/kisspeptin analog KISS1-305 or the investigational agent TAK-448 suppresses hypothalamic pituitary gonadal function and depletes plasma testosterone in adult male rats.

Metastin/kisspeptin, a hypothalamic peptide, plays a pivotal role in controlling GnRH neurons. Here we studied the effect of chronic sc administration of two kisspeptin analogs, KISS1-305 and TAK-448, on hypothalamic-pituitary-gonadal function in male rats in comparison with a GnRH analogue leuprolide or bilateral orchiectomy (ORX). The prototype polypeptide, KISS1-305 (1-4 nmol/h), caused substantial elevations of plasma LH and testosterone, followed by abrupt reductions of both hormone levels. Notably, testosterone levels were reduced to castrate levels within 3 d and remained depleted throughout the 4-wk dosing period, an effect that was faster and more pronounced than leuprolide (1 nmol/h) dosing. KISS1-305 also reduced genital organ weight more profoundly than leuprolide. In mechanistic studies, chronic KISS1-305 administration only transiently induced c-Fos expression in GnRH neurons, suggesting that GnRH-neural response was attenuated over time. Hypothalamic GnRH content was reduced to 10-20% of control at 3 wk without any changes in Gnrh mRNA expression. Dosing with the investigational peptide TAK-448 was also studied to extend our understanding of hypothalamic-pituitary functions. Similar to ORX, TAK-448 (0.1 nmol/h) depleted testosterone and decreased GnRH content by 4 wk. However, in contrast to ORX, TAK-448 decreased gonadotropin levels in pituitary and plasma samples, implying the suppression of GnRH pulses. These results suggest that chronic administration of kisspeptin analogs disrupts endogenous kisspeptin signals to suppress intrinsic GnRH pulses, perhaps by attenuating GnRH-neural response and inducing continuous GnRH leakage from the hypothalamus. The potential utility of kisspeptin analogs as novel agents to treat hormone-related diseases, including prostate cancer, is discussed.

Trypsin resistance of a decapeptide KISS1R agonist containing an Nω-methylarginine substitution.

Metastin/kisspeptin is an amidated peptide with 54 amino acid residues isolated from human placental tissues as a ligand of the orphan G-protein-coupled receptor KISS1R that is expressed throughout the central nervous system and in a variety of endocrine and gonadal tissues. Compared to the full-length metastin protein, the N-terminal truncated peptide metastin(45-54) has 3-10 times higher receptor affinity and enhanced ability to increase intracellular calcium concentration which is essential for activation of protein kinases involved in intracellular signaling in a number of pathways that affect reproduction and cell migration. However, metastin(45-54) is rapidly inactivated in serum. In this study, we designed and synthesized a number of metastin(45-54) analogs and evaluated their agonistic activity and trypsin resistance. Among analogs with substitutions of arginine at position 53, N(ω)(-)methylarginine analog 8 showed 3-fold more potent agonistic activity compared with metastin(45-54). Furthermore, analog 8 was shown to resist trypsin cleavage between positions 53 and 54. This substitution may be useful in the development of other Arg-containing peptides for which the avoidance of cleavage is desired.