KR100822782B1 - Analogues of peptide synthesized and produced from Gaegurin 5 showing antiobesity activity - Google Patents

Abstract

The present invention relates to an anti-obesity agent containing a peptide derivative synthesized and prepared from openulin 5 that specifically binds to human melanocortin-4-receptor (MC4R). May be useful for the discovery and analysis of melanocyte cortin-4-receptors, which play a pivotal role in appetite control and obesity induction, and is safe for the human body because it is not cytotoxic and has low hemolytic activity against human red blood cells. It can be usefully used as a prophylactic and therapeutic agent.

Eoffrin 5, anti-obesity agent, melanocortin-4-receptor,

Description

Antialogues of peptide synthesized and produced from Gaegurin 5 showing antiobesity activity}

1 is a diagram showing a sequence list of the opening 5 and its derivatives,

2 is a diagram showing the binding stability of TAMRA-NDP-αMSH to the human MC4 receptor membrane,

3 is a diagram showing the substitution of TAMRA-NDP-αMSH by ligand,

4 shows substitution of TAMRA-NDP-αMSH by frog peptides.

The present invention relates to a novel use of peptide derivatives synthesized and prepared from openulin 5.

Recently, as the standard of living improves, the hygiene environment is improved, and the dietary life is westernized, and the average life span is extended. As a result, geriatric disease has emerged as the biggest medical task today, and obesity, which is a major cause of geriatric disease, is rapidly increasing. Obesity refers to a phenomenon in which excess calories are accumulated in the form of fat in the body by ingesting excessive calories compared to calories consumed. Obesity is thought to be caused by a variety of causes, including genetic effects, environmental effects of westernized diets, and psychological effects of stress, but the exact cause and mechanisms are not clearly established. However, because obesity can act not only as a problem of obesity itself, but also as a cause of diseases such as cardiovascular disease and diabetes (Manson et al., New England J. Med., 333, pp677-685, 1995; Kopleman PG, Nature, 404 pp635-643, 2000; Must et al., JAMA , 282 , pp1523-1529, 1999). There is much interest in the treatment of obesity worldwide.

On the other hand, cholesterol is mainly produced in the liver and is present in the blood in the form of small round particles called lipoproteins, which carry cholesterol throughout the body. There are two types of lipoproteins, low density lipoprotein (LDL) and high density lipoprotein (HDL). HDL carries cholesterol from other tissues. On the other hand, LDL is produced mainly in the liver and carries cholesterol to other tissues of the human body, so a large amount of LDL accumulates a lot of cholesterol in blood vessels to promote atherosclerosis, and about 70% of blood cholesterol is present as harmful cholesterol (LDL). Kim, Kyoung-hwan, Lee Woo-joo, Pharmacology Lecture, 4th edition, p466-481, 2001).

In fact, cholesterol is an essential ingredient necessary for the normal functioning of the human body, nutrients necessary to make cells, and a component of various hormones such as corticosteroids, male hormones, and female hormones. It can help you digest fatty foods. In addition, it is a nutrient necessary for the human body, such as a component of a biological membrane and used as a starting material for hormonal synthesis. However, excessive intake of such cholesterol accumulates in blood vessels and is known to cause heart disease. So far, there is no preventive method other than low cholesterol diet, and taking drugs such as cholesterol-lowering drugs are effective, but their use is extremely limited because of causing side effects such as liver dysfunction due to inhibition of cholesterol synthase.

Homeostasis of fat is maintained by the balance between production and breakdown of fat. ADD1 / SREBP1 is a transcription factor that regulates lipoogenesis and regulates the synthesis and absorption of fatty acids, triglycerides, cholesterol, phospholipids (Horton JD et al., J. Clin . Invest., 109 , pp 1125-1131, 2002). SREBPs are synthesized as an inert precursor bound to the endoplasmic reticulum membrane, and after the N terminus is cut off, it is activated and moved to the nucleus and binds to the SRE (sterol regulatory elements) of the regulatory gene promoter. Among these isomers, SREBP1c mainly regulates the synthesis of neutral lipids, while SREBP2 is known to be involved in cholesterol synthesis. Genes regulated by SREBP1c are ATP citrate lyase (ACL), acetyl CoA carboxylase (ACC), fatty acid synthase (FAS) and stearoyl-CoA desarurase (SCD), and others (Osborn TF et al., J. Biol . Chem ., 275 , pp 32379-32382, 2000; Soazig L. L et al., J. Biol . Chem ., 277 , pp 35625-35634, 2002).

Current strategies for developing obesity treatments include reducing meals, suppressing caloric intake, promoting exothermic reactions, regulating energy metabolism, and regulating signaling through the nervous system (Kopleman PG, Nature , 404 , pp 635-643, 2000). Attempts have been made for the treatment of obesity by developing drugs to function more than one function for such a strategy, but it is not easy to develop drugs that have both safety and efficacy at the same time.

Therefore, attempting to find an ingredient that meets the strategy for treating obesity from natural products with proven safety may be a more efficient approach than developing a therapeutic agent from a synthetic formulation.

Melanocortin 4 receptor (MC4R) is an important regulator of energy homeostasis and food absorption, and has recently attracted great attention as a mediator of antipyretic, hyperthermic action. Cells expressing MC4R and its messenger RNA are predominantly present in the central nervous system, and MC4R is a major melanocortin receptor subtype of the cortex, hypothalamus, brain and spinal cord. MC4R agonists lower food absorption and MC4R antagonists increase food absorption. MC4R is involved in other bioactive processes such as insulin, luteinizing hormone and prolactin release and obesity.

MC4R is also important for the regulation of melanocyte stimulating hormone peptides, agouti proteins, and agoti related peptide (AgRP) effects. MTV, an MC4R agonist, reduced food uptake by 4 hours and significantly increased water uptake in the first 4 hours after administration in food-derived mice. Application to MTII COS-1 cells increased the phosphorylation of mitogen-activated protein kinase (MAPK) and the formation of cAMP. In the presence of 0.7 nM agoti, various concentrations of alpha melanocyte stimulating hormone (αMSH) antagonized the activation of αMSH in human MC4R. AgRP at 1 nM or higher concentration-dependently inhibited cAMP accumulation induced by MCMS-mediated αMSH.

MC4R inhibits the hypothalamic leptin signal for food absorption, and HS014, a specific MC4R antagonist, antagonizes the inhibitory effect of leptin on food absorption. Human MC4R is a 333 amino acid long GPCR (GTP-binding protein-coupled receptor) with 7 transmembrane (TM) domains, and in L-cells containing genes encoding MC4R, αMSH is positively dependent on cAMP concentration in cells. dose-dependently. NDP-αMSH and αMSH specifically bind to the MC4R receptor and their relative Ki values are 2.93 nM and 900 nM, respectively.

We have identified potential modulators of MC4 receptors from Korean frog peptide derivatives with the development of FP binding assays for human MC4 receptors using MC4R membrane preparation and fluorescent NDP-αMSH. Based on the experiments, we demonstrated that cAMP is produced intracellularly in response to stimulation of various peptide derivatives and αMSH, and MC4R interacts with the antibiotic peptide Rana Rugosa derivatives indiscriminately to inhibit obesity. It was confirmed that the present invention was completed.

It is an object of the present invention to provide derivatives of peptides designed and synthesized from openingulin 5 that specifically recognize human melanocottin-4-receptors and pharmaceutical compositions for the prevention and treatment of obesity-related diseases comprising the same.

In order to achieve the above object, the present invention is to prevent obesity-related diseases comprising a peptide derivative represented by FLGWLFKVASK (A4W-GGN5N11) and FLGALFKWASK (V8W-GGN5N11) and pharmaceutically acceptable carriers designed and synthesized from And it provides a pharmaceutical composition for treatment.

The peptide derivative is characterized in that it is synthesized by replacing a specific site of the peptide with tryptopan (Tryptopan).

In addition, the peptide derivative is characterized in that it binds to the human melanocortin 4 receptor.

The obesity-related diseases include overeating, hypereating and bulimia, hypertension, diabetes, increased plasma insulin levels, insulin resistance, hemostatic abnormalities, hyperlipidemia, metabolic syndrome, insulin resistance syndrome, obesity related gastroesophageal reflux, arteriosclerosis, hypercholesterolemia, Hyperuricemia, lower back pain, cardiac hypertrophy and left ventricular hypertrophy.

Hereinafter, the present invention will be described in detail.

Peptide derivatives designed and synthesized from openingulin 5 of the present invention can be obtained by the following method.

Peptide derivatives of the present invention can be synthesized and prepared through an engineering modification of an open chain 5 having the shortest length (24 residues) among the peptides isolated from a Korean frog, Om frog, (frog 1 to 6 open chains). have.

Specifically, in order to synthesize an optimal peptide derivative, it can be synthesized using a solid phase synthesis method using a Fmoc amino group protective container with the opening molecules 4 and 5 as the parent molecules, and preferably, When the C-terminal residues of 4 were removed one by one, the antimicrobial activity of the peptide derivatives was verified, and when the C-terminal 14 residues of the opening 4 were removed, leaving only 23 residues of the N-terminal, all the antibiotics disappeared. The substitution of Trp (tryptophan) residue at position 16 of the openulin 4 derivative was performed to synthesize the derivative of openulin 4 (DI6W-GGN4N23) and to verify its antimicrobial activity. Recognizing the importance, it can be used for the synthesis of derivatives of openulin 5.

As a result of verifying the anti-obesity activity by removing the C-terminal residues of the open-air 5 one by one in the same manner as above, it was confirmed that the least residue having the anti-obesity activity was 13 residues, as mentioned above. Since the usefulness of Trp in the antiobesity peptide derivative development is expected from the study of the derivatives of openingulin 4, a total of 11 types of Trp substituents of openingulin 5 were substituted by replacing the Trp residues with 11 residues inactive segments instead of 13 residues active segment of openingulin 5 Among them, A4W-GGN5N11 in which Trp was introduced at position 4 and V8W-GGN5N11 derivatives in which Trp was introduced at position 8 were examined. After confirming that it exhibits similar anti-obesity activity to gurin 5, the other amino acid, preferably the hydrophobic residue Leu (leucine), is hydrophilic to determine whether only Trp is specific at positions 4 and 8 Hi may be replaced with three amino acids synthesized by amino acid substitutions of one Gurin 5 of the seven kinds of Phe (phenyl alanine) with a directional ring (aromatic ring), such as Lys (lysine), and Trp having a zwitterion.

In addition, derivatives of the peptides of the present invention can be obtained not only by chemical methods using a peptide synthesizer, but also include DNA or RNA sequences encoding peptides, and gene clones suitable for expressing the peptides can be used for genetic manipulation. It can also be obtained by genetic recombination techniques to obtain the peptides expressed by making them and transplanting them into suitable cells.

Among the peptide derivatives of the present invention obtained as described above, in order to find an optimal antiobesity peptide having excellent molecular weight and stability, the antiobesity activity and hemolytic activity of the peptide derivatives were examined. It was confirmed that derivatives such as anti-obesity activity and hemolytic activity similar to that of 5.

GGN5N13: F-L-G-A-L-F-K-V-A-S-K-V-L

A4W-GGN5N11: F-L-G-W-L-F-K-V-A-S-K

V8W-GGN5N11: F-L-G-A-L-F-K-W-A-S-K

On the other hand, among the derivatives of openingulin 5, the following derivatives showed similar antiobesity activity as that of openingulin 5, but it was confirmed that hemolytic activity was high.

AL4-GGN5N11: F-L-F-L-L-F-K-V-A-S-K

V8L-GGN5N11: F-L-G-A-L-F-K-L-A-S-K

W4,8-GGN5N11: F-L-G-W-L-F-K-W-A-S-K

Based on whether or not the peptide derivatives of the present invention have structural similarity to antiviral 5, antiobesity activity and hemolytic activity, the derivatives thereof in order to have similar structure, antiobesity activity and hemolytic activity Should have at least 11 residues out of 23 amino acid residues of open 5 and also that the derivatives must be amphiphilic via Trp substitution at the position between the hydrophilic end and the hydrophobic start side in order to have sufficient antiobesity activity. From the above results, it was confirmed that the optimal anti-obesity peptide derivatives for the development of anti-obesity and therapeutic agents were A4W-GGN5N11 and V8W-GGN5N11.

The pharmaceutical composition for preventing and treating obesity-related diseases of the present invention comprises 0.1 to 50% by weight of the compound, based on the total weight of the composition.

Pharmaceutical compositions comprising the compounds of the present invention may further comprise suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions.

Pharmaceutical dosage forms of the compounds of the present invention may be used in the form of their pharmaceutically acceptable salts, and may be used alone or in combination with other pharmaceutically active compounds as well as in a suitable collection.

The pharmaceutical compositions comprising the compounds according to the present invention may be prepared in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral preparations, suppositories, and sterile injectable solutions, respectively, according to conventional methods. Can be formulated and used. Carriers, excipients and diluents that may be included in the composition comprising the compound include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate , Cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. When formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants are usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations may contain at least one excipient such as starch, calcium carbonate, sucrose, or the like. ) Or lactose, gelatin and the like are mixed. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Oral liquid preparations include suspensions, solvents, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

Preferred dosages of the compounds of the present invention depend on the condition and weight of the patient, the extent of the disease, the form of the drug, the route of administration and the duration, but may be appropriately selected by those skilled in the art. However, for the desired effect, the compound of the present invention is preferably administered at 0.0001 to 100 mg / kg, preferably at 0.001 to 10 mg / kg. Administration may be administered once a day or may be divided several times. The dosage does not limit the scope of the invention in any aspect.

Hereinafter, the present invention will be described in detail by the following Examples and Experimental Examples.

However, the following Examples and Experimental Examples are only illustrative of the present invention, and the content of the present invention is not limited by the following Experimental Examples.

Example 1. Synthesis and Purification of Peptides

In order to synthesize an optimal anti-obesity peptide having an amphiphilic structure, a new peptide having a much smaller size was used as a parent molecule, which is a peptide isolated from the epidermis of domestic om frog, using a standardized Fmoc-method. Automatically synthesized using a solid phase method (Wellings, DA, and Atherton, E., Methods Enzymol ., 289 , pp 44-67, 1997) using a peptide synthesizer (Model 90, Advanced Chemtech, Inc. United States of America). . In other words, after adding 70 mM Resin (Resin Resin) to the reaction vessel (equivalent to 3 equivalents of amino acid for the resin, 2 equivalents of HOBT (1-Hydroxybenzotriazole) for the amino acid and 2 equivalents for the amino acid) DIC (1,3-Diisopropylcarbodiimide) was dissolved in an amino acid vessel (amino acid vessel) and dissolved in 10 ml of dimethylformamide (DMF), and the resin of the reaction vessel was swelled using DMF. The Fmoc group of the resin was removed using 25% piperidine / DMF and then the resin was washed off with DMF, Dichloromethane (DCM) and Isopropanol (IPA). The amino acid dissolved in the amino acid container was transferred to the reaction container and reacted with the resin for 2 to 3 hours. In the case of attaching several amino acids, the above procedure was carried out in sequence to bind amino acids in sequence, and then the Fmoc group attached to the N-terminus of the last amino acid was removed using 25% piperidine / DMF. Remove the protecting group attached to the lysine or serine residues, and add 20% of 10% Trifluoro acetic acid (DFA) / DCM to separate the synthesized peptide from the resin.

After the reaction, the solid resin was filtered off and the reaction solution was distilled into a round flask. After all the solvents have evaporated, 10 ml of 20% acetonitrile (0.1% TFA) is added dropwise to dissolve again, and then filtered by centrifugation or a filter. The supernatant is lyophilized and reversed phase HPLC (WE J55B5) using a C-18 column is performed. , HITACHI, mobile phase: acetonitrile, fixed phase: C-18 resin, flow rate: 1 ml / min) was purified. For 45 minutes using acetonitrile / water mixed solvent containing 0.1% trifluor acetic acid at a flow rate of 1 ml / min, the fractions of which only the desired peptides were detected in the eluate were collected and freeze-dried. After obtaining, the molecular weight was measured by mass spectroscopy (Mass Spectroscopy) to obtain the results of A4W (MW: 1295.75) and V8W (MW: 1267.72). The sequence listing of the peptide derivatives of synthesized openulin 5 is shown in FIG. 1.

Reference Example 1. Reagents and Instruments

1-1. material

αMSH and other commercial peptides were purchased from Bachem, and human recombinant MC4 receptor membranes were prepared from PerkinElmer Life and Analytical Sciences from stable HEK-293 cells and BODIPY-TMR NDP-αMSH. NDP-αMSH labeled with amino acid-modified TAMRA (5-carboxymethylrhodamine) was purchased from AnaSpec. Prazosin, yohimbine, and propranolol were received from Professor Ryu Pan-dong, Seoul National University. Korean frog peptide derivatives were synthesized by solid-phase methods using standard Fmoc chemistry. Chemical libraries (libraries) were obtained from HPLC separations from Korean herbal extracts. All other reagents were purchased from Sigma Chemical Co.

1-2. device

Fluorescence spectroscopy was performed with Wallac Victor 3VTM (PerkinElmer Life and Analytical Sciences). Excitation filters, 544 (15) nm and divergence filters, 595 (60) nm were supplied from the factory. In the absence of the receptor membrane, the mP value for 1.0 nM TAMRA-NDP-αMSH in 50 mM HEPES with 0.1% Pluronic was 99 ± 4 mP. Primary and secondary receptor-ligand binding assays. Human MC4R membrane homogenate was diluted with binding buffer to the extent necessary for membrane concentration. (50 mM HEPES buffer, pH 7.4, 2 mM CaCl ₂ , variable concentrations of detergents) Typically binding assays are performed with a final volume of 40 μl: 20 μl of substituted ligand and 10 μl of fluorescent tracer And 10 μl of diluted receptor membrane were added in turn, and the reaction was incubated at room temperature for 0.5-24 hours until FP was measured according to each experiment.

1-3. Data analysis

FP binding data were analyzed using GraphPad Prism® software. Competitive binding data were fitted to the one-site competitive binding equation using nonlinear regression curve fitting. IC50 values were determined through nonlinear regression analysis on the inhibition curve. Data is the average of 4-6 repetitions, and all errors are ± 1 standard deviation units in which the data are scattered except for the substitution curve, where error is ± SE. The affinity (IC 50 value) obtained by inhibiting FP binding was converted to Ki value using the Cheng-Prusoff equation, and the Kd value for MC4R was obtained by radioligand binding analysis. Suggested by the membrane manufacturer. The concentration of [125I] NDP-αMSH used was 0.18 nM. MC4R cDNA was obtained from human brain tissue that exhibited high expression rates during normal cloning. Total RNA was prepared using acid salt (guanidine isothiocyanate), and polyadenylated RNA was prepared using Poly ATract mRNA Isolation System III (Promega). In addition, cDNA was generated by RT-PCR using two primers specific to MC4Rdp. (sense, 5'-CTCAAGCTTCGACTGAACTCCACCCACCGTGGG-3 'and antisense, 5'-CGGTGGATCCCGGGCTTAATATCTGCTAGACAAGTCACA-3'). The 999-bp open reading frame (ORF) of MC4R based on NM005912 was sequenced in both directions. cDNA was cloned into pEGFP-N1 to express MC4R in cultured mammalian cells and analyzed by adenylyl cyclase. Cyclic AMP responsive element (CRE) from luciferase reporter analysis was performed as described below. COS-7 cells were reporter plasmid CRE-Luc (Stratagene), and were temporarily infected with plasmids expressing human MC4R using Lipofectamine reagent (Invitrogen). To level infection efficiency, DNA for pCMV-β-galactosidase (Promega) was infected together. After stimulation with various concentrations of αMSH or pure peptides for 6 hours, whole cell extracts were isolated and luciferase activity was determined according to Promega's protocol. Each value was leveled by galactosidase activity.

Experimental Example 1. MC4 receptor analysis using fluorescence polarization assay

1-1. Experiment preparation

In order to demonstrate the efficiency of fluorescence polarization assay (FP) in HTS assay for MC4 receptor, the MC4 receptor assay was used in the present invention. Human recombinant MC4R membrane preparation, TAMRA-NDP-α-MSH, assay buffer and various substituted ligands were prepared in 384-well microplates, and the performance of MC4 receptors on two nonionic surfactants Parameters for describing the are shown in Table 1 below. MC4 receptor with 5.2 pmol / mg Bmas per well and 1.0 nM of TAMRA-NDP-αMSH or BODIPY-TMR NDP-αMSH were used.

1-2. Experiment result

Commonly used surfactants, 2.5% PEG and BODIPY-TMR tracers, showed a Z value of 0.26 due to high standard deviations, although the modified FP assay window was high at 118 mP. Therefore, to further optimize the HTS assay, a simpler buffer system with 0.1% Pluronic F-127 was performed, indicating that the concentration determines the optimal FP assay window and good Z values. Binding of TAMRA-NDP-αMSH (0.5, 0.75, 1.0, 1.5, 2.0 nM / well) to 5.0 μg human MC4R membrane protein was measured with and without 2 μM NDP-αMSH. The most optimal concentration of TAMRA-NDP-αMSH (1.0 nM / well) was 5.9 times higher than the known Kd value (0.17 nM) in radioligand analysis. The range of fluorophore concentrations (0.9-1.0 nM) with FP performance was very narrow. High fluorophore concentrations above 2 nM TAMRA-NDP-α-MSH resulted in poor performance due to the large number of unbound fluorophores, which serve to reduce the assay window. A time curve for the binding reaction between TAMRA-NDP-αMSH and MC4R is shown in FIG. 2. The equilibrium took almost 60-90 minutes at room temperature to reach the maximum substitution signal, and the combined and substituted signals showed a good analysis window and were stable for at least 6 hours (79-86 mP). Tracer concentrations of 1.0 nM / well showed Z values greater than 0.5 when observed for more than 8 hours. The accuracy of the analysis decreased slowly between 7 and 8 hours, but was stable in the Z range of 0.51-0.63. However, when the plate was stored overnight at room temperature, the assay window fell below 45 mP and the Z value was lower than 0.2. The overnight slowing of the binding signal was due to the increased nonspecific binding of MC4R to the surface of the microplate by hydrophobic and ionic reactions.

 Surfactants  Protein / well (μg) Bound signal (mp) Gamma (σ ^a ) value in combined signal Displaced signal (mp) Gamma (σ ^b ) value in substitution signal  Z 'value  Modified FP Analysis Window 2.5% polyethylene glycol (PEG ^c )  5.0  238  19  120  10  0.26  118 0.1% Pluronic F-127 ^d  5.0  173  5.0  90  5.7  0.61  84 a: Intra-assay precision was determined by averaging six replicates of two signal values associated with binding. b: The bound peptide was completely substituted using 2 μM NDP-αMSH. c: BODIPY-TMR-NDP-αMSH (1 nM / well) was used. d: TAMRA-NDP-αMSH (1 nM / well) was used.

Experimental Example  2. Substitution Study

2-1. human MC4R For TAMRA - NDP -α MSH Substitution

The substitution curve of TAMRA-NDP-αMSH for human MC4R by using αMSH and other MC4R ligands is shown in FIG. 3. The FP analysis system has a high analysis window of> 80 mPs and excellent Z values under various experimental conditions. The high accuracy of the current FP method is clear when viewing errors at each data point, indicating that the method is suitable for HTS. Receptor concentration in membrane preparation was determined according to literature concentration (5.0-6.25 μg protein) and manufacturer recommended concentration (6.4 μg / well) in FP binding assays. Membrane proteins of various concentrations (2.5, 5.0, 6.25 μg) were titrated in the presence of 1.0 nM TAMRA-NDP-αMSH and 2μM NDP-αMSH. A large amount of MC4R membrane (5.0 μg), representing 78% of radioisotope filtration assays, showed good results. 1-10 μM NDP-αMSH, MTII and SHU9119 were almost substituted with the tracer TAMRA-NDP-αMSH, but 18 μM αMSH was required to be replaced with the tracer. Since the Kd value determined by the FP analysis is proportional, the Kd value determined by the radioligand analysis is absolute, and according to the present invention, the KD value determined by the radioligand analysis is reported in the manufacturer's data sheet to calculate the KI value of the NPFF ligand. Kd value of NDP-αMSH (0.17 nM) was used.

2-2. Comparison of affinity of receptors by ligand

For HTS analysis, the consistency of the data was tested by ligand-receptor binding assays on three different days. Table 2 shows the empirically determined Ki values for all ligands used in the FP experiments and shows a list of comparative Ki values obtained by radioisotope filtration analysis and FP analysis using BODIPY-TMR NDP-αMSH. In FP analysis with TAMRA-NDP-αMSH, SHU9119 (Ki = 0.109 nM) showed the highest affinity for the ligand, whereas αMSH (Ki = 77.22 nM) showed the lowest affinity. NDP-αMSH (Ki = 0.661 nM) and MTII (Ki = 0.185 nM) showed high affinity. In Table 1 and FIG. 3, the maximum mP values were relatively varied. It was observed that the maximum mP value and the signal analysis window value decreased with time at 0.1% Pluronic. In the present invention, 0.9 nM tracer and 6.25 μg membrane protein were used in the experiments of FIGS. 3 and 2. Various mPs up to 30% could be observed.

 Inhibitor TAMRA-NDP- MSH Human [ ¹²⁵ I] Ligand Filtration Human BODIPY-TMR NDP-MSH Human ^a Ki (nM) ^b Ki (nM) ^c Ki (nM) MSH 77.22 ± 10.06 900 ± 97 458 NDP-MSH 0.661 ± 0.091 2.93 ± 0.34 0.68 MTⅡ 0.1850.029 6.60 ± 0.82 ND SHU9119 0.109 ± 0.013 0.360 ± 0.059 0.63 FP affinity expressed as a: Ki was obtained from competition binding data using TAMRA-NDP-MSH as a human MC4 receptor membrane preparation and tracer. Results were obtained by means ± S.E. From three separate experiments. b: data (from Biochem. Biophys. Res. Commun. 301: 399-405 (2003) c: data (from J. Recept. Signal Transd. Res. 22: 335-345 (2002); ND = not determined)

Experimental Example  3. Binding Specificity Analysis

3-1. Human MC4 Receptor Binding of Uvein Five Peptide Derivatives

Table 3 shows Korean frog and herbal extracts searched and identified for human recombinant MC4 receptors. The 370 library was tested twice in two separate experiments to examine the correspondence to percent reactivity. Two 384-well plates were each treated with 40 μl per well and analyzed respectively. Percent reactivity was calculated as the percentage of mP value of unknown library against 2 μM NDP-αMSH, a positive control. An interesting well was defined as showing over 705 inhibition. Hit ratio was calculated as the percentage of the library number showing over 70% inhibition upon retest. In the limit of 70% inhibition, two hits (0.54% hit rate), FLGWLFKVASK and FLGWLFKWASK, were identified by primary search from peptide derivatives derived from Korean frogs, Om Frogs.

Experimented Compound % Inhibition rate Interesting wells Hit rate Retest rate 370 > 70% 22 5.9% 0.54%

Nonlinear analysis of substitution data in FIG. 4 showed inhibition constants (Ki) of 2.21 and 2.54 for FLGWLFKVASK and FLGWLFKWASK. Three other peptide derivatives were also tested to study the structure-activity correlation. Table 4 shows that the ranking of ligand binding affinity for the MC4 receptor is consistent with the ability obtained in function and antibiotic assays. FLGALFKVASK showed the highest Ki value at 32.21μΜ, while FLGFLFKVASK showed the lowest Ki value at 1.41μΜ. Interestingly, substitution of Gly at position 4 with Phe or Trp and Val at position 8 with Trp showed an increase in performance in binding assays, indicating hydrophobicity at positions 4 and 8 in 11 peptides. Hydrophobic phenylalanine and amphipathic tryptophan appear to be important for binding to MC4 receptors.

Ligand Ki, μM EC ₅₀ Antibiotics ^b , μg / ml αMSH 0.077 ± 0.01 ^a 1.469 ± 0.605nM N.D FLGALFKVASK 32.21 ± 2.97 N.D > 200 FLGFLFKVASK 1.41 ± 0.07 N.D 12.5 FLGWLFKVASK 2.21 ± 0.37 88.68 ± 5.215μM 6.3 FLGALFKWASK 3.17 ± 0.14 N.D 12.5 FLGWLFKWASK 2.54 ± 0.46 16.40 ± 6.566μM 3.2 Affinity, denoted Ki, was obtained from human MC4 receptor membrane preparation (6.25 μg / well) and competition binding data using TAMRA-NDP-αMSH as tracer. Intra-assay precision was determined by averaging five replicates of two signal values associated with binding. The bound tracer was completely substituted using 10 μM NDP-αMSH, 164 μM peptide derivative. Results were obtained by means ± SE from three separate experiments. EC ₅₀ is the concentration of agonist that increases 50% of intracellular cAMP production in COS-7 cells. Antibiotics completely inhibit cell growth of Bacillus subtilis The lowest peptide concentration, μg / ml, is indicated as the minimal inhibitory concentration. a: The data refer to Table 2. b: HS Won et al., J. Biol. Chem . 279 , pp 14784-14791, 2004 ND has not been determined.

3-2 human MC4  Functional Overview of the Receptor

In order to measure the functional activity of the human MC4 receptor on the unopened peptide derivatives, CRE-luciferase activity was measured with and without the peptide. It is already known that human MC4R has increased adenylyl cyclase activity in COS-1 and HEK-293 cells. As shown in Table 4, the EC50 value of αMSH is determined to be 1.469 nM, which is similar to the previously reported value. Of the five peptides, two peptide derivatives, FLGWLFKVASK and FLGWLFKWASK, showed EC 50 values of 88.68 μM and 16.40 μM, respectively, while the other three peptides did not show significant luciferase activity.

Examples of the pharmaceutical composition of the present invention will be described, but the present invention is not intended to limit the present invention, but is only intended to be described in detail.

Formulation Example 1 Preparation of Powder

20 mg of peptide derivative of Example 1

Lactose 100 mg

Talc 10 mg

The above ingredients are mixed and filled in an airtight cloth to prepare a powder.

Formulation Example 2 Preparation of Tablet

10 mg of peptide derivative of Example 1

Corn starch 100 mg

Lactose 100 mg

2 mg magnesium stearate

After mixing the above components, tablets are prepared by tableting according to a conventional method for preparing tablets.

Formulation Example 3 Preparation of Capsule

10 mg of peptide derivative of Example 1

3 mg of crystalline cellulose

Lactose 14.8 mg

Magnesium Stearate 0.2 mg

According to a conventional capsule preparation method, the above ingredients are mixed and filled into gelatin capsules to prepare capsules.

Formulation Example 4 Preparation of Injection

10 mg of peptide derivative of Example 1

Mannitol 180 mg

Sterile distilled water for injection 2974 mg

_{Na 2 HPO 4 · 12H 2 O} 26 mg

According to the conventional method for preparing an injection, the amount of the above ingredient is prepared per ampoule (2 ml).

Formulation Example 5 Preparation of Liquid

20 mg of peptide derivative of Example 1

10 g of isomerized sugar

5 g of mannitol

Purified water

According to the conventional method of preparing a liquid solution, each component is added and dissolved in purified water, lemon flavor is added to the mixture, and then the above ingredients are mixed, purified water is added to adjust the total amount to 100 ml, and then filled in a brown bottle. The solution is prepared by sterilization.

As described above, the peptide derivatives designed and synthesized from the openulin 5 of the present invention are useful for detecting and analyzing human melanocortin 4 receptors by binding to the melanocortin receptors, which play a pivotal role in appetite control and obesity induction. It can be used, and because it has no cytotoxicity and little hemolytic activity against human erythrocyte cells, it can be usefully used as an agent for preventing and treating obesity-related diseases that are safe for human body.

Claims (5)

Human melanocorpeic acid represented by FLGWLFKVASK (A4W-GGN5N11) incorporating Trp at position 4, synthesized by substituting tryptopan for the amino acid terminal portion of the peptide designed and synthesized from aperturerin 5 designated FLGALFKVASKVLPSVKCATK-KC. A pharmaceutical composition for preventing and treating hypertension, diabetes mellitus or atherosclerosis, comprising a peptide derivative that binds to the Tin 4 receptor and a pharmaceutically acceptable carrier. delete delete delete         Human melanocorpeic acid represented by FLGALFKWASK (V8W-GGN5N11) incorporating Trp at position 8, synthesized by substituting tryptopan for the amino acid terminal portion of the peptide designed and synthesized from openulin 5 designated FLGALFKVASKVLPSVKCATK-KC. A pharmaceutical composition for preventing and treating hypertension, diabetes mellitus or atherosclerosis, comprising a peptide derivative that binds to the Tin 4 receptor and a pharmaceutically acceptable carrier.

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