A DR8 cyclic peptide analogue and its application

Abstract

The present invention belongs to the technical field of biochemistry, and specifically relates to a DR8 cyclic peptide analog and its application. The structure of the DR8 cyclic peptide analog is R1-dAsp-His-Asn-Cys-Pro-Gln-dAla-Cys-CONH2, wherein R1 is a C1-C30 chain alkyl group, and the 4th amino acid and the 8th amino acid are cyclized through a chain alkyl group. The present invention conducts research on the biological activity of the DR8 cyclic peptide analog, including constructing in vitro cell models and in vivo animal models of renal fibrosis to evaluate indexes such as the in vitro anti-fibrotic activity, inhibitory effect and in vivo therapeutic effect of the DR8 cyclic peptide analog, as well as studying the cytotoxicity, serum stability and pharmacokinetics of the DR8 cyclic peptide analog. The results show that compared with the polypeptide DR3penA, the DR8 cyclic peptide analog obtained in the present invention has better activity indexes and stability in all aspects and can be used to prepare drugs for preventing kidney fibrosis diseases.

Description

Technical Field

[0001] The invention belongs to the technical field of biochemistry, and specifically relates to a DR8 cyclic peptide analog and application thereof. Background Art

[0002] Renal fibrosis is an important driver and pathological manifestation of the development of chronic kidney disease. The main manifestation of renal fibrosis is the deposition of extracellular matrix in the renal interstitium, which leads to renal scarring and decreased or even loss of renal function, and eventually develops into end-stage renal disease. However, its treatment methods are limited and the mortality rate is high. Although the underlying mechanisms of renal fibrosis have been comprehensively studied, there is no approved treatment for clinical use. Therefore, the development of new and effective drugs to treat renal fibrosis is of great significance in clinical practice.

[0003] DR8 (DHNNPQIR) is an antioxidant peptide extracted from rapeseed protein, which can remove reactive oxygen species in cells and reduce the damage of free radicals to the body. Existing technology uses DR8 to treat pulmonary fibrosis and renal fibrosis. The results show that DR8 can effectively improve pulmonary and renal fibrosis. The activity and stability of DR3penA, an analogue obtained by designing and modifying DR8, are significantly improved. However, DR3penA still has problems such as short half-life in vivo, low bioavailability, and the efficacy needs to be improved. Summary of the invention

[0004] In view of the above problems, the object of the present invention is to provide a DR8 cyclic peptide analog.

[0005] Another object of the present invention is to provide a method for preparing the DR8 cyclic peptide analog.

[0006] Another object of the present invention is to provide the application of the DR8 cyclic peptide analog.

[0007] According to a specific embodiment of the present invention, a DR8 cyclic peptide analog has the following general structure:

[0008] R1-dAsp-His-Asn-Cys-Pro-Gln-dAla-Cys-CONH2, where

[0009] R1 is a C1-C30 alkyl group,

[0010] The 4th amino acid and the 8th amino acid are cyclized via a chain alkyl group.

[0011] The DR8 cyclic peptide analogue of the present invention overcomes the problems of poor stability and low bioavailability of traditional polypeptides by modifying the N-terminal chain alkyl acid of DR8 and the cyclization strategy of the 4th amino acid and the 8th amino acid, while showing significant anti-fibrosis activity, excellent serum stability and low cost advantages.

[0012] Preferably, R1 is a C6-C16 linear alkyl group, or R1 is a C10-C12 linear alkyl group, or R1 is a C12-C18 linear alkyl group.

[0013] Preferably, the 4th amino acid and the 8th amino acid are cyclized through a substituted or unsubstituted C4-C12 linear alkyl group, or the 4th amino acid and the 8th amino acid are cyclized through a substituted or unsubstituted C4-C10 linear alkyl group, or the 4th amino acid and the 8th amino acid are cyclized through a substituted or unsubstituted C4-C8 linear alkyl group.

[0014] Preferably, the cysteine at the 4th position and the cysteine at the 8th position are cyclized through a butane chain, a hexane chain, or an octane chain.

[0015] The "linear alkyl group" in the sense of the present invention preferably refers to the following groups: methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, 2-methylbutyl, n-pentyl, sec-pentyl, neopentyl, cyclopentyl, n-hexyl, neohexyl, n-heptyl, n-neoheptyl, n-nonyl, n-decyl, etc.

[0016] In the present invention, the term "substituted or unsubstituted" means being substituted or unsubstituted by one or more substituents selected from hydrogen, deuterium, halogen atoms, hydroxyl groups, nitrile groups, nitro groups, amino groups, amidino groups, hydrazino groups, hydrazone groups, carboxyl groups or their carboxylates, sulfonic acid groups or their sulfonates, phosphoric acid groups or their phosphates, aliphatic hydrocarbon groups or alkyl groups, or being substituted or unsubstituted by a substituent formed by linking two or more of the above-exemplified substituents.

[0017] In many cases, the general substituents are selected from the group consisting of deuterium, halogen, alkyl, cycloalkyl, heteroalkyl, heterocycloalkyl, aralkyl, alkoxy, aryloxy, amino, silyl, alkenyl, cycloalkenyl, heteroalkenyl, alkynyl, aryl, heteroaryl, acyl, carboxylic acid, ether, ester, nitrile, isonitrile, thio group, sulfinyl group, sulfonyl group, and phosphino group.

[0018] The "halogen" and "halogen atom" in the sense of the present invention are used interchangeably and refer to fluorine, chlorine, bromine, or iodine.

[0019] The aliphatic hydrocarbon group or alkyl group substituted by the above groups preferably refers to the following groups: methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, 2-methylbutyl, n-pentyl, sec-pentyl, neopentyl, cyclopentyl, n-hexyl, neohexyl, cyclohexyl, n-heptyl, cycloheptyl, n-octyl, cyclooctyl, 2-ethylhexyl, cyclohexenyl, heptenyl, cycloheptenyl, octenyl, or cyclooctenyl.

[0020] The DR8 cyclic peptide analog according to the specific embodiment of the present invention has the following structure:

[0021]

[0022] In the present invention, cysteine is linked by a hexane chain, thereby changing the steric hindrance of the polypeptide, forming a hydrophobic microenvironment around itself as a hydrophobic side chain, promoting the interaction with non-polar free radicals and unsaturated fatty acids, increasing the interaction between the peptide and lipid targets or the lipid bilayer of the membrane, and facilitating the polypeptide to enter the cell interior to better exert its function. In addition, in the present invention, a hexane chain is introduced to form a ring between two cysteines at the 4th and 8th positions, which can effectively prevent recognition by proteases in the body, has higher drug stability, can extend the half-life in the body, and helps to improve the persistence and efficacy of the prepared anti-fibrotic drug.

[0023] The DR8 cyclic peptide analog screened by chemical modification in the present invention has better biological activity against kidney fibrosis diseases; compared with the polypeptide DR3penA and the control drug, the dosage is significantly reduced and the stability is improved; compared with small molecule compounds, the analog has low toxicity and a large safety window; compared with macromolecular proteins, the preparation cost is more economical and the generalizability is higher.

[0024] The present invention also provides the use of the above-mentioned DR8 cyclic peptide analog in the preparation of a drug for treating fibrosis diseases.

[0025] Preferably, the fibrosis diseases include renal fibrosis diseases, pulmonary fibrosis diseases, and hepatic fibrosis diseases.

[0026] The DR8 cyclic peptide analog of the present invention has an improvement effect on kidney fibrosis diseases and can be used to directly or indirectly treat diseases characterized by kidney fibrosis. Among them, kidney fibrosis diseases include obstructive nephropathy, diabetic nephropathy, renal sclerosis, nephritis, kidney tumors, and kidney fibrosis diseases caused by various factors such as bad living habits, adverse drug reactions, and pathogenic microorganism infections.

[0027] In addition to the improvement effect on kidney fibrosis diseases, the DR8 cyclic peptide analog of the present invention also has a similar improvement effect on pulmonary fibrosis diseases and hepatic fibrosis diseases.

[0028] The present invention also provides a drug for treating fibrosis diseases, which uses the DR8 cyclic peptide analog as the main component, and the drug also includes a pharmaceutically acceptable carrier or excipient.

[0029] Another object of the present invention is to provide a composition which contains the above-mentioned DR8 cyclic peptide analog and further includes a pharmaceutically acceptable carrier or excipient. The carrier can be a carrier that can reduce drug degradation and loss and reduce side effects, such as micelles, microemulsions, gels, etc.; the excipient can be a material added to make the drug into a suitable dosage form, such as a buffer, an excipient for freeze-drying, etc. The liquid formulation is generally a buffer solution, an isotonic solution and an aqueous solution. Specifically, a pharmaceutically acceptable carrier and / or excipient is added with the DR8 cyclic peptide analog as the active ingredient to support the pharmaceutical composition.

[0030] The pharmaceutical combination of the present invention is suitable for various administration routes, such as oral administration, transdermal administration, intravenous administration, intramuscular administration, topical administration, nasal administration, etc. According to the administration route adopted, the polypeptide analog pharmaceutical combination of the present invention can be supported in various suitable dosage forms, which contain at least one effective amount of the polypeptide analog of the present invention and at least one pharmaceutically acceptable pharmaceutical carrier.

[0031] Examples of suitable dosage forms are tablets, capsules, sugar-coated tablets, granules, oral solutions and syrups, ointments and patches for the skin surface, aerosols, nasal sprays, and sterile solutions that can be used for injection. The pharmaceutical composition containing the polypeptide analog of the present invention can be made into a solution or a lyophilized powder for parenteral administration, and the powder can be reconstituted with a suitable solvent or other pharmaceutically acceptable carriers before use. The liquid formulation is generally PBS buffer solution, physiological saline isotonic solution and aqueous solution.

[0032] The dosage of the polypeptide analog of the present invention in the pharmaceutical composition can vary within a wide range, and those skilled in the art can easily determine it according to some objective factors such as the type of disease, the severity of the condition, the patient's weight, the dosage form, the administration route, etc.

[0033] The present invention provides a preparation method of the above-mentioned DR8 cyclic peptide analog, which includes the following steps:

[0034] Step 1: Using the Fmoc solid-phase synthesis method, synthesize the linear polypeptide resin corresponding to the DR8 cyclic peptide analog from the carboxyl terminus to the amino terminus;

[0035] Step 2: Cut the linear polypeptide resin synthesized in Step 1, remove the full protection of the peptide chain, and collect the cutting solution containing the DR8 linear analog;

[0036] Step 3: Purify the cutting solution obtained in Step 2 to obtain the DR8 linear analog;

[0037] Step 4: Dissolve the DR8 linear analog obtained in Step 3 in 250 μL of 1 M Tris-HCl (pH = 9.0), and dissolve the same molar amount of thianthrenium salt in 750 μL of DMSO; mix well and stir at room temperature for 1 h for reaction;

[0038] Step 5: Purify the reaction solution obtained in Step 4 to obtain the DR8 cyclic peptide analog.

[0039] Preferably, in Step 1, the resin used in the Fmoc solid-phase synthesis method is MBHA resin.

[0040] Preferably, in Step 2, the polypeptide resin synthesized in Step 1 is cleaved using a cleavage solution, and the cleavage solution is composed of TFA: Tis: H2O: βME in a volume ratio of 90:6.5:2.5:1.

[0041] Advantages of the present invention:

[0042] The present invention provides a DR8 cyclic peptide analog, and studies on the biological activity of the DR8 cyclic peptide analog are carried out, including constructing in vitro cell models and in vivo animal models of renal fibrosis to evaluate indicators such as the in vitro anti-fibrotic activity, inhibitory effect and in vivo therapeutic effect of the DR8 cyclic peptide analog, and also including studies on the cytotoxicity, serum stability and pharmacokinetics of the DR8 cyclic peptide analog. The results show that compared with the polypeptide DR3penA, the DR8 cyclic peptide analog obtained in the present invention has better activity indicators and stability in all aspects. Thus, it is proved that the DR8 cyclic peptide analog of the present invention can be used to prepare drugs for preventing kidney fibrosis diseases. Description of the Drawings

[0043] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0044] Figure 1 Survival rate of mouse embryonic fibroblasts by the DR8 cyclic peptide analog.

[0045] Figure 2 Results of the effect of the DR8 cyclic peptide analog on the expression of Fibronectin and α-SMA in the mouse embryonic fibroblast model detected by Western blot and its corresponding statistical chart.

[0046] Figure 3 Results of the effect of the cyclic peptide 79 on the expression of Fibronectin and α-SMA in the mouse embryonic fibroblast model detected by Western blot and its corresponding statistical chart.

[0047] Figure 4Results of the effects of cyclic peptide 79 on the expressions of Collagen I, MMP2, and Vimentin in a mouse embryonic fibroblast model detected by Western blot and their corresponding statistical charts.

[0048] Figure 5 mRNA expression levels of Fibronectin, Collagen I, α-SMA, TGF-β1, Vimentin, and MMP2 in a mouse embryonic fibroblast model detected by qPCR for cyclic peptide 79.

[0049] Figure 6 Survival rate of cyclic peptide 79 on human renal tubular epithelial cells.

[0050] Figure 7 Results of the effects of cyclic peptide 79 on the expressions of Fibronectin and α-SMA in a human renal tubular epithelial cell model detected by Western blot and their corresponding statistical charts.

[0051] Figure 8 Results of the effects of cyclic peptide 79 on the expressions of Collagen I, MMP2, E-cadherin, and Vimentin in a human renal tubular epithelial cell model detected by Western blot and their corresponding statistical charts.

[0052] Figure 9 mRNA expression levels of Fibronectin, Collagen I, α-SMA, E-cadherin, Vimentin, and MMP2 in a human renal tubular epithelial cell model detected by qPCR for cyclic peptide 79.

[0053] Figure 10 Results of the effects of cyclic peptide 79 and polypeptide DR3penA on the expressions of Fibronectin and α-SMA in a human renal tubular epithelial cell model detected by Western blot and their corresponding statistical charts.

[0054] Figure 11 Results of the effects of cyclic peptide 79 and polypeptide DR3penA on the expressions of Fibronectin, Collagen I, E-cadherin, Vimentin, MMP2, and α-SMA in a mouse kidney detected by Western blot and their corresponding statistical charts.

[0055] Figure 12 Results of the effects of cyclic peptide 79 and polypeptide DR3penA on the expressions of Fibronectin, Collagen I, TGF-β1, α-SMA, E-cadherin, Vimentin, and MMP2 in a mouse kidney detected by qPCR.

[0056] Figure 13 Results of the effects of cyclic peptide 79 and Captopril on the expressions of Fibronectin, Collagen I, E-cadherin, MMP2, Vimentin and α-SMA in the kidneys of mice detected by Western blot and their corresponding statistical charts.

[0057] Figure 14 Results of the effects of cyclic peptide 79 and Captopril on the expressions of Fibronectin, Collagen I, TGF-β1, α-SMA, E-cadherin, Vimentin and MMP2 in the kidneys of mice detected by qPCR.

[0058] Figure 15 Results of H&E and immunohistochemical staining of pathological sections of the kidneys of mice in each group after administration of cyclic peptide 79 for 7 days.

[0059] Figure 16 Results of the in vitro serum stability of cyclic peptide 79 and polypeptide DR3penA.

[0060] Figure 17 Results of the in vivo half-lives of cyclic peptide 79 and polypeptide DR3penA. Detailed implementation manners

[0061] To make the objectives, technical solutions and advantages of the present invention clearer, the technical solutions of the present invention will be described in detail below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other implementation manners obtained by those of ordinary skill in the art based on the embodiments of the present invention without making creative efforts fall within the scope protected by the present invention.

[0062] The specific meanings of the abbreviations used in the present invention are as follows:

[0063] dAla (abbreviated as a) is D-alanine, Ile (abbreviated as I) is isoleucine, Cys (abbreviated as C) is cysteine, Pro (abbreviated as P) is proline, Gln (abbreviated as Q) is glutamine, Arg (abbreviated as R) is arginine, Asn (abbreviated as N) is asparagine, dAsn (abbreviated as n) is D-asparagine, Asp (abbreviated as D) is aspartic acid, dAsp (abbreviated as d) is D-aspartic acid, His (abbreviated as H) is histidine, α-(4-pentenyl)-Ala (abbreviated as X) is α-(4-pentenyl)-alanine.

[0064] Materials and methods:

[0065] The experimental conditions and methods described in the following examples are conventional conditions and methods unless otherwise specified, and the reagents and instruments described are all commercially available.

[0066] Among them, Western blot results were analyzed by grayscale analysis of protein expression using EvolutionCapt software, and statistics and analysis were performed using GraphPadPrism 8.0 software. The data were expressed as mean ± SD, and significant differences were analyzed using one-way ANOVA and Tukey test. Compared with the control group, **P < 0.01, *P < 0.05; compared with the model group, ##P < 0.01, #P < 0.05.

[0067] Example 1 Design and Synthesis of Cyclic Peptide

[0068] Step 1: The linear peptide was synthesized by Fmoc solid phase synthesis from the carboxyl end to the amino end. The amino acid sequence is as follows:

[0069] C12-dAsp-His-Asn-Cys-Pro-Gln-dAla-Cys-CONH2.

[0070] The synthesis specifically comprises the following steps:

[0071] (1) Activation of MBHA resin: Weigh the resin, add appropriate amount of DCM and swell on a shaker for 30 min. After draining, add DMF to wash three times, each time for 3 min.

[0072] (2) Indole test: Add ninhydrin: pyridine: phenol = 1:2:1 indole test reagent into a test tube, dip a small amount of resin into it, and boil it in boiling water for 3 minutes. If the indole test result is yellow, it indicates that the resin is normal;

[0073] (3) Resin deprotection: Add DMF solution containing 3% redistilled piperidine to remove the protecting group, remove the residual reagent, add DMF to wash, each time for 3 minutes, repeat 4 times, and remove the residual reagent;

[0074] (4) Indole test: Add ninhydrin: pyridine: phenol = 1:2:1 indole test reagent to a test tube, take a small amount of resin into the test tube, and boil it in boiling water for 3 minutes. If the indole test result is blue-purple, it proves that the protecting group has been removed;

[0075] (5) Amino acid condensation reaction: weigh 3 times the amount of the amino acid to be condensed and HOBT in a beaker, dissolve in a small amount of DMF, add 6 times excess DIEA to fully dissolve, and finally add 3 times the amount of HBTU and immediately pour into the resin, stir for 1 hour, drain the solvent, wash with DMF for 3 minutes, and repeat 3 times;

[0076] (6) Indene test: If the result of the indene test is yellow, it indicates successful condensation.

[0077] (7) Repeat steps (3), (4), (5), and (6) and condense them in sequence according to the order of the amino acid sequence in the compound (Cys, dAla, Gln, Pro, Cys, Asn, His, dAsp, C12-chain alkyl acid) until all the amino acids of the compound to be synthesized are condensed to obtain the polypeptide resin.

[0078] Step 2: Cleave the polypeptide chain: After the peptide chain is synthesized, deprotect it, wash the resin with DCM for 3 min and repeat 2 times; wash with methanol for 3 min; wash with DCM for 3 min; finally wash with methanol for 3 min and repeat 2 times. Drain the resin until it becomes powdery.

[0079] Prepare the cleavage solution of TFA: Tis: H2O: βME = 90:6.5:2.5:1 (V / V / V / V), add it to the resin for cleavage for 3 h, and collect the cleavage solution; rotary evaporate the cleavage solution, then precipitate it with pre-cooled ice ether, extract it with deionized water, collect the aqueous phase with a separatory funnel and dispense it into a 50 mL beaker, freeze it overnight at -80 °C in a refrigerator and then perform freeze-drying treatment to obtain the crude peptide.

[0080] Step 3: Preparation and purification of linear polypeptide:

[0081] (1) Weigh about 40 mg of the crude peptide and dissolve it in deionized water to make a polypeptide solution. After it is completely dissolved, remove the insoluble substances in the polypeptide solution with a 0.45 μm filter, and add 0.1% TFA to the elution solvent (acetonitrile and deionized water).

[0082] (2) Use a C18 reverse preparative column for high-performance liquid chromatography to rinse with 100% acetonitrile until the baseline is stable, equilibrate with an initial concentration of 30% acetonitrile, and inject the sample after setting the flow rate gradient.

[0083] (3) After injection, detect the absorption peak at 220 nm, collect the main peak, finally label the collected components, seal them with plastic wrap, pierce the vent holes, freeze them overnight at -80 °C in a refrigerator and then perform freeze-drying treatment to obtain the linear polypeptide compound.

[0084] (4) After freeze-drying, dissolve a small amount of the compound, use a C18 reverse analytical column, elute it with 5%-95% acetonitrile / deionized water for 30 min, integrate the peak area of the chromatogram at 220 nm to calculate the purity. The purity > 95%, and use mass spectrometry to characterize and identify the separated product, confirm that the measured m / z value of the protonated molecular ion peak is 1068.4831, and the theoretical m / z is 1068.4892, and confirm that the synthesized and purified product is the target product.

[0085] Step 4: Preparation and purification of cyclic peptide:

[0086] (1) The linear polypeptide obtained in Step 3 was dissolved in 250 μL of 1 M Tris-HCl (pH = 9.0), and the same molar amount of thianthrenium salt was dissolved in 750 μL of DMSO; the two were mixed well and stirred at room temperature for 1 h for reaction;

[0087] The structure of the thianthrenium salt.

[0088] (2) The reaction solution obtained was purified by high-performance liquid chromatography to obtain the DR8 cyclic peptide analog, with the structure as follows:

[0089]

[0090] (3) After freeze-drying, a small amount of the compound was dissolved. The C18 reverse analysis column was eluted with 5%-95% acetonitrile / deionized water for 30 min. The purity was statistically analyzed by integrating the peak area of the chromatogram at 220 nm. The purity of the compound was >95%. The separated product was characterized and identified by mass spectrometry. It was confirmed that the measured m / z value of the protonated molecular ion peak was 1150.5771, and the theoretical m / z was 1150.5748. It was confirmed that the synthesized and purified product was the target product.

[0091] Table 1 Structures, theoretical m / z, measured m / z, and purities of the cyclic peptides synthesized in Example 1

[0092]

[0093] Referring to a similar synthesis method as described in the above example, the compounds in Table 2 were prepared:

[0094] Table 2 Structures, theoretical m / z, measured m / z, and purities of the compounds synthesized by referring to Example 1

[0095]

[0096] The DR8 cyclic peptide analog in Example 2 was used as a test substance for cytotoxicity evaluation against NIH-3T3 cells

[0097] The NIH-3T3 cell line was selected to study and observe the cytotoxicity of cyclic peptide DR8 (labeled as cyclic peptide 79) and each polypeptide analog shown in Table 2 against NIH-3T3 cells.

[0098] The NIH-3T3 cells were seeded into 96-well plates and cultured in DMEM medium containing 10% FBS + 1% double antibody at 37 °C and 5% CO2 for 24 h. Then, 0, 10, 20, 40, 80, and 160 μM of the test substance were added. After incubation for 48 h, 10 μL of CCK-8 solution was added and incubated for 2 h. The absorbance value at 450 nm was measured by an enzyme-linked immunosorbent assay reader.

[0099] Figure 1The viability of NIH-3T3 cells was shown. The results indicated that within the concentration range of 0 - 160 μM, after the DR8 cyclic peptide analogs 30, 36, 31, 37, 66, and 67 acted on the cells, the cell viability remained at a basically the same level as that of the control group, showing no significant difference, indicating no significant toxicity to the cells. Within the concentration range of 10 - 160 μM, after the DR8 cyclic peptide analogs 68, 69, 70, 71, and 83 acted on the cells, the cell viability was significantly decreased compared with the control group, indicating greater toxicity to the cells. Within the concentration range of 0 - 40 μM, after the cyclic peptide DR8 acted on the cells, the cell viability remained at a basically the same level, showing no significant difference, indicating that the cyclic peptide DR8 had no significant toxicity to the cells at this concentration. At the concentrations of 80 and 160 μM, the cyclic peptide DR8 significantly decreased the cell viability, indicating that the cyclic peptide DR8 above 80 μM had significant toxicity to the cells.

[0100] Example 3 Detection of the Activity of DR8 Cyclic Peptide Analogs against In Vitro Fibrosis of NIH-3T3 Cells

[0101] The NIH-3T3 cell line was selected for the experiment to study and observe the effects of the cyclic peptide DR8 (labeled as cyclic peptide 79) and each polypeptide analog shown in Table 2 on the expression of fibronectin and α-smooth muscle actin (α-SMA) in NIH-3T3 cells induced by transforming growth factor TGF-β1.

[0102] NIH-3T3 cells were seeded in 6-well plates with DMEM (Gibco) medium containing 10% FBS and 1% double antibiotics, cultured at 37 °C under 5% CO2 for 24 h, then changed to serum-free medium and cultured for 12 h. After that, 5 ng / mL TGF-β1 and 10 μM cyclic peptide DR8 or each polypeptide analog shown in Table 2 were added to act on the cells for 48 h, and then the total cell proteins were extracted. The protein expression levels of fibronectin and α-SMA were detected by Western blot.

[0103] Control group (labeled as Ctrl): TGF-β1 and DR8 cyclic peptide analogs were not added to the medium;

[0104] Induction group (labeled as TGF-β1): 5 ng / mL TGF-β1 was added to the medium;

[0105] DR8 cyclic peptide analog group (labeled as cyclic peptide 79 or the name of each polypeptide analog shown in Table 2): 5 ng / mL TGF-β1 and 10 μM DR8 cyclic peptide analog (the cyclic peptide 79 prepared in Example 1 and each polypeptide analog shown in Table 2) were added to the medium.

[0106] Figure 2After the NIH-3T3 cells were treated with the TGF-β1 and DR8 cyclic peptide analog group, the protein expression results of Fibronectin, α-SMA and GAPDH were detected by Western blot, and the relative expression levels of Fibronectin and α-SMA proteins were analyzed.

[0107] It can be seen from Figure 2 that both the cyclic peptide DR8 and each polypeptide analog shown in Table 2 can significantly inhibit the expression of Fibronectin and α-SMA proteins. Among them, the activity of analog 67 cyclized by a hexane chain is better than that of analogs 66 and 37 cyclized by a butane chain and an octane chain. Combining Figure 1 it was found that with the extension of the fatty acid chain in analogs 68, 69, 70 and 71, while the activity increased, the cytotoxicity also increased significantly. The cyclic peptide DR8 synthesized by combining the cyclization strategy and the fatty acid modification strategy has good activity and low cytotoxicity. Subsequently, taking the cyclic peptide DR8 as an example, the anti-fibrotic effect was further investigated.

[0108] Example 4 examines the activity of DR8 cyclic peptide analogs against in vitro fibrosis of NIH-3T3 cells

[0109] The NIH-3T3 cell line was selected for the experiment to study and observe the effects of cyclic peptide DR8 (labeled as cyclic peptide 79) on the expression of Fibronectin, α-SMA, type I collagen (Collagen I), matrix metalloproteinase (MMP2) and vimentin in TGF-β1-induced NIH-3T3 cells.

[0110] NIH-3T3 cells were inoculated into 6-well plates with DMEM (Gibco) medium containing 10% FBS and 1% double antibody, cultured at 37 °C and 5% CO2 for 24 h, then changed to serum-free medium and cultured for 12 h. After that, 5 ng / mL TGF-β1 and 1, 2, 5 and 10 μM cyclic peptide DR8 were added to act on the cells for 48 h, and then the total cell protein and total RNA were extracted. The protein expression levels of Fibronectin, α-SMA, Collagen I, MMP2 and Vimentin were detected by Western blot, and the mRNA expression levels of Fibronectin, Collagen I, α-SMA, TGF-β1, Vimentin and MMP2 were detected by qPCR.

[0111] Control group (labeled as Ctrl): TGF-β1 and cyclic peptide DR8 were not added to the medium;

[0112] Induction group (labeled as TGF-β1): 5 ng / mL TGF-β1 was added to the medium;

[0113] Cyclic peptide DR8 group (labeled as cyclic peptide 79): Add 5 ng / mL TGF-β1 and 1, 2, 5, and 10 μM of cyclic peptide DR8 (cyclic peptide 79 prepared in Example 1) to the culture medium.

[0114] Figure 3 After TGF-β1 and the cyclic peptide DR8 group acted on NIH-3T3 cells, the protein expression results of Fibronectin, α-SMA, and GAPDH were detected by Western blot, and the relative expression levels of Fibronectin and α-SMA proteins were analyzed. From Figure 3 It can be seen that TGF-β1 stimulated NIH-3T3 cells to highly express Fibronectin and α-SMA proteins, and cyclic peptide DR8 could inhibit the expression of Fibronectin and α-SMA proteins in a dose-dependent manner, and the effect was the strongest at 2 μM.

[0115] Figure 4 After TGF-β1 and the cyclic peptide DR8 group acted on NIH-3T3 cells, the protein expression results of Vimentin, MMP2, Collagen I, and GAPDH were detected by Western blot, and the relative expression levels of Vimentin, MMP2, and Collagen I proteins were analyzed. From Figure 4 It can be seen that TGF-β1 stimulated NIH-3T3 cells to highly express Vimentin, MMP2, and Collagen I proteins, and cyclic peptide DR8 could effectively inhibit the expression of Vimentin, MMP2, and Collagen I proteins.

[0116] Figure 5 After TGF-β1 and the cyclic peptide DR8 group acted on NIH-3T3 cells, the mRNA expression levels of Fibronectin, Collagen I, α-SMA, TGF-β1, Vimentin, and MMP2 were detected by qPCR. From Figure 5 It can be seen that TGF-β1 stimulated the significant increase in the mRNA levels of Fibronectin, Collagen I, α-SMA, TGF-β1, Vimentin, and MMP2 in NIH-3T3 cells, and cyclic peptide DR8 could effectively inhibit the increase in the mRNA levels of these genes.

[0117] From Figure 3 , Figure 4 and Figure 5 It can be seen that the cyclic peptide DR8 obtained in Example 1 could inhibit the expression of fibrosis proteins and genes induced by TGF-β1 in NIH-3T3 cells in a dose-dependent manner.

[0118] Example 5 investigated the cytotoxicity of DR8 cyclic peptide analogs to HK-2 cells

[0119] The HK-2 cell line was selected to study and observe the detection of the cytotoxicity of cyclic peptide DR8 on HK-2 cells. HK-2 cells were inoculated into 96-well plates and cultured in DMEM / F12 medium containing 10% FBS + 1% double antibody at 37 °C and 5% CO2 for 24 h. After adding cyclic peptide DR8 at 0, 10, 20, 40, 80, and 160 μM and incubating for 24 h, 10 μL of CCK-8 solution was added and incubated for 2 h, and the absorbance at 450 nm was measured using an enzyme-linked immunosorbent assay (ELISA) reader.

[0120] Figure 6 Regarding the survival rate of HK-2 cells, the results showed that within the concentration range of 0 - 160 μM, after the action of cyclic peptide DR8 on the cells, the cell survival rate remained at a basically the same level with no significant difference, indicating that cyclic peptide DR8 has no significant toxicity to the cells.

[0121] Example 6 investigated the activity of the DR8 cyclic peptide analog on the in vitro fibrosis of HK-2 cells

[0122] The HK-2 cell line was selected to study and observe the effects of cyclic peptide DR8 on the expression of Fibronectin, Collagen I, E-cadherin, MMP2, Vimentin, and α-SMA in HK-2 cells induced by TGF-β1. HK-2 cells were inoculated into 6-well plates with DMEM / F12 (Gibco) medium containing 10% FBS and 1% double antibody and cultured at 37 °C and 5% CO2 for 24 h. After changing to serum-free medium and culturing for 12 h, 5 ng / mL TGF-β1 and 1, 2, 5, and 10 μM cyclic peptide DR8 were added to act on the cells for 24 h, and then total cell protein and total RNA were extracted. The protein expression levels of Fibronectin, α-SMA, Collagen I, MMP2, E-cadherin, and Vimentin were detected by Western blot, and the mRNA expression levels of Fibronectin, Collagen I, α-SMA, E-cadherin, Vimentin, and MMP2 were detected by qPCR.

[0123] Control group (labeled as Ctrl): TGF-β1 and cyclic peptide DR8 were not added to the medium;

[0124] Induction group (labeled as TGF-β1): 5 ng / mL TGF-β1 was added to the medium;

[0125] Cyclic peptide DR8 group (labeled as cyclic peptide 79): 5 ng / mL TGF-β1 and 1, 2, 5, and 10 μM cyclic peptide DR8 (cyclic peptide 79) were added to the medium.

[0126] Figure 7 After TGF-β1 and the cyclic peptide DR8 acted on HK-2 cells, the protein expression results of Fibronectin, α-SMA, and GAPDH were detected by Western blot, and the relative protein expression levels obtained were analyzed. As Figure 7 shown, TGF-β1 stimulated HK-2 cells to highly express Fibronectin and α-SMA proteins, and the cyclic peptide DR8 could inhibit the expression of Fibronectin and α-SMA proteins in a dose-dependent manner, with the strongest effect at 5 μM.

[0127] Figure 8 After TGF-β1 and the cyclic peptide DR8 acted on HK-2 cells, the protein expression results of Collagen I, MMP2, E-cadherin, Vimentin, and GAPDH were detected by Western blot, and the relative protein expression levels obtained were analyzed. As Figure 8 shown, TGF-β1 stimulated HK-2 cells to highly express Vimentin, MMP2, and Collagen I proteins, and the cyclic peptide DR8 could effectively inhibit the expression of Vimentin, MMP2, and Collagen I proteins and upregulate the expression of E-cadherin.

[0128] Figure 9 After TGF-β1 and the cyclic peptide DR8 acted on HK-2 cells, the mRNA expression levels of Fibronectin, Collagen I, α-SMA, E-cadherin, Vimentin, and MMP2 were detected by qPCR. As Figure 9 shown, TGF-β1 stimulated a significant increase in the mRNA levels of Fibronectin, Collagen I, α-SMA, Vimentin, and MMP2 in HK-2 cells, and the cyclic peptide DR8 could effectively inhibit the increase in the mRNA levels of these genes and upregulate the mRNA level of E-cadherin.

[0129] As Figure 7 , Figure 8 and Figure 9 shown, the cyclic peptide DR8 obtained in Example 1 could inhibit the expression of fibrosis-related proteins and genes induced by TGF-β1 in HK-2 cells in a dose-dependent manner.

[0130] Example 7 investigated the in vitro inhibitory effect of the DR8 cyclic peptide analog and DR3penA on fibrosis

[0131] The HK-2 cell line was selected to study and observe the effects of the test substances, the DR8 cyclic peptide analog and DR3penA, on the expression levels of different cell fibrosis-related proteins.

[0132] Seed HK-2 cells in a 6-well plate and culture them in DMEM / F12 medium containing 10% FBS + 1% penicillin-streptomycin at 37°C and 5% CO2 for 24 h. Then change to serum-free medium and culture for 12 h. Add 5 ng / mL TGF-β1 and 1 or 5 μM of the test compound and co-treat the HK-2 cells for 24 h, and then extract the total cellular proteins. Detect the protein expression levels of Fibronectin and α-SMA by Western blot.

[0133] Control group (labeled as Ctrl): Neither TGF-β1 nor the test compound was added to the medium.

[0134] Induction group (labeled as TGF-β1): Add 5 ng / mL TGF-β1 to the medium.

[0135] Experimental group (labeled as the test compound name DR3penA or cyclic peptide 79): Add 5 ng / mL TGF-β1 and 1 or 5 μM of the test compound to the medium.

[0136] Figure 10 After treating HK-2 cells with TGF-β1 and the test compound, the protein expression results of α-SMA, Fibronectin, and GAPDH detected by Western blot and the relative protein expression levels analyzed are shown. The results show that the relative expression levels of the two proteins, Fibronectin and α-SMA, in HK-2 cells treated with the cyclic peptides DR8 and DR3penA in the experimental group are significantly lower than those in the TGF-β1 induction group, indicating that the cyclic peptides DR8 and DR3penA can significantly inhibit the effect of TGF-β1-induced renal fibrosis in HK-2 cells. Compared with DR3penA, after treating HK-2 cells with 1 and 5 μM of the cyclic peptide DR8, the relative protein expression levels of α-SMA and Fibronectin are significantly lower, indicating that the cyclic peptide DR8 has stronger anti-fibrotic activity than DR3penA.

[0137] Example 8 examines the therapeutic effects of DR8 cyclic peptide analogs and DR3penA on renal fibrosis in mice

[0138] (1) Test compounds: Cyclic peptides DR8 and DR3penA.

[0139] (2) Randomly divide 30 male C57BL / 6J mice (6 weeks old, weighing about 20 g, purchased from Lanzhou Veterinary Research Institute) into 6 groups with 5 mice in each group.

[0140] Grouping: Sham operation group (Sham), model group (UUO), cyclic peptide 79 group (0.5 mg / kg), DR3penA group (0.5 mg / kg).

[0141] (3) Establishment of renal fibrosis model: Unilateral ureteric obstruction (UUO) was used to induce renal fibrosis in mice.

[0142] After anesthesia by intraperitoneal injection of 1% sodium pentobarbital solution, the left ureter of the model mice was isolated. The ureter near the renal pelvis was ligated with 4-0 suture, and the middle part of the ligated ureter was cut. The UUO-induced renal fibrosis model was characterized by hydronephrosis, thinning of the renal cortex, dilation of renal tubules, and infiltration of inflammatory cells. After treatment of the UUO model mice with the test substance, pathological analysis of their kidneys was performed. In the sham operation group, only the left ureter of the mice was isolated without ligation.

[0143] (4) Cyclic peptide DR8 group: The model mice were intraperitoneally injected with 0.5 mg / kg cyclic peptide DR8 daily.

[0144] DR3penA group: The model mice were intraperitoneally injected with 0.5 mg / kg compound DR3penA daily.

[0145] UUO group: The mice were model mice. The UUO group mice and the sham operation group were intraperitoneally injected with an equal amount of sterile PBS solution. Samples were taken after 7 days of administration.

[0146] Figure 11 The statistical chart of the expression levels of Fibronectin, Collagen I, α-SMA, MMP2, E-cadherin, Vimentin, and GAPDH proteins in the kidney tissues of mice in each group after 7 days of administration and the relative expression levels obtained by analysis showed that in the UUO model mice, after treatment with the test substance, the levels of fibrosis-related proteins in the kidneys were significantly decreased, indicating that the test substance had a good effect on improving renal fibrosis. In addition, the therapeutic effect of cyclic peptide DR8 was significantly better than that of DR3penA.

[0147] Figure 12 The gene expression levels of Fibronectin, Collagen I, α-SMA, MMP2, E-cadherin, and Vimentin in the kidney tissues of mice in each group after 7 days of administration showed that in the UUO model mice, after treatment with the test substance, the gene levels of fibrosis-related proteins in the kidneys were significantly decreased, indicating that the test substance had a good effect on improving renal fibrosis. In addition, the therapeutic effect of cyclic peptide DR8 was significantly better than that of DR3penA.

[0148] Example 9 investigated the improvement and treatment effects of DR8 cyclic peptide analog and captopril on renal fibrosis in mice

[0149] (1) Test substance: Cyclic peptide DR8.

[0150] (2) Thirty-six male C57BL / 6J mice (6 weeks old, weighing approximately 20 g, purchased from Lanzhou Veterinary Research Institute) were randomly divided into 6 groups, with 6 mice in each group.

[0151] Grouping: Sham operation group (Sham), model group (UUO), Captopril positive control group (Captopril, 20 mg / kg), drug administration groups: cyclic peptide DR8 (0.05 mg / kg), cyclic peptide DR8 (0.25 mg / kg), cyclic peptide DR8 (1.25 mg / kg).

[0152] (3) The renal fibrosis model was established in the same way as in Example 8.

[0153] (4) In the drug administration groups, the model mice were intraperitoneally injected with 0.05, 0.25 or 1.25 mg / kg of cyclic peptide DR8 daily; in the Captopril group, the model mice were intraperitoneally injected with 20 mg / kg of Captopril; the mice in the UUO group were model mice, and the mice in the UUO group and the sham operation group were intraperitoneally injected with an equal amount of sterile PBS solution. Samples were taken after 7 days of drug administration.

[0154] Figure 13 For the statistical chart of the expression levels of Fibronectin, Collagen I, α-SMA, MMP2, E-cadherin, Vimentin and GAPDH proteins in the kidney tissues of mice in each group after 7 days of drug administration and the relative expression levels obtained by analysis, it can be seen that in the UUO model mice, after treatment with cyclic peptide DR8, the levels of fibrosis-related proteins in the kidney were significantly decreased, indicating that cyclic peptide DR8 has a good effect on improving renal fibrosis.

[0155] Figure 14 For the gene expression levels of Fibronectin, Collagen I, α-SMA, MMP2, E-cadherin, Vimentin in the kidney tissues of mice in each group after 7 days of drug administration, it can be seen that in the UUO model mice, after treatment with cyclic peptide DR8, the levels of fibrosis-related proteins in the kidney were significantly decreased, indicating that cyclic peptide DR8 has a good effect on improving renal fibrosis. From the above results, it can be seen that cyclic peptide DR8 can inhibit and treat UUO-induced renal fibrosis and kidney injury, and the treatment effect is significantly better than that of the positive drug Captopril. In addition, the dosage of cyclic peptide DR8 (1.25 mg / kg) is lower than that of Captopril (20 mg / kg), showing a significant protective effect.

[0156] Figure 15 For the H&E and immunohistochemical staining results of the pathological sections of the kidney tissues of mice in each group after 7 days of drug administration in Example 7.

[0157] H&E staining: Bake the paraffin sections in an oven at 60 °C for 1 h. Dewaxing and hydration: Xylene I (20 min) → Xylene II (20 min) → Absolute ethanol I (2 min) → Absolute ethanol II (2 min) → 95% ethanol I (2 min) → 95% ethanol II (2 min) → 80% ethanol (2 min) → Rinse with warm water twice (2 min each). Staining: Dropwise stain with hematoxylin (3 min) → Rinse with tap water twice (2 min each) → Differentiate with 0.5% hydrochloric acid alcohol (2 s) → Wash thoroughly with tap water → Blue with dilute ammonia water (5 - 10 s) → Wash thoroughly with tap water → Stain with 5% eosin (5 s). Dehydration and clearing: 95% ethanol I (15 s) → 95% ethanol II (15 s) → Absolute ethanol I (15 s) → Absolute ethanol II (15 s) → Air dry → Clear with xylene twice (2 min each) → After air drying, seal the sections with neutral balsam.

[0158] Immunohistochemistry: Bake the paraffin sections in an oven at 60 °C for 1 h. Dewaxing and hydration: Xylene I (20 min) → Xylene II (20 min) → Absolute ethanol I (2 min) → Absolute ethanol II (2 min) → 95% ethanol I (2 min) → 95% ethanol II (2 min) → 80% ethanol (2 min) → Rinse with warm water twice (2 min each). Antigen retrieval → Block endogenous peroxidase (10 min) → Primary antibody (overnight at 4 °C) → Reaction enhancer (20 min) → Enhanced goat anti-rabbit IgG polymer labeled with beauty (20 min) → DAB chromogenic reaction → Counterstain with hematoxylin → Wash thoroughly with tap water. Dehydration and clearing: 95% ethanol I (15 s) → 95% ethanol II (15 s) → Absolute ethanol I (15 s) → Absolute ethanol II (15 s) → Air dry → Clear with xylene twice (2 min each) → After air drying, seal the sections with neutral balsam.

[0159] Figure 15 The H&E staining results showed that compared with the sham operation group, the kidneys in the UUO group were significantly damaged, showing pathological changes such as dilation of the renal tubular lumen, exfoliation of renal tubular epithelial cells, and infiltration of inflammatory cells. After treatment with cyclic peptide DR8 for 7 days, the dilation of the renal tubular lumen was significantly reduced, and the infiltration of inflammatory cells decreased, indicating obvious therapeutic and improvement effects. Figure 15 The immunohistochemical staining results showed that Fibronectin and α-SMA were highly expressed in the kidneys of the UUO group. After treatment with cyclic peptide DR8 for 7 days, the expression levels decreased significantly, indicating that renal fibrosis in mice was treated and improved.

[0160] Example 10 Compare the serum stability of DR8 cyclic peptide analog and polypeptide DR3penA

[0161] After anesthetizing C57BL / 6J mice, blood was collected, left standing at room temperature for 30 min, centrifuged at 5000 rpm / min for 30 min, the upper-layer serum was aspirated and stored at -80 °C. Before the experiment, the frozen serum was thawed and incubated at 37 °C for 2 min. An appropriate amount of the test substance was weighed and prepared into a stock solution with a concentration of 10 mM. 855 μL of mouse serum was mixed well with 45 μL of 10 mM test substance and incubated at 37 °C. 70 μL of samples were taken at different time points (0 h, 3 h, 6 h, 10 h, 24 h, 48 h, 72 h), and immediately 70 μL of ice-cold acetonitrile was added. After vortex oscillation, it was left standing on ice for 10 min. The samples were centrifuged at 13000 g for 15 min, and the supernatant was collected.

[0162] Using a C18 reverse-phase analytical column, gradient elution was performed with 5%-95% acetonitrile / deionized water for 30 min, and the absorption peak at 220 nm was detected. By integrating the peak area of the test substance and comparing it with the corresponding peak area of the test substance at 0 min, the degradation rate of the test substance at different time points was determined.

[0163] Figure 16 It is the content curves of cyclic peptide DR8 and polypeptide DR3penA in serum. The content of cyclic peptide DR8 in serum was still higher than 50% at 24 h, and its half-life was 27.03 h, while the half-life of DR3penA was only 2.91 h, indicating that the stability of cyclic peptide DR8 was significantly better than that of DR3penA.

[0164] Example 11 Comparison of the pharmacokinetics of DR8 cyclic peptide analog and polypeptide DR3penA

[0165] C57BL / 6J mice were injected with 2.4 mmol / kg of cyclic peptide DR8 (2.8 mg / kg) and DR3penA (2.5 mg / kg) via the tail vein. Blood was collected from the eyeballs at different time points (0 min, 5 min, 10 min, 20 min, 30 min, 45 min, 60 min, 120 min, 240 min), mixed well with 200 μL of 2 mg / kg heparin sodium, centrifuged at 2000 rpm at 4 °C for 10 min, 100 μL of the upper-layer plasma was taken, 300 μL of methanol was added and vortexed well, centrifuged at 15000 rpm at 4 °C for 10 min. After concentrating 300 μL of the supernatant, the precipitate was resuspended with 100 μL of 5% acetonitrile solution, centrifuged at 15000 rpm at 4 °C for 5 min, and the supernatant was aspirated for LC-MS detection.

[0166] Figure 17 It is the plasma concentration-time curves of cyclic peptide DR8 and DR3penA in vivo. The concentration of cyclic peptide DR8 in vivo was much higher than that of DR3penA, and its half-life was 1.08 h, while the half-life of DR3penA was 0.19 h, indicating that the action time and concentration of cyclic peptide DR8 in vivo were significantly improved compared with DR3penA.

[0167] As described above, it is only the specific implementation manner of the present invention. However, the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention can easily think of changes or substitutions, which should all be covered by the protection scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims described above.

Claims

1. A DR8 cyclic peptide analogue, characterized in that: Its structure is as follows:

2. Use of the DR8 cyclic peptide analogue according to claim 1 in the preparation of a medicament for treating renal fibrosis.

3. A drug for treating renal fibrosis, characterized in that: The drug has the DR8 cyclic peptide analogue described in claim 1 as a main component.

4. The drug for treating renal fibrosis according to claim 3, characterized in that: The medicine also includes a pharmaceutically acceptable carrier or excipient.

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