A cyclic peptide and its use

Abstract

The present application relates to the field of biomedicine and cosmetics, and more specifically, to a cyclic peptide containing HMGB1 as the main target. The cyclic peptide obtained in the present application can significantly prevent or treat atopic dermatitis by inhibiting inflammatory response, thereby alleviating the rash and itching phenotype.

Description

Technical Field

[0001] The present application relates to the fields of biomedicine and cosmetics, and in particular, to a cyclic peptide and its use. Background Art

[0002] Atopic dermatitis is one of the most common diseases in dermatology, with eczematous rashes as the main manifestations, such as papules, blisters, dry desquamation, often accompanied by exudative crusting and severe and persistent itching reactions. According to the global burden of disease analysis, the current global burden of disease study shows that the prevalence of atopic dermatitis in children in high-income countries is about 20%, and the prevalence in adults is as high as 10%. Atopic dermatitis ranks 15th among all non-fatal diseases, and has the highest disease burden among skin diseases measured by disability-adjusted life years (DALYs). There are about tens of millions of patients in my country. The onset of atopic dermatitis is caused by the interaction between genetic and environmental factors. Patients often have genetic susceptibility, and there are many factors such as immune disorders, skin barrier dysfunction, and skin flora disorders. The pathogenesis is very complex. The existing topical drugs and care products are not ideal for controlling the disease, which brings great difficulties and challenges to anti-inflammatory and skin barrier repair.

[0003] High mobility group protein B1 (HMGB1) is a highly conserved nuclear protein widely distributed in mammalian cells. With the discovery of its pro-inflammatory effect, HMGB1 has become one of the hot spots in critical care medicine research in recent years. A review report published by Cui Lixia et al. revealed the important role of HMGB1 in many skin diseases. For example, in atopic dermatitis, the expression levels of HMGB1 in serum and lesions were higher than those in the control group, and were positively correlated with the severity of dermatitis. In the mouse model of psoriasis vulgaris, the expression level of HMGB1 was significantly increased; in addition to its important role in skin diseases, related studies have shown that HMGB1 protein is also involved in physiological processes such as pigment regulation, inhibition of inflammation, regulation of immunity, and improvement of aging. Currently known HMGB1 antagonists include but are not limited to: cisplatin, quercetin, ethyl pyruvate, glycyrrhizin, triptolide, etc. However, most of the above-mentioned antagonists are chemical drugs or traditional Chinese medicines, which have limited effects, unclear mechanisms, and certain side effects. In summary, HMGB1, as an important target, provides researchers with a research object for the treatment of various diseases.

[0004] Based on the defects of the prior art, the inventors of the present application combined computer-assisted molecular simulation technology to screen a cyclic peptide that is effective in treating or alleviating atopic dermatitis. By constructing a mouse atopic dermatitis model, the inventors verified the important role of the cyclic peptide in effectively alleviating dermatitis and reducing related inflammatory factors, and achieved significant technical effects. Summary of the invention

[0005] The present invention relates to a therapeutic agent effective in treating dermatitis;

[0006] In a first aspect, the present invention relates to a cyclic peptide, the amino acid sequence of which is CIPRFYLFYC.

[0007] In some embodiments, the cyclic peptide is formed by a disulfide bond between the first and last two cysteines to form a ring.

[0008] In some embodiments, the amino acid side chain groups, the amino terminus or the carboxyl terminus of the cyclic peptide can be modified by hydroxylation, carboxylation, carbonylation, methylation, acetylation, phosphorylation, esterification or glycosylation to obtain derivatives of the cyclic peptide without affecting its biological activity.

[0009] In a second aspect, the present invention relates to a pharmaceutical composition comprising the cyclic peptide and a pharmaceutically acceptable carrier.

[0010] In some embodiments, the pharmaceutically acceptable carrier is selected from isotonic saline, ethanol, phosphate buffered saline, balanced salt solution, and dimethyl sulfoxide (DMSO).

[0011] In other embodiments, the composition may be administered orally, by inhalation, rectally, vaginally, topically, nasally, ophthalmically, or parenterally.

[0012] In other embodiments, the composition is a dosage form selected from the group consisting of an oral dosage form, a parenteral dosage form, a buccal dosage form, a sublingual dosage form, a nasal dosage form, an inhaler, a nebulizer, a topical dosage form, a transdermal dosage form, and a suppository.

[0013] In a third aspect, the present invention relates to use of the above-mentioned cyclic peptide or pharmaceutical composition in the preparation of a drug for treating HMGB1-mediated diseases.

[0014] In some embodiments, the disease is dermatitis.

[0015] In other embodiments, the disease is dermatitis caused by bacterial, fungal or viral infection.

[0016] In other embodiments, the dermatitis is atopic dermatitis.

[0017] In other embodiments, the disease is dermatitis not caused by bacterial, fungal or viral infection.

[0018] In a fourth aspect, the present invention relates to use of the cyclic peptide or the pharmaceutical composition in preparing skin care products.

[0019] In a fifth aspect, the present invention relates to use of the cyclic peptide or the pharmaceutical composition in preparing cosmetics.

[0020] In a sixth aspect, the present invention relates to use of the cyclic peptide or the pharmaceutical composition in preparing a drug for treating dermatitis.

[0021] In a sixth aspect, the present invention relates to the use of the cyclic peptide and an antiviral agent or an antibacterial agent in the combined preparation of a drug for treating dermatitis.

[0022] The inventors of this application simulated and predicted the structure of HMGB1 protein and its related binding proteins based on computer-assisted molecular simulation technology, and then annotated and analyzed the main interaction domains of HMGB1 to screen potential binding sequences. The polypeptide structure was further analyzed and optimized by molecular dynamics simulation technology. The main design principle is: analyze the secondary structure of the polypeptide sequence in a low energy state, and theoretically enhance the stability of the polypeptide in a solution environment through strategies such as amino acid replacement and cyclization. The specific potential HMGB1 binding activity and good stability obtained by the above method were synthesized and experimentally determined. The relevant sequence was verified by experiments to have excellent therapeutic efficacy in treating or relieving dermatitis, and is expected to become a monomer functional ingredient with specific good anti-inflammatory activity in cosmetic applications.

[0023] In the seventh aspect, the present invention relates to the dosage of the cyclic peptide. In some embodiments, the relevant dosage includes: 0.0001 μM-100 μM, preferably 1-20 μM, more preferably 5-12 μM, and most preferably 10 μM. In other embodiments, the relevant dosage is preferably 1-10 nM, more preferably 2-8 nM. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 :A) Prediction of the binding mode of cIY-8 polypeptide (CIPRFYLFYC) and HMGB1. B) Schematic diagram of the two-dimensional structure of cIY-8 polypeptide. C) Schematic diagram of the tertiary structure of cIY-8 polypeptide. D) Comparison of human and mouse sequences and structural homology of HMGB1 protein (human source: Homo sapiens is shown in blue; mouse source: Mus musculus is shown in green)

[0025] Figure 2 : Experimental results of atopic dermatitis in mice.

[0026] Note: Figure 2 The upper part is the MC903 group, where only MC903 was used; Figure 2 In addition to the MC903 group, the lower half also used the cyclic peptide therapeutic agent cIY8 of the present application.

[0027] Figure 3 :Results of pruritus experiment on atopic dermatitis in mice.

[0028] Figure 4:The results of cytokine experiments in a mouse atopic dermatitis model.

[0029] Figure 5A : Laser confocal microscopy results.

[0030] Figure 5B : Results of competitive binding experiment between cIY8 and HMGB1. DETAILED DESCRIPTION

[0031] Unless otherwise defined, all scientific and technical terms used herein have the same meanings as understood by those of ordinary skill in the art. For definitions and terms in the art, professionals can refer specifically to Current Protocols in Molecular Biology (Ausubel). The abbreviations for amino acid residues are the standard 3-letter and / or 1-letter codes used in the art to refer to one of the 20 commonly used L-amino acids.

[0032] Although the numerical ranges and parameter approximations shown in the broad scope of the present application, the numerical values ​​shown in the specific examples are recorded as accurately as possible. However, any numerical value is bound to contain a certain error, which is caused by the standard deviation in their respective measurements. In addition, all ranges disclosed herein should be understood to cover any and all sub-ranges contained therein. For example, the range of "1 to 10" recorded should be considered to include any and all sub-ranges between the minimum value 1 and the maximum value 10 (including the endpoints); that is, all sub-ranges starting with a minimum value of 1 or greater, such as 1 to 6.1, and sub-ranges ending with a maximum value of 10 or less, such as 5.5 to 10. In addition, any reference referred to as "incorporated herein" should be understood to be incorporated in its entirety.

[0033] Example 1: Screening and obtaining of cIY8 cyclic peptide

[0034] Extracellular HMGB1 can bind to TLR4 and induce the expression and production of various cytokines and chemokines. HMGB1 transmits downstream inflammatory signals through TLR4 and TLR2. HMGB1 consists of two structurally similar box structures, including A-box and B-box domains. Related studies have revealed the contribution of HMGB1's A-box and B-box domains alone in TLR4 signaling activation. For example, the antibody mAb 2G7 can block HMGB1 A-box and downstream signaling pathways.

[0035] Based on the above information, the inventors of the present application identified a new peptide inhibitor molecule cIY8 targeting HMGB1 through a peptide-protein complex prediction method and a molecular force field-based simulation method.

[0036] 1. Download the 3D structures of HMGB1 and related proteins from the RCSB Protein Database (RCSB PDB) (https: / / www.RCSB.org) and the AlphaFold protein structure database, which provides more than 200 million predicted protein structures (https: / / AlphaFold.ebi.ac.uk / ). AlphaFold2 Multimer generated the predicted binding sites of peptide-protein complexes ( Figure 1 A);

[0037] 2. The predicted alignment error and predicted local distance difference test were analyzed using ALPHAPICKLE software, and the molecular interactions of complex structures were simulated by computer simulation using Chimera and PyMol software. Molecular dynamics simulations of peptides and peptide-protein complexes were performed using GROMACS software, and 3.0 ns dynamics simulations were performed in simulated aqueous solution at 300K;

[0038] 3. Analyze the structural information of peptides and peptide-protein complexes using Chimera and PyMol software. Use Chemical Sketch to generate a two-dimensional schematic diagram and a three-dimensional simulation diagram of the peptide chemical structure ( Figure 1 B. Figure 1 C); Based on the similarity of the amino acid sequences of specific regions of mouse and human HMGB1 ( Figure 1 D) The biological effects of cIY8 cyclic peptide can be demonstrated through mouse functional experiments.

[0039] Example 2: Construction of atopic dermatitis model and drug efficacy verification

[0040] 2.1 Mouse pruritus experiment

[0041] In order to verify the relevant drug efficacy, an atopic dermatitis model was constructed and the drug efficacy was verified. Studies have shown that topical application of MC903 can cause atopic dermatitis syndrome in mice (Wang Can et al., Establishment of atopic dermatitis mouse model induced by calcipotriol, 2016); therefore, MC903 (calcipotriol) commonly used in the art was used for atopic dermatitis phenotype modeling. The cyclic peptide of the present application was named: cIY8 in this experiment. In addition, in order to better avoid the influence of individual factors of mice, each mouse was treated with MC903 alone in the left ear and MC903+cIY8 double treatment in the right ear.

[0042] The specific steps are as follows:

[0043] 1. Eight-week-old C57 / 6J mice were used for the experiment, with 3 mice in each group.

[0044] 2. Use a pipette gun to apply 20ul / mouse MC903 (2nM) to the left ear of each mouse; for the right ear: apply 20ul / mouse of a mixture of MC903 (2nM) + HMGB1 inhibitor cIY8 (100μM), and administer the drug once a day for 7 consecutive days.

[0045] 3. On the 8th day, animal behavior analysis was performed. The number of itching times on both sides of the mice within 1 hour was recorded using a Nikon camera. A positive reaction was considered when the mice raised their hind legs and scratched the back of their necks. The number of itching times on both sides was counted and the mice were photographed at the same time.

[0046] 4. Based on the analysis of gross skin lesions, the experimental results show that (see Figure 2 ), the skin of the treated mice: ear edema, thickening and erythema were significantly lower than those of the non-treated group. In addition, the number of scratches on the treated side was significantly lower than that on the non-treated side (see Figure 3 ), the result has a statistically significant difference.

[0047] The experimental results show that cIY8 can effectively reduce skin lesions and itching symptoms caused by dermatitis.

[0048] 2.2 Cytokine detection experiment

[0049] The expression of cytokines in mice after administration was detected. The specific steps are as follows:

[0050] TRIzol (Invitrogen) was used to extract total RNA from lesional skin, and then The One-Step gDNA Removal and cDNA Synthesis SuperMix Kit was used to reverse transcribe it into cDNA. qRT-PCR was then performed. The total volume was 20uL, including 10uL PowerUp SYBR Green (Applied Biosystems), 1uL forward substrate, 1uL reverse primer and 2uL cDNA. After cycling at 95°C for 30 seconds, run at 95°C for 5 seconds, run at 58-65°C for 34 seconds, and cycle 40 times. GAPDH was used as an internal reference, MC903 was the modeling group, MC903+cIY8 was the drug-treated group, and the untreated group was Healthycontrol. For related primers, please refer to Table 1.

[0051] Table 1: Primers used in this experiment

[0052]

[0053] The experimental results show that ( Figure 4): Compared with the modeling group, cIY8 can significantly inhibit the expression of various inflammatory cytokines (IL-1α, IL-4, IL-6, IL-13, IL-31) in the lesions of atopic dermatitis model mice, which explains the mechanism why cIY8 can relieve dermatitis and reduce itching.

[0054] 2.3 Experiment on HMGB1 targeted inhibition mechanism

[0055] 1. Human fibroblasts (HFF-1P27) were seeded in confocal microplates and allowed to adhere for 24 hours.

[0056] 2. HMGB1 (500 ng / ml) and cIY8 (concentration gradient, specifically: 10 μM, 1 μM, 500 nM, 100 nM) were incubated at room temperature for 30 minutes and then added to the cells. The cells were cultured in an incubator at 37°C for 1 hour and then the samples were collected.

[0057] 3. Follow the instructions of the Bio-Tech NFκB p65 Activation Detection Kit (Cat. No. SN368).

[0058] 4. After staining, take photos using a Leica DMI8 laser confocal microscope and select 3 fields of view for each sample.

[0059] 5. Use Image J image processing software to perform data statistics and analysis. The images are displayed at 20X image field of view.

[0060] The experimental results show that ( Figure 5A-Figure 5B ): cIY8 can competitively bind to HMGB1, and this competitive binding is concentration-dependent.

[0061] The above experimental results show that the cyclic peptide obtained in the present application can effectively and competitively bind to HMGB1, inhibit its downstream signaling pathway, relieve inflammatory response, effectively treat or relieve itching symptoms caused by atopic dermatitis, and achieve unexpected technical effects.

[0062] The above is an explanation of the embodiments of the present invention. However, the present invention is not limited to the above embodiments. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A cyclic peptide, whose amino acid sequence is CIPRFYLFYC, wherein the cyclic peptide is formed by a disulfide bond between the first and last cysteines.

2. A pharmaceutical composition comprising the cyclic peptide according to claim 1 and a pharmaceutically acceptable carrier.

3. The pharmaceutical composition of claim 2, wherein the pharmaceutically acceptable carrier is selected from isotonic saline, ethanol, phosphate buffered saline, balanced salt solution and dimethyl sulfoxide (DMSO).

4. Use of the cyclic peptide according to claim 1 or the pharmaceutical composition according to any one of claims 2 to 3 in the preparation of a medicament for treating atopic dermatitis.

5. Use of the cyclic peptide according to claim 1 or the pharmaceutical composition according to any one of claims 2 to 3 in the preparation of cosmetics for treating atopic dermatitis.

6. Use of the cyclic peptide according to claim 1 or the pharmaceutical composition according to any one of claims 2 to 3 in preparing a skin care product for treating atopic dermatitis.

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