New use of a functional polypeptide

Abstract

The application discloses a new use of a functional polypeptide, and the functional polypeptide is Ezy-1 of a sea spurs peach sponge peptide, which can be used for preparing an anti-aging medicine, or used in combination with a chemotherapy medicine to prepare an anti-aging medicine, or used as an effective component of an anti-aging medicine, or used for reformation of the functional polypeptide as a precursor of the anti-aging medicine, or used for preparing an mTOR activity inhibitor. The new use of the functional polypeptide can be used for significantly targeting and combining with an mTOR activation protein Rictor, interfering with assembly of an mTOR complex, inhibiting mTOR activation, delaying aging formation, and has a significant anti-aging function.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology. Specifically, it relates to a novel use of a functional polypeptide. Background Technology

[0002] Aging is a leading risk factor for many chronic diseases, primarily manifested as a decline in hormone regulation levels, a gradual decline in the function of various organs, and the emergence of degenerative diseases in these organs. The accumulation of senescent cells is a key factor driving aging and the development of age-related diseases, and forms the basis of overall aging. Inhibiting the production of senescent cell phenotypes has become a new strategy for delaying population aging and preventing age-related diseases; however, effective drugs for delaying aging are currently lacking.

[0003] Enhancing immune function is one of the most effective ways to combat aging. As the most basic substances for maintaining cell activity and supplying cell energy, peptides are one of the most obvious ways to regulate immune function. Peptides can delay the aging of specific immune organs and immune cells, delay the aging of non-specific immune tissues and immune cells, repair damaged specific immune organs and immune cells, and activate the body's immune function.

[0004] Currently, representative theories regarding the mechanisms of aging include the free radical theory and the mitochondrial DNA damage theory. Human aging is always accompanied by immune dysfunction; therefore, improving immune function is one of the most effective ways to combat aging. Aging is a leading risk factor for many chronic diseases, primarily manifested as a decline in hormone regulation levels, a gradual decline in the function of various organs, and the gradual emergence of degenerative diseases. The accumulation of senescent cells is a key factor driving aging and the development of age-related diseases, and the foundation of aging. Inhibiting the production of senescent cell phenotypes has become a new strategy for delaying population aging and preventing age-related diseases. Currently, there is a lack of effective drugs to delay aging. Peptides, as the most basic substances for maintaining cellular activity and supplying cellular energy, are one of the most significant ways to regulate immune function. Peptides can delay the aging of specific immune organs and immune cells, delay the aging of non-specific immune tissues and immune cells, repair damaged specific immune organs and immune cells, and activate human immune function.

[0005] Eucheuma, also known as chicken foot seaweed or chicken glue seaweed, is a type of algae with thick, fleshy, cylindrical, flattened, or radiating or lateral branching thallus. The genus comprises about 20 species, with approximately 5 found in China. It is a tropical seaweed with high medicinal value. Functional peptides purified from Eucheuma have significant anti-aging properties. However, there are currently no literature reports on the anti-aging effects of Eucheuma-derived peptides, and no reports have been found domestically or internationally of using Eucheuma-derived peptides as anti-aging drugs.

[0006] The model animal *Caenorhabditis elegans* not only has advantages such as a short lifespan and simple experimental operation, but also its key signaling pathways have high homology with those of humans. Therefore, it is an ideal animal model for studying the activity of natural products and traditional Chinese medicine. Thus, the aging state of humans can be simulated through the life-extending experiment of *Caenorhabditis elegans*. The mTOR signaling pathway is closely related to cellular senescence. Activation of this pathway can promote cellular senescence, while inhibition of its activity can slow down the aging process. Targeting and inhibiting the mTOR pathway is a strategy for screening anti-aging drugs. The mTOR complex is assembled from multiple proteins with the mTOR protein as its core. Existing mTOR complex inhibitors mainly target and inhibit the core protein mTOR, but these inhibitors exhibit strong toxic side effects. Summary of the Invention

[0007] Therefore, the technical problem to be solved by the present invention is to provide a new use for a functional polypeptide. Through experiments, it has been confirmed that the independently developed functional polypeptide (patent number: 201711141671X) can significantly target and bind to the mTOR activator protein Rictor, interfere with the assembly of the mTOR complex, inhibit mTOR activation, delay the formation of aging, and has a significant anti-aging function. It can be used to prepare anti-aging drugs or active ingredients of drugs or drug prodrugs.

[0008] To solve the above-mentioned technical problems, the present invention provides the following technical solution:

[0009] A functional polypeptide, which is isolated and purified from Euphorbia milii and named Euphorbia milii peptide EZY-1, has an amino acid sequence as shown in SEQ ID NO:1.

[0010] An anti-aging drug comprising a safe and effective amount of the above-mentioned Euphorbia lactea peptide EZY-1.

[0011] A functional polypeptide composition comprising EZY-1 peptide and at least one excipient, wherein the composition is in any therapeutically acceptable dosage form and in any therapeutically acceptable dosage and administration method.

[0012] An mTOR activity inhibitor comprising the aforementioned *Erythrina variegata* peptide EZY-1, wherein the *Erythrina variegata* peptide EZY-1 targets and binds to Rictor, the activating protein of the mTOR complex, thereby inhibiting mTOR activity by interfering with the assembly of the mTOR complex.

[0013] A functional polypeptide having the amino acid sequence shown in SEQ ID NO:1, for use in the preparation of anti-aging drugs.

[0014] The application of a functional polypeptide having the amino acid sequence shown in SEQ ID NO:1 in the preparation of the active ingredient of an anti-aging drug.

[0015] The use of a functional polypeptide having the amino acid sequence shown in SEQ ID NO:1 in the preparation of a precursor for an anti-aging drug.

[0016] A functional polypeptide having the amino acid sequence shown in SEQ ID NO:1, and its application in the preparation of anti-aging drugs in combination with chemotherapy drugs.

[0017] The technical solution of the present invention achieves the following beneficial technical effects:

[0018] 1. The functional polypeptide described in this invention can target and bind to Rictor, the activating protein of the mTOR complex, thereby inhibiting mTOR activity by interfering with the completion of mTOR complex assembly and avoiding toxic side effects.

[0019] 2. The functional polypeptides described in this invention can be used to prepare drugs for delaying aging, or active ingredients of drugs, or drug precursors.

[0020] 3. The anti-aging drug of the present invention contains functional polypeptides and can be made into oral capsules or other products in various forms. It can be used alone as a finished product or as an active ingredient combined with other adjuvants to form a composition. Attached Figure Description

[0021] Figure 1 A schematic diagram of the inhibition of epithelial cell senescence phenotype and inhibition of senescence-related signaling pathways by the Euphorbia lactea peptide EZY-1 of this invention;

[0022] Figure A: EZY-1 peptide from *Euphorbia hirta* inhibits the senescent phenotype of epithelial cells; β-galactosidase activity was detected.

[0023] B. *Euphorbia milii* peptide EZY-1 inhibits the mTOR signaling pathway;

[0024] C. The *Euphorbia milii* peptide EZY-1 targets and binds to the mTOR-activated component Rictor;

[0025] D. Mammalian two-hybrid systemic analysis: EZY-1 inhibits the binding of mTOR and Rictor.

[0026] Figure 2 Schematic diagram of the invention showing how the peptide EZY-1 from *Euphorbia milii* extends the lifespan of *C. elegans*.

[0027] Figure 3 yes Figure 2 A schematic diagram showing the point values ​​corresponding to the survival rate and time of *C. elegans*. Detailed Implementation

[0028] Example 1

[0029] Eucheuma, also known as chicken foot seaweed or chicken glue seaweed, is a type of algae with thick, fleshy, cylindrical, flattened or compressed, and branching radially or laterally. There are about 20 species in this genus, with about 5 species in China. It is a tropical seaweed with high medicinal value.

[0030] This invention provides a novel use for a functional polypeptide, which is isolated and purified from Euphorbia milii and named Euphorbia milii peptide EZY-1. Euphorbia milii peptide EZY-1 has the amino acid sequence shown in SEQ ID NO:1, which is RTGACFCVIYNGILYP.

[0031] Example 2

[0032] Based on the amino acid sequence shown in SEQ ID No. 1 described in Example 1, a polypeptide with the same or similar function after substitution, deletion, or addition of one or more amino acids.

[0033] A polypeptide having the same or similar function after substitution, deletion, or addition of one or more amino acids based on the amino acid sequence shown in SEQ ID No. 1.

[0034] Based on SEQ ID NO:1, one or more amino groups at positions 1, 2, 8, 9, 11, 13, and 14 are substituted with amino groups having the same or similar functions, specifically, substitutions can be selected in one or more of the following ways:

[0035] The first amino acid, R, can be replaced with K because R and K are both basic amino acids and have similar functions.

[0036] The second amino acid, T, can be replaced with S because R and K are both hydroxyl-containing amino acids and have similar functions.

[0037] The 8th amino acid, V, can be replaced with I or L. The reason is that V, I, and L are all branched-chain amino acids and have similar functions.

[0038] The 9th amino acid I can be replaced with V or L because V, I and L are both branched-chain amino acids and have similar functions.

[0039] The 11th amino acid N can be replaced with Q because N and Q are both amino acids containing aminoamide groups and have similar functions.

[0040] The 13th amino acid, I, can be replaced with V or L. The reason is that V, I, and L are all branched-chain amino acids and have similar functions.

[0041] The 14th amino acid, L, can be replaced with V or I. The reason is that V, I, and L are all branched-chain amino acids and have similar functions.

[0042] Example 3

[0043] The mTOR signaling pathway is closely related to cellular senescence. Activation of this pathway can promote cellular senescence, while inhibition of its activity can slow down the aging process. Targeting and inhibiting the mTOR pathway is a strategy for screening drugs to delay aging. The mTOR complex is composed of multiple proteins with the mTOR protein as its core. Existing mTOR complex inhibitors mainly target and inhibit the core protein mTOR, and these inhibitors exhibit strong toxic side effects.

[0044] EZY-1, derived from *Euphorbia milii*, targets and binds to Rictor, the activator of the mTOR complex. By interfering with the assembly of the mTOR complex, EZY-1 inhibits mTOR activity, thus avoiding toxic side effects and achieving the function of delaying aging.

[0045] EZY-1, a peptide derived from *Euphorbia milii*, is obtained by separation and purification from *Euphorbia milii*. The applicant has already disclosed the purification and separation method of *Euphorbia milii* peptide in the application of *Euphorbia milii* peptide in the prevention and treatment of pulmonary fibrosis in the prior Chinese patent application 201710302276.9, which will not be repeated here. Alternatively, it can be obtained by artificially synthesizing it using amino acids as raw materials, according to the amino acid sequence described in SEQ ID No. 1, using a peptide synthesis instrument, and then purifying it by high performance liquid chromatography.

[0046] Example 4

[0047] Based on Examples 1 and 2, the present invention also provides the use of the functional polypeptide, which can be used to prepare anti-aging drugs, or as an effective component of anti-aging drugs, or as a precursor for anti-aging drugs.

[0048] A drug for delaying aging, the drug comprising a safe and effective amount of the aforementioned Euphorbia lactea peptide EZY-1.

[0049] Application of a functional polypeptide, Euphorbia lactea peptide EZY-1, in the preparation of anti-aging drugs.

[0050] Application of a functional polypeptide, Euphorbia lactea peptide EZY-1, in the preparation of effective components for anti-aging drugs.

[0051] Application of a functional polypeptide, Euphorbia lactea peptide EZY-1, in the preparation of a precursor for anti-aging drugs.

[0052] Application of a functional polypeptide, Euphorbia lactea peptide EZY-1, in combination with chemotherapy drugs in the preparation of anti-aging drugs.

[0053] A functional polypeptide composition comprising EZY-1 peptide and various pharmaceutical excipients, wherein the composition is in any pharmacologically acceptable dosage form, and in any pharmacologically acceptable dosage and administration method, and its application in delaying aging can be achieved through the following methods:

[0054] The functional polypeptide compound of this invention is administered orally at a dose of 10–50 mg / kg once daily.

[0055] Example 5

[0056] The functional peptides provided by this invention were tested in the following experiments to inhibit cell senescence:

[0057] Epithelial cell senescence phenotype experiment

[0058] (1) Before inoculating epithelial cells, sterile cell sheets were placed in a 6-well culture plate, and 1×105 epithelial cells were added to each well. The plates were cultured overnight at 37°C and 5% CO2 to allow the epithelial cells to grow on the cell sheets.

[0059] (2) In a clean bench, aspirate the culture medium from the 6-well culture plate, add 1×PBS, wash the cells once, and then add 2% formaldehyde or 0.2% glutaraldehyde fixative and fix for 3-5 minutes at room temperature.

[0060] (3) Remove 2% formaldehyde or 0.2% glutaraldehyde fixative, add 1×PBS, and wash the cells 3 times for 3 minutes each time;

[0061] (4) Remove 1×PBS, add X-Gal solution to immerse the cell slides, prepare cell spread sheets, incubate at 37°C for 4-8 hours, and cover the 6-well culture plate with plastic wrap to prevent the staining solution from evaporating.

[0062] (5) Remove the cell slide, rinse twice with deionized water, fix again with 2% formaldehyde or 0.2% glutaraldehyde fixative for 4 minutes, and rinse with running water.

[0063] (6) Dehydrate and clear the cell smears in the following order: 2 min 95% ethanol → 2 min 95% ethanol → 2 min 100% ethanol → 5 min 100% ethanol → 2 min xylene → 2 min xylene.

[0064] (7) Neutral resin sealing;

[0065] (8) Observe the morphology of senescent cells under a regular optical microscope;

[0066] (9) The activity of mTOR, a aging-related pathway, was detected by Western blotting.

[0067] Test results as follows Figure 1As shown, A represents the inhibition of epithelial cell senescence phenotype by *Erythrina variegata* peptide EZY-1, and the detection of β-galactosidase activity; B represents the inhibition of the mTOR signaling pathway by *Erythrina variegata* peptide EZY-1; C represents the targeted binding of *Erythrina variegata* peptide EZY-1 to the mTOR-activating component Rictor; and D represents the mammalian two-hybrid system analysis showing the inhibition of mTOR and Rictor binding by EZY-1.

[0068] Functional peptides can target and bind to Rictor, the activating protein of the mTOR complex, thereby inhibiting mTOR activity by interfering with the completion of mTOR complex assembly and avoiding toxic side effects.

[0069] Example 6

[0070] Experiments to extend the lifespan of *C. elegans*

[0071] (1) Nematode collection

[0072] S1. Select uncontaminated NGM culture medium with a high oviposition period using a microscope. Wash the plate with 2 mL of M9 buffer. Add M9 buffer to a 10 mL glass centrifuge tube using a pipette. If there are many nematodes remaining on the plate, wash again until there are no obvious nematodes remaining on the plate.

[0073] S2. Let the centrifuge tube containing the washing solution stand for 5 minutes. After the nematodes settle, remove the supernatant.

[0074] S3. Take 5 ml of M9 buffer to wash the precipitated nematodes, discard the supernatant, and repeat four times until the M9 buffer solution is clear.

[0075] S4. Mix 10% hypochlorous acid and 1M sodium hydroxide in equal proportions to prepare a lysis buffer. Pour the lysis buffer into the cleaned precipitate of *C. elegans*, mix thoroughly, and shake rapidly for 5 minutes, or shake with a vortex shaker for 1 minute, until all *C. elegans* are lysed. Observe under a microscope for any adult worm residues.

[0076] S5. Collect the eggs, centrifuge at 1200 rpm for 1 min, discard the supernatant, and retain the eggs. If the precipitate is yellow, it indicates incomplete lysis and some insect bodies remain; if the precipitate is milky white, it is the eggs.

[0077] S6. Add 5 mL of M9 buffer and mix thoroughly. Centrifuge at 1200 rpm for 1 min and discard the supernatant.

[0078] The precipitates after discarding the supernatant in S7 and S6 were washed four times with M9 buffer, and the supernatant was discarded.

[0079] S8. Add ≥2mL of M9 buffer solution to the culture dish, which does not completely cover the culture dish, and repeatedly pipette to disperse the eggs. Transfer the liquid culture medium containing the eggs to the culture dish.

[0080] S9. Under a microscope, the field of view is filled with eggs. The eggs are spread out and evenly distributed. The eggs are placed in a 20℃ biochemical incubator and cultured for 16 hours.

[0081] S10. Inoculate the larvae hatched from S9 onto a culture medium coated with E. coli OP50. Observe under a microscope. Use a pipette to collect larvae from areas with a large number of hatched larvae and drop them onto different locations on the culture medium coated with E. coli OP50. After drying, invert the culture dish and incubate it in a 20°C biochemical incubator.

[0082] (2) Life Extension Experimental Methods

[0083] W1. Synchronize the collected nematodes and grow them on NGM plates to the specified stage, then wash with M9 buffer or S medium.

[0084] W2. Adjust the microscopic level to ensure that the average concentration of *C. elegans* in the liquid is 50 worms / 100 μL;

[0085] W3. Add 100 μL of *C. elegans* to each well of a 96-well culture plate, with an average of 50 worms per well.

[0086] W4. Centrifuge to collect OP50 cells, dilute to 4% (w / w), and add 50 μL to each well of a 96-well plate.

[0087] W5. Add the test material to each well of a 96-well cell culture plate, 50 μL, and incubate at 20°C until the exposure ends.

[0088] W6. The time when the tested nematodes begin to grow from eggs is recorded as day 0. The time when the last nematode in each experimental group dies is the longest lifespan of the nematodes in that experimental group.

[0089] The results are as follows Figure 2 and Figure 3 As shown: In the control group, all nematodes died after 19 days of culture, while the 80 μg EZY-1 group still had a 100% survival rate, the 40 μg EZY-1 group had a survival rate of 37.5%, and the 10 μg EZY-1 group had a survival rate of 12.5%. The longest lifespan of nematodes given the 40 μg EZY-1 group was 35 days, at which point the nematode survival rate was 6.25%. At the end of the experiment, 38 days later, no nematode death was observed in the nematodes given the 80 μg high-concentration group of EZY-1, and the survival rate was 100%. Therefore, EZY-1 can significantly prolong the lifespan of nematodes.

[0090] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of the claims of this patent application.

Claims

1. Application of Euphorbia lactea peptide EZY-1 in the preparation of anti-aging drugs, wherein the amino acid sequence of Euphorbia lactea peptide EZY-1 is SEQ ID NO:

1.

2. Application of Euphorbia lactea peptide EZY-1 in the preparation of effective components of anti-aging drugs, wherein the amino acid sequence of Euphorbia lactea peptide EZY-1 is SEQ ID NO:1.

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