CN110590921A - Human Kv1.3 type potassium ion channel activity inhibition peptide mimic, and preparation method and application thereof - Google Patents
Human Kv1.3 type potassium ion channel activity inhibition peptide mimic, and preparation method and application thereof Download PDFInfo
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Abstract
The invention belongs to the field of biological medicine, and particularly relates to a human Kv1.3 type potassium ion channel activity inhibition peptidomimetic, and a preparation method and application thereof. The molecular structure of the human Kv1.3 type potassium ion channel activity inhibition peptide mimic is as follows:the invention provides a peptide mimic (natural truncated polypeptide derivative) which is derived from pathogenic fungi T.benhamiae and has human Kv1.3 potassium ion channel inhibitory activity through artificial design and preparation. The peptide mimic disclosed by the invention has better activity of inhibiting human Kv1.3 potassium ion channels at a micromolar concentration level. The theoretical molecular weight of the human Kv1.3 type potassium ion channel activity inhibition peptide mimic is 1594.97, and the human Kv1.3 type potassium ion channel activity inhibition peptide mimic has the activity of inhibiting human Kv1.3 potassium ion channels.
Description
Technical Field
The invention belongs to the field of biological medicine, and particularly relates to a human Kv1.3 type potassium ion channel activity inhibition peptidomimetic, and a preparation method and application thereof.
Background
Potassium ion channels, an important class of membrane proteins, are widely present in cells of different species, including microorganisms, plants, and animals. Currently, in human, more than 80 genes encoding potassium ion channels have been found, distributed in various organs and tissues in the body, and regulating various physiological activities and functions of the body, including: the maintenance of resting potential, the excitation of cardiac and neural cells, the transmission of neurotransmitters, muscle contraction and the secretion of hormones. More importantly, potassium ion channels are related to a plurality of diseases such as immunoregulation, cardiovascular diseases, tumors and the like.
Among the potassium channel families, the voltage-gated potassium channels (Kv) are the most abundant one, and about 40 Kv channels exist in the human body, and can be divided into twelve subfamilies according to their sequence similarity and functional characteristics, as shown in fig. 7.
Among the many voltage-gated potassium channels, Kv1.3 is currently the most widely studied one of the channel proteins, which is abundant in the nervous system and immune system, and affects various physiological activities such as the body's sensitivity to insulin, cell proliferation and apoptosis.
The kv1.3 channels are distributed primarily in nerve cells and immune cells, including T cells, B cells, microglia, dendritic cells, and macrophages. When potassium ion channels on the surface of T cells are studied, it is found thatEMThe expression level of Kv1.3 channel is greatly changed during the activation process of cells, and the T is self-reactiveEMCells are an important etiological agent of autoimmune diseases, and thus the relationship between Kv1.3 channels and the occurrence of various physiological diseases is revealed, includingAutoimmune diseases such as psoriasis, and chronic diseases such as allergic contact dermatitis, ulcerative colitis and asthma. The relationship between Kv1.3 channel and cancer is a popular field studied in recent years, and Kv1.3 expression level is found to be significantly increased in breast cancer biopsy compared with normal tissue, and researchers also find that the Kv1.3 channel expression level is changed in the occurrence of prostate cancer, renal cancer, bladder cancer and lung cancer, and the change is different from breast cancer, and the Kv1.3 channel expression level shows a trend of decreasing in the process of the occurrence of related cancer.
Kv1.3 is also distributed in non-excitable cell fat cells and plays an important role in the metabolism of organisms, and Kv1.3 knockout mice show an unexpected phenotype, do not have obvious immunodeficiency, and show the phenomena of enhanced sensitivity to smell and increased basal metabolic rate. The research finds that in an obesity mouse model with deletion of melanocortin-4 gene, the knockout of Kv1.3 gene can reduce obesity symptoms and body weight of the mouse by increasing activity and overall metabolism. The important role of Kv1.3 in weight control also makes it an important target for the potential to treat obesity, type II diabetes and related metabolic diseases.
Therefore, for the diseases related to the up-regulation of human Kv1.3, the detection of the diseases by using tool reagents and the development of inhibitors thereof for treating the diseases are particularly important. Therefore, the invention truncates the protein (NCBI ID: XP _003013237.1) secreted and expressed by trichophyton pathogenic fungi (Trichophyton benhamiae) and prepares the peptide mimic based on the short peptide (the amino acid sequence of the short peptide is KILQTLPSLFR). So as to obtain the peptide-like/natural polypeptide derivative with human Kv1.3 potassium ion channel inhibition activity and excellent property.
Disclosure of Invention
An object of the present invention is to provide a human Kv1.3 type potassium channel activity inhibitory peptidomimetic which has a good inhibitory activity against human Kv1.3 potassium channels at a micromolar concentration level.
The second purpose of the invention is to provide a preparation method of the human Kv1.3 type potassium ion channel activity inhibition peptide mimic, which has the advantages of convenient preparation and low production cost and is suitable for industrial large-scale production.
The invention also aims to provide application of the human Kv1.3 type potassium ion channel activity inhibition peptide mimic.
The scheme adopted by the invention for realizing one of the purposes is as follows: a human Kv1.3 type potassium ion channel activity inhibition peptide mimic, the molecular structure of which is:
preferably, the human Kv1.3 type potassium channel activity inhibiting peptide mimic is truncated from the protein NCBI ID: XP-003013237.1 secreted and expressed by Trichophyton pathogenic fungi, using a short peptide with the amino acid sequence KILQTLPSLFR as a template.
The second scheme adopted by the invention for achieving the purpose is as follows: the preparation method of the human Kv1.3 type potassium ion channel activity inhibition peptidomimetic comprises the steps of firstly preparing a peptide fragment KILQTLPSLFRC by a polypeptide solid phase synthesis method, then forming a covalent disulfide bond by a sulfydryl of a carbon-terminal cysteine side chain and a sulfydryl of a compound 3-sulfydryl-1-propanesulfonic acid sodium salt, and finally purifying to finally obtain the human Kv1.3 type potassium ion channel activity inhibition peptidomimetic.
Preferably, the method comprises the following steps: (1) peptide fragment KILQTLPSLFRC prepared by polypeptide solid phase synthesis method: adopting 9-fluorenylmethoxycarbonyl to protect amino end group, 4-methyl diphenylmethylamine resin as solid phase carrier, HOBt/DCC as condensing agent, extending peptide chain from carboxyl end to amino end, adopting mixed liquor of 95% trifluoroacetic acid, 3% water and 2% triisopropyl silane in mass percentage respectively to crack the mixture from MBHA resin, precipitating ethyl ether, purifying by RP-HPLC, and using C as product18Gradient elution is carried out on the reversed-phase preparation column to obtain an intermediate peptide segment KILQTLPSLFRC; the mobile phase adopted by the gradient elution is trifluoroacetic acid, water and acetonitrile, VTrifluoroacetic acid+VWater (W)+VAcetonitrile100 percent, the volume ratio of trifluoroacetic acid is 1.25 thousandth, the volume ratio of acetonitrile is 0 to 55 percent, the balance is water, and the elution flow rate is 0.5 mL/min;
(2) to the prepared intermediate peptide fragment KILQTLPSLFRC and a small molecular compound 3-mercapto-1-propanesulfonic acid sodium salt are taken as raw materials, the raw materials are dissolved in Tris-HCl buffer solution with the concentration of 0.1M and the pH value of 9.0 according to the mass ratio of 4:1, then the obtained solution is placed in a shaking table to be incubated for a certain time at the temperature of 26 +/-2 ℃ and 80rpm, then the supernatant is obtained by centrifugation, purified by RP-HPLC, and the C is used18Gradient elution is carried out on the reversed-phase preparation column to obtain the human Kv1.3 type potassium ion channel activity inhibition peptide mimic; the mobile phase adopted by the gradient elution is trifluoroacetic acid, water and acetonitrile, VTrifluoroacetic acid+VWater (W)+VAcetonitrile100 percent, the volume ratio of trifluoroacetic acid is 0.15 percent, the volume ratio of acetonitrile is 0 to 65 percent, the balance is water, and the elution flow rate is 0.5 mL/min.
The scheme adopted by the invention for realizing the third purpose is as follows: the human Kv1.3 type potassium ion channel activity inhibition peptidomimetic is applied to tool reagents or/and medicaments, and the action target of the human Kv1.3 type potassium ion channel activity inhibition peptidomimetic is a human Kv1.3 type potassium ion channel.
Preferably, the tool agent is a solution and/or a lyophilized powder; the medicine is tablet, injection, sterile powder for injection, powder, granule, capsule, oral liquid, ointment or cream; the medicine is introduced into muscle, endothelium, subcutaneous, vein or mucosa tissue by oral administration, injection, nasal drop, eye drop, physical or chemical mediated method, or is mixed or coated with other substances and then introduced into human body.
A composition comprising the human kv 1.3-type potassium channel activity-inhibiting peptidomimetic of claim 1 or 2 or the human kv 1.3-type potassium channel activity-inhibiting peptidomimetic prepared by the preparation method of claim 3 or 4.
Preferably, the human Kv1.3 type potassium ion channel activity inhibiting peptide mimetic further comprises at least one of pharmaceutically acceptable salts, hydrates, solvates and acceptable carriers.
The application of the composition in preparing a tool reagent and/or a medicament.
Preferably, the tool agent is a solution and/or a lyophilized powder; the medicine is tablet, injection, sterile powder for injection, powder, granule, capsule, oral liquid, ointment or cream; the medicine is introduced into muscle, endothelium, subcutaneous, vein or mucosa tissue by injection, oral administration, nasal drop, eye drop, physical or chemical mediated method, or is mixed or coated with other substances and then introduced into human body.
The peptide mimic related by the invention is derived from the protein (NCBI ID: XP _003013237.1) of human pathogenic fungus T.benhamiae, and the sequence is as follows:
the carbon end of the peptide segment KILQTLPSLFR (amino acid sequence in box) in the full-length protein is introduced with a cysteine (the carboxyl of arginine R and the amino of cysteine C form a peptide bond), namely the polypeptide sequence is KILQTLPSLFRCAnd then the introduced sulfhydryl of cysteine and sulfhydryl in the structure of the small molecular compound 3-sulfhydryl-1-propanesulfonic acid sodium salt form covalent disulfide bond connection, and the molecules with brand new structures are spliced.
The peptidomimetic molecule can be obtained by separating and purifying by Reverse phase-high performance liquid chromatography (RP-HPLC), has a theoretical molecular weight of 1594.97, and has activity of inhibiting human Kv1.3 potassium ion channels. The peptide mimic has the advantages of simple preparation method and technical route, strong stability, easy control of product quality, capability of meeting the requirements of mass preparation and large-scale industrial production and the like.
The invention has the following advantages and beneficial effects: the invention provides a peptide mimic (natural truncated polypeptide derivative) which is derived from pathogenic fungi T. benhamiae and has human Kv1.3 potassium ion channel inhibitory activity through artificial design and preparation. The peptide mimic disclosed by the invention has better activity of inhibiting human Kv1.3 potassium ion channels at a micromolar concentration level. The theoretical molecular weight of the human Kv1.3 type potassium ion channel activity inhibition peptide mimic is 1594.97, and the human Kv1.3 type potassium ion channel activity inhibition peptide mimic has the activity of inhibiting human Kv1.3 potassium ion channels.
The preparation method of the human Kv1.3 type potassium ion channel activity inhibition peptide mimic has the advantages of simple technical route, strong stability, easy control of product quality, capability of meeting the requirements of mass preparation and large-scale industrial production and the like.
The human Kv1.3 type potassium ion channel activity inhibition peptidomimetic has the human Kv1.3 type potassium ion channel inhibition effect, and shows low hemolytic activity and stability under high temperature, high salt and acid-base environments. Can be used as a molecular probe and provides a template molecule and a new choice for the research and development of new drugs.
Drawings
FIG. 1A is an HPLC chart of a human Kv1.3 type potassium channel activity inhibiting peptidomimetic prepared in example 1 of the present invention;
FIG. 1B is a mass spectrum of a human Kv1.3 type potassium channel activity inhibitory peptidomimetic prepared in example 1 of the present invention;
FIG. 2 is a graph showing the results of inhibition of human Kv1.3 type potassium channel current by the human Kv1.3 type potassium channel activity-inhibiting peptidomimetics of example 2 of the present invention;
FIG. 3 is a result of hemolytic activity of human Kv1.3 type potassium channel activity inhibitory peptide mimic of example 3 of the present invention;
FIG. 4 is the results of the activity of human Kv1.3 type potassium channel activity inhibiting peptidomimetics of example 4 of the present invention in salt environments of various concentrations;
FIG. 5 is a graph showing the results of the activity of the human Kv1.3 type potassium channel activity inhibiting peptidomimetics of example 5 of the present invention in various temperature environments;
FIG. 6 is the results of the activity of the human Kv1.3 type potassium channel activity inhibiting peptidomimetics of example 6 of the present invention in various pH environments;
FIG. 7 is a schematic representation of a subfamily of voltage-gated potassium ion channels in the background of the invention.
Detailed Description
The following examples are provided to further illustrate the present invention for better understanding, but the present invention is not limited to the following examples.
EXAMPLE 1 preparation of human Kv1.3 type Potassium channel Activity inhibitory Peptidomimetics
(1) Synthesis of intermediate peptide fragment KILQTLPSLFRC: protecting an amino end group by using 9-Fluorenylmethoxycarbonyl (FMOC), selecting 4-methyl-benzhydrylamine resin (4-methyl-benzhydrylamine resin HCl, MBHA resin) as a solid phase carrier, selecting HOBt/DCC as a condensing agent, and extending a peptide chain from a carboxyl end to an amino end; cracking the mixture from MBHA resin by using a mixed solution of 95% trifluoroacetic acid, 3% water and 2% triisopropyl silane in percentage by mass, repeatedly precipitating diethyl ether for many times, and purifying by using preparative RP-HPLC; use of C18Gradient elution is carried out on a reversed-phase preparation column (20mm multiplied by 250mm, 5 mu m) to obtain an intermediate peptide segment KILQTLPSLFRC, and the intermediate peptide segment KILQTLPSLFRC is freeze-dried for later use; the mobile phase adopted by the gradient elution is trifluoroacetic acid, water and acetonitrile, VTrifluoroacetic acid+VWater (W)+VAcetonitrile100 percent, the volume ratio of trifluoroacetic acid is 1.25 thousandth, the volume ratio of acetonitrile is 0 to 55 percent, the balance is water, and the elution flow rate is 0.5 mL/min.
(2) The prepared intermediate peptide segment KILQTLPSLFRC and the small molecular compound 3-mercapto-1-propanesulfonic acid sodium salt are taken as raw materials. Dissolving 1mg of intermediate peptide fragment and 0.25mg of the small molecular compound by using 1.5mL of Tris-HCl buffer solution with the concentration of 0.1M (the pH value is 9.0), and accelerating the dissolution of the intermediate peptide fragment and the small molecular compound by using a liquid transfer gun to gently blow and suck the solution; the EP tube containing the polypeptide solution was incubated in a constant temperature shaker at 26. + -. 2 ℃ and 80rpm for about 36 h. Centrifuging at room temperature 10000 rmp for 4min, collecting supernatant, and purifying by preparative RP-HPLC; use of C18Gradient elution is carried out by reversed phase preparative column (20mm is multiplied by 250mm, 5 μm); the mobile phase adopted by the gradient elution is trifluoroacetic acid, water and acetonitrile, VTrifluoroacetic acid+VWater (W)+VAcetonitrile100 percent, trifluoroacetic acid with the volume ratio of 0.15 percent, acetonitrile with the volume ratio of 0 to 65 percent and the balance of water, and the elution flow rate of 0.5mL/min, and preparing the human Kv1.3 type potassium ion channel activity inhibition peptide mimetic. RT of 11.41min and its purity by RP-HPLC detection>95% (FIG. 1A), and is lyophilized for use. Identified by ESI-QTOF-MS,the molecular weight of the peptide is consistent with the corresponding theoretical molecular weight (1594.97-23 ═ 1571.97) of the prepared peptide mimic, and M/z 1572.0[ M-Na [, N-Na ]]-(FIG. 1B).
Example 2 measurement of inhibitory Activity of human Kv1.3 type Potassium ion channel
Transfection experiments were performed in 24-well cell culture plates using TurboFect Transfection Reagent, with the steps: adding 95 mu L of Opti-MEM into a sterilized 1.5mL EP tube, then adding 1 mu g of ultrapure potassium channel plasmid and 0.5 mu g of ultrapure EGFP-N1 plasmid, uniformly mixing by using a pipette gun, and standing at room temperature for 3 min; adding 2 μ L transfection reagent, mixing well with pipette, standing at room temperature for 15 min; dropping the mixed solution into cells cultured by a 24-pore plate, slowly shaking to uniformly mix the mixed solution, and then putting the mixed solution into an incubator for culturing; after transfection for 5 hours, cells in a 24-well plate are digested and transferred to a 6-well plate containing a glass slide, and after culturing for 24 hours, an electrophysiological experiment can be performed, the peptide mimic prepared in example 1 is prepared into a solution with the concentration of 1 mu M, the peptide mimic is slowly and uniformly added to the periphery of a single cell to be detected through a drug delivery system matched with a patch clamp instrument, and the current of a human Kv1.3 potassium ion channel before and after drug delivery is measured.
The electrode used for the electrophysiological experiment is drawn by an electrode drawing instrument PC-100 manufactured by NARISHIGE company, and then polished by a polisher, so that the tip of the electrode is smoother, and the water inlet resistance of the polished electrode is 2-4M omega. The potassium channel electrophysiological data were collected using EPC 10 amplifier and Patchmaster software manufactured by HEKA, Germany, and the inhibitory rate of the peptidomimetic on human Kv1.3 potassium channel current at a concentration of 1. mu.M was about 64.4%, as shown in FIG. 2.
EXAMPLE 3 determination of hemolytic Activity of human Kv1.3 type Potassium channel Activity inhibitory peptidomimetics
Fresh blood was obtained from a healthy donor, human red blood cells were separated and suspended, and the hemolytic activity of the human Kv1.3 type potassium channel activity inhibitory peptidomimetic prepared in example 1 was measured using sterilized physiological saline as a negative control and 1% Triton X-100 as a positive control.
The method comprises the following specific steps: collecting whole blood with EDTA anticoagulation tube, slightly inverting to fully anticoagulate blood, centrifuging at room temperature 500rpm for 10min, discarding upper layer plasma and retaining lower layer red blood cells; adding 4 times volume of sterilized normal saline, slightly inverting to make the centrifuge tube suspend the bottom red blood cells, centrifuging at room temperature at 500rpm for 10min, discarding the supernatant, retaining the precipitated red blood cells, repeating the operation for 3 times until the supernatant is colorless; preparing erythrocyte into 2% (v/v) cell suspension by using sterilized normal saline; dissolving the human Kv1.3 type potassium channel activity inhibiting peptidomimetic with sterilized physiological saline to prepare solutions with concentrations of 512. mu.g/mL, 256. mu.g/mL, 128. mu.g/mL, 64. mu.g/mL, 32. mu.g/mL, 16. mu.g/mL, 8. mu.g/mL, 4. mu.g/mL and 2. mu.g/mL; mixing 100 μ L of the human Kv1.3 type potassium channel activity-inhibiting peptidomimetic solution and 100 μ L of the red blood cell suspension and adding to a 96-well plate, the final concentrations of the human Kv1.3 type potassium channel activity-inhibiting peptidomimetic being 256 μ g/mL, 128 μ g/mL, 64 μ g/mL, 32 μ g/mL, 16 μ g/mL, 8 μ g/mL, 4 μ g/mL, 2 μ g/mL and 1 μ g/mL, respectively; the negative control group is red blood cells suspended by sterilized physiological salt, and the positive control group is red blood cells suspended by sterilized physiological salt containing 1% TritonX-100; putting the sample into a constant temperature shaking table, and incubating for 60min at 37 ℃ and 85 rpm; the samples were centrifuged at 2500 rpm for 8min at room temperature, and 100. mu.L of supernatant per well was transferred to another 96 well plate; the absorbance at 490nm was measured with a full wavelength microplate reader and the percentage of hemolysis was calculated by the following formula, where H is the absorbance at 490 nm:
hemolysis rate (%) - (H)sample-Hnegative)/(Hpositive-Hnegative)×100％。
As shown in FIG. 3, the inhibitory peptide mimics of human Kv1.3 potassium channel activity according to the present invention showed no significant hemolysis (hemolysis < 6%) at final concentrations up to 256. mu.g/mL.
Example 4 Activity of human Kv1.3 type Potassium ion channel inhibits the Activity of peptidomimetics in salt environments of varying concentrations
Human Kv1.3 type potassium channel activity-inhibiting peptidomimetics prepared in example 1 were dissolved in sterilized NaCl solutions at concentrations of 0mM, 150mM, 200mM, 300mM, 400mM, and 500mM, respectively, to prepare human Kv1.3 type potassium channel activity-inhibiting peptidomimetics solutions at a concentration of 0.5. mu.M. The inhibitory activity of the human Kv1.3 type potassium channel activity inhibitory peptide mimics on the human Kv1.3 channel was determined in the environment of different salt concentrations according to the method of example 2, and the relative activity of the human Kv1.3 type potassium channel activity inhibitory peptide mimics of the present invention in the environment of different salt concentrations is shown in FIG. 4, in comparison with the inhibitory rate in example 2.
The data in FIG. 4 show that human Kv1.3 inhibitory activity of the human Kv1.3 type potassium channel activity inhibitory peptidomimetics of the present invention gradually decreased with increasing salt concentration, but similar inhibitory activity (relative activity > 95%) was observed at salt concentrations below 200 mM. And the salt concentration under physiological conditions is 150mM, which shows that the human Kv1.3 type potassium channel activity of the invention inhibits the activity of the peptidomimetic stably under the condition of the physiological salt concentration. The relative activity of the human Kv1.3 type potassium channel activity of the present invention to inhibit peptidomimetics is still > 50% even at salt concentrations as high as 500 mM.
Example 5 Activity of human Kv1.3 type Potassium ion channel inhibits the Activity of peptidomimetics in various temperature environments
The human Kv1.3 type potassium channel activity inhibitory peptide mimics prepared in example 1 were dissolved in sterile PBS to prepare human Kv1.3 type potassium channel activity inhibitory peptide mimics at a concentration of 0.5. mu.M, and incubated at constant temperature conditions of 25 ℃, 30 ℃, 35 ℃, 40 ℃, 45 ℃, 50 ℃ and 55 ℃ for 2 hours, respectively. The activity of inhibiting human Kv1.3 channel after incubation under different temperature environments was measured according to the method in example 2, and the relative activity of the human Kv1.3 type potassium channel activity inhibiting peptide mimics of the present invention under different temperature environments is shown in FIG. 5, in comparison with the results (inhibition rate) in example 2.
The data in figure 5 show that the inhibitory activity of the human Kv1.3 type potassium channel activity inhibiting peptidomimetics decreases with increasing temperature, with 51% relative activity after incubation in an environment at temperatures up to 55 ℃. It is worth noting that the activity of the human Kv1.3 type potassium ion channel activity inhibition peptide mimic of the invention for inhibiting the human Kv1.3 channel activity is not obviously reduced (the relative inhibition activity is more than or equal to 95 percent) within the range of 25-35 ℃, so the human Kv1.3 type potassium ion channel activity inhibition peptide mimic of the invention has better stability at room temperature and physiological environment temperature.
Example 6 Activity of human Kv1.3 type Potassium ion channels inhibits the Activity of peptidomimetics in different pH environments
The human Kv1.3 type potassium ion channel activity inhibition peptidomimetic prepared in example 1 is adjusted in pH value by HCl or NaOH to prepare sterile PBS solutions with pH values of 1, 3, 5, 7, 9, 11 and 13 respectively, and the human Kv1.3 type potassium ion channel activity inhibition peptidomimetic is dissolved by the sterile PBS solutions with different pH values respectively to prepare a human Kv1.3 type potassium ion channel activity inhibition peptidomimetic solution with the concentration of 0.5 mu M. Changes in inhibitory activity against human Kv1.3 channel after incubation in different pH environments were measured according to the method of example 2, and the results of the inhibitory rate of example 2 are used as a control, and the relative activity of the human Kv1.3 type potassium channel activity inhibitory peptide mimics after incubation in different pH environments for 2 hours is shown in FIG. 6.
The data in fig. 6 show that at pH7, the relative activity of the human kv 1.3-type potassium ion channel activity-inhibiting peptidomimetics in inhibiting human kv1.3 channels is almost indistinguishable from that in example 2 (control). In the physiological pH range +/-2 (the physiological pH is about 7.4, namely the pH value is in the range of 5-9), the relative activity of the human Kv1.3 channel activity inhibition peptide mimic for inhibiting the human Kv1.3 channel is more than 85 percent. The relative activity of the peptidomimetic decreases as the acidity and basicity continue to increase, but the basic environment (-20% relative activity at pH 13) has a greater effect on the inhibition of the relative activity of the peptidomimetic by human kv1.3 type potassium channel activity than does the acidic environment (-68% relative activity at pH 1).
EXAMPLE 7 tablets
0.3g of the human Kv1.3 type potassium ion channel activity inhibition peptidomimetic prepared in example 1, 5g of starch and 5g of dextrin are mixed, medicinal grade polyvinylpyrrolidone (PVP) with the mass concentration of 30% is used as a binding agent, and the mixture is granulated and tableted to obtain the tablet.
EXAMPLE 8 Freeze-dried powder for injection
2g of the human Kv1.3 type potassium ion channel activity inhibiting peptide mimic and 30g of mannitol, which are prepared in example 1, are taken and placed in a container, a proper amount of PBS buffer solution (0.1M, pH7.4) is added for dissolution, water for injection is added to 600mL, shaking is carried out evenly, 10-15 g of needle activated carbon is added, stirring is carried out for 30-60 minutes at room temperature, rough filtration is carried out, filtration sterilization is carried out by using a 0.22 mu M filter membrane, subpackaging is carried out, 1mL is carried out in each bottle, a quick freezing method is adopted, the temperature is reduced by 8-10 ℃ per minute, the temperature is reduced to-45 ℃, the maintenance is carried out for 2.5 hours, vacuum pumping is carried out, the temperature is slowly increased under a vacuum state, the temperature increasing speed is 4-8 ℃ per hour, the temperature is stopped increasing to 30 ℃, the.
While the foregoing is directed to the preferred embodiment of the present invention, other and further embodiments of the invention may be devised without departing from the basic scope thereof, and the scope thereof is determined by the claims that follow.
Claims (10)
1. A human Kv1.3 type potassium ion channel activity inhibiting peptide mimic, characterized in that its molecular structure is:
2. the human Kv1.3 type potassium channel activity inhibiting peptidomimetic according to claim 1, wherein: the human Kv1.3 type potassium ion channel activity inhibition peptide mimic is truncated from a protein NCBIID XP-003013237.1 secreted and expressed by trichophyton pathogenic fungi, and a short peptide with an amino acid sequence of KILQTLPSLFR is used as a template.
3. A method for producing a human kv 1.3-type potassium channel activity-inhibiting peptidomimetic according to claim 1 or 2, comprising: firstly, preparing a peptide segment KILQTLPSLFRC by a polypeptide solid phase synthesis method, then forming a covalent disulfide bond by a sulfydryl of a carbon-terminal cysteine side chain and a sulfydryl of a compound 3-sulfydryl-1-propanesulfonic acid sodium salt, and finally purifying to finally obtain the human Kv1.3 type potassium ion channel activity inhibition peptidomimetic.
4. Human Kv1.3 type according to claim 3The preparation method of the potassium ion channel activity inhibition peptide mimic is characterized by comprising the following steps: (1) peptide fragment KILQTLPSLFRC prepared by polypeptide solid phase synthesis method: adopting 9-fluorenylmethoxycarbonyl to protect amino end group, 4-methyl diphenylmethylamine resin as solid phase carrier, HOBt/DCC as condensing agent, extending peptide chain from carboxyl end to amino end, adopting mixed liquor of 95% trifluoroacetic acid, 3% water and 2% triisopropyl silane in mass percentage respectively to crack the mixture from MBHA resin, precipitating ethyl ether, purifying by RP-HPLC, and using C as product18Gradient elution is carried out on the reversed-phase preparation column to obtain an intermediate peptide segment KILQTLPSLFRC; the mobile phase adopted by the gradient elution is trifluoroacetic acid, water and acetonitrile, VTrifluoroacetic acid+VWater (W)+VAcetonitrile100 percent, the volume ratio of trifluoroacetic acid is 1.25 thousandth, the volume ratio of acetonitrile is 0 to 55 percent, the balance is water, and the elution flow rate is 0.5 mL/min;
(2) dissolving prepared intermediate peptide KILQTLPSLFRC and micromolecular compound 3-mercapto-1-propanesulfonic acid sodium salt as raw materials in a Tris-HCl buffer solution with the concentration of 0.1M and the pH value of 9.0 according to the mass ratio of 4:1, then placing the obtained solution in a shaking table to incubate for a certain time at the temperature of 26 +/-2 ℃ and at the speed of 80rpm, then centrifuging to take supernatant, purifying by RP-HPLC, and using C18Gradient elution is carried out on the reversed-phase preparation column to obtain the human Kv1.3 type potassium ion channel activity inhibition peptide mimic; the mobile phase adopted by the gradient elution is trifluoroacetic acid, water and acetonitrile, VTrifluoroacetic acid+VWater (W)+VAcetonitrile100 percent, the volume ratio of trifluoroacetic acid is 0.15 percent, the volume ratio of acetonitrile is 0 to 65 percent, the balance is water, and the elution flow rate is 0.5 mL/min.
5. Use of the human Kv1.3 type potassium channel activity-inhibiting peptidomimetics according to claim 1 or 2 or the human Kv1.3 type potassium channel activity-inhibiting peptidomimetics prepared by the preparation method according to claim 3 or 4, characterized in that: the human Kv1.3 type potassium ion channel activity inhibition peptidomimetic is applied to a tool reagent or/and a medicament, and the action target of the human Kv1.3 type potassium ion channel activity inhibition peptidomimetic is a human Kv1.3 type potassium ion channel.
6. Use of a human Kv1.3 type potassium channel activity inhibiting peptidomimetic according to claim 5 wherein: the tool reagent is solution and/or freeze-dried powder; the medicine is tablet, injection, sterile powder for injection, powder, granule, capsule, oral liquid, ointment or cream; the medicine is introduced into muscle, endothelium, subcutaneous, vein or mucosa tissue by oral administration, injection, nasal drop, eye drop, physical or chemical mediated method, or is mixed or coated with other substances and then introduced into human body.
7. A composition characterized by: comprising the human Kv1.3 type potassium channel activity-inhibiting peptidomimetic according to claim 1 or 2 or the human Kv1.3 type potassium channel activity-inhibiting peptidomimetic prepared by the preparation method according to claim 3 or 4.
8. The composition of claim 7, wherein: also comprises at least one of pharmaceutically acceptable salts, hydrates, solvates and acceptable carriers of the human Kv1.3 type potassium ion channel activity inhibition peptide mimics.
9. Use of a composition according to claim 7 or 8, wherein: the composition is applied to the preparation of a tool reagent and/or a medicament.
10. Use of a composition according to claim 9, characterized in that: the tool reagent is solution and/or freeze-dried powder; the medicine is tablet, injection, sterile powder for injection, powder, granule, capsule, oral liquid, ointment or cream; the medicine is introduced into muscle, endothelium, subcutaneous, vein or mucosa tissue by injection, oral administration, nasal drop, eye drop, physical or chemical mediated method, or is mixed or coated with other substances and then introduced into human body.
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