CN114106134A - Polypeptide and application thereof in analgesia - Google Patents

Abstract

The invention provides a polypeptide and an application thereof in analgesia, wherein the polypeptide has the amino acid sequence shown in SEQ ID NO: 1, wherein the amino acids at positions 50 and 55 are connected by a C-S bond. The polypeptide of the invention has good stability in vivo, and has better inhibition effect on ASIC1a channel compared with natural polypeptide Mambalgin-1, thereby better exerting analgesic effect, and having high scientific research value and clinical application value.

Description

Polypeptide and application thereof in analgesia

Technical Field

The present invention relates to the field of medicine. In particular, the invention relates to polypeptides and their use in analgesia.

Background

The acid sensitive ion channel is mammalian cell sensitive extracellular H⁺The ion channels of concentration play an important role in other nerve activities such as pain sensation, touch sensation, taste sensation and the like. Acid-sensitive ion channel subtype 1a (abbreviated as ASIC1a) is widely distributed in the central and peripheral nervous systems and plays an unappreciable role in pain perception in particular. In recent years, it has been found that the amabalagin-1 polypeptide from Heimanban venom can play a good analgesic role by inhibiting ASIC1 in central and peripheral nervous systems, and intrathecal injection of the mambalagin-1 can reduce pain response by inhibiting acid sensitive ion channels containing ASIC1a subunits. However, although all natural toxin polypeptides have high activity and specificity, most of the natural polypeptides have the defects of easy degradation and short half-life, and have great defectsLimiting its clinical application.

Therefore, Mambalgin-1 is still under study.

Disclosure of Invention

The present invention aims to solve at least to some extent at least one of the technical problems of the prior art. Therefore, the invention provides the polypeptide, the medicine, the application of the polypeptide in preparing the medicine and the method for improving the stability of the natural polypeptide, the polypeptide has good stability in vivo, and has better inhibition effect on an ASIC1a channel compared with the natural polypeptide Mambalgin-1, thereby better exerting the analgesic effect, and the scientific research value and the clinical application value are high.

In one aspect of the invention, the invention features a polypeptide. According to an embodiment of the invention, the polypeptide has the amino acid sequence of SEQ ID NO: 1, wherein the amino acids at positions 50 and 55 are connected by a C-S bond.

LKCYQHGKVVTCHRDMKFCYHNTGMPFRNLKLILQGCSSSCSETENNKCCSTDRCNK(SEQ ID NO：1)

The inventor researches modification modes such as mutation, cyclization and side chain modification on the natural polypeptide Mambalgin-1 to maintain or enhance the structural stability. Research shows that some modification methods cannot achieve the purpose of improving stability, and some modification methods can improve stability but affect the effect of inhibiting the ASIC1a channel, so how to improve stability and inhibit effect simultaneously becomes the technical bottleneck of the modification process. On the basis, intensive research shows that the amino acids at the 50 th site and the 55 th site of the natural polypeptide Mambalgin-1 (the amino acid sequence is shown as SEQ ID NO: 1) are connected through an S-S bond, and the S-S bond is easily oxidized in vivo, so that the structure of the polypeptide is changed, the function of the polypeptide is damaged, the polypeptide is easily degraded, and the half life period is short. Furthermore, the inventor replaces the S-S bond with the C-S bond, finds that the structure of the polypeptide in vivo is not easy to change, the stability is obviously improved, and compared with the natural polypeptide, the polypeptide with the C-S bond has a better inhibition effect on an ASIC1a channel, so that the analgesic effect is better exerted, and the scientific research value and the clinical application value are high.

According to an embodiment of the invention, the above-mentioned polypeptide may also have the following additional technical features:

according to an embodiment of the invention, the amino acids at positions 3 and 19 are linked by an S-S bond, the amino acids at positions 12 and 36 are linked by an S-S bond, and the amino acids at positions 41 and 49 are linked by an S-S bond. These amino acid sequences are identical to those of the natural polypeptide Mambalgin-1, and the inventors have found that it is only necessary to replace the S-S bonds at positions 50 and 55 with C-S bonds without replacing all the S-S bonds in the natural polypeptide with C-S bonds, whereby not only the stability of the polypeptide but also the inhibitory effect thereof can be improved. When S-S bonds at other positions are substituted, the inhibitory effect of the polypeptide is adversely affected.

In yet another aspect of the invention, a medicament is provided. According to an embodiment of the invention, the medicament comprises: the aforementioned polypeptide. As mentioned above, the polypeptide has good stability in vivo, and has better inhibition effect on ASIC1a channel compared with the natural polypeptide Mambalgin-1, thereby better exerting analgesic effect.

According to an embodiment of the invention, the medicament further comprises: pharmaceutically acceptable adjuvants. Therefore, the use effect of the medicine can be further improved by adding the auxiliary materials.

According to an embodiment of the invention, the dosage form of the drug is oral or injectable. This makes it possible to exert an analgesic effect more effectively.

In a further aspect of the invention, the invention provides the use of a polypeptide as hereinbefore described in the manufacture of a medicament. According to an embodiment of the invention, the medicament is for analgesia. The polypeptide in the medicine has good stability in vivo, and can fully exert analgesic effect in vivo.

According to an embodiment of the invention, the drug inhibits acid sensitive ion channels subtype 1a of the central and peripheral nervous system. The polypeptide in the medicine has strong stability in vivo, can better act on the acid sensitive ion channel subtype 1a of the central nervous system and the peripheral nervous system, and plays a role in inhibiting, thereby playing a role in easing pain.

In yet another aspect of the invention, the invention features a method of increasing the stability of a native polypeptide having the amino acid sequence of SEQ ID NO: 1, wherein the amino acids at positions 3 and 19 are connected by an S-S bond, the amino acids at positions 12 and 36 are connected by an S-S bond, the amino acids at positions 41 and 49 are connected by an S-S bond, and the amino acids at positions 50 and 55 are connected by an S-S bond. According to an embodiment of the invention, the method comprises: replacing S-S bonds of amino acids at positions 50 and 55 in the amino acid sequence with C-S bonds. The research finds that the amino acids at the 50 th position and the 55 th position of the natural polypeptide Mambalgin-1 (the amino acid sequence is shown as SEQ ID NO: 1) are connected through an S-S bond, and the S-S bond is easily oxidized in vivo, so that the structure of the polypeptide is changed, the function of the polypeptide is damaged, the polypeptide is easily degraded, and the half life period is short. Furthermore, the inventor replaces the S-S bond with the C-S bond, finds that the structure of the polypeptide in vivo is not easy to change, the stability is obviously improved, and compared with the natural polypeptide, the polypeptide with the C-S bond has a better inhibition effect on an ASIC1a channel, so that the analgesic effect is better exerted, and the scientific research value and the clinical application value are high.

Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.

Drawings

The above and/or additional aspects and advantages of the present invention will become apparent and readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings of which:

FIG. 1 shows an analytical chromatogram after renaturation of a native polypeptide Mambalgin-1 according to one embodiment of the invention;

FIG. 2 shows a diagram of mass spectrometric identification of a native polypeptide after mambragin-1 renaturation according to one embodiment of the present invention;

figure 3 shows a graph of drug concentration effect according to one embodiment of the present invention.

Detailed Description

The scheme of the invention will be explained with reference to the examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples, where specific techniques or conditions are not indicated, are to be construed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.

Example 1

Firstly, synthesizing a natural polypeptide Mambalgin-1 analogue:

1. solid phase synthesis of manbarbus venom fragment Ma-1-40-NHNH₂And Ma-41-57-NH₂

a. Microwave-assisted solid-phase synthesis of polypeptide:

adding 0.25mmol of corresponding Ma-1-40-NHNH₂Fmoc-hydrazine resin and Ma-41-57-NH₂And the rink amide AM resin is respectively placed in a microwave polypeptide synthesizer of Liberty Blue to carry out solid-phase polypeptide synthesis based on an Fmoc method. DMF was used to dissolve the amino acids and condensation reagents (DIC and Oxyma) and to wash the resin. Fmoc-Gly (Dmb) -OH (Dmb:2, 4-dihydroxyphenyl) was introduced at glycine positions 7 and 36 to aid solid phase synthesis. All amino acids and condensing agents were used in 4 times the amount of resin. The coupling condition of arginine, cysteine and histidine is 10min at room temperature, the reaction is 5min at 50 ℃, and the coupling condition of other amino acids is 75 ℃ for 10 min. For Ma-1-40-NHNH₂The coupling efficiency was ensured by carrying out the coupling reaction twice for all amino acids after 30 amino acids. Removal of the Fmoc protecting group was performed using 20% piperidine/DMF solution (0.1M Oxyma addition to prevent racemization during deprotection). For Ma-41-57-NH₂The diamino diacid is introduced at position 55 (structure below).

After the coupling is completed, the reaction is continued in a microwave synthesizer until the coupling is completed to the 51 th amino acid, and the Fmoc protecting group of the 51 st amino acid is reserved. The method comprises the steps of removing Alloc and allyl protection groups of a mixture (molar ratio is 1:5) of tetrakis (triphenylphosphine) palladium and phenyl silane serving as a removal reagent, removing Fmoc protection groups of the mixture by using a DMF solution containing 20% piperidine as the removal reagent, and performing condensation cyclization reaction on a 50 th diamino diacid building block and a 51 th amino acid by using a PyAOP/DIEA cyclization condensation system for 4 hours, so that the original 50-55 th disulfide bond is replaced, and finally, the replacement is continuously coupled to the 41 th amino acid.

b. Preparation of crude peptide:

after the reaction of the microwave synthesizer is finished, taking out the corresponding Ma-1-40-NHNH₂And Ma-41-57-NH₂The resin was placed in a polypeptide synthesis tube. The resin was washed 5 times with DMF and 5 times with DCM and the resin was pumped dry using an air pump. 10mL of a solution containing trifluoroacetic acid/water/thioanisole/ethanedithiol 85: 5: 5: 3(v/v/v, 0.55g phenol) cleavage reagent was added to the resin and reacted for 4 h. The polypeptide is cleaved from the resin and all side chain amino acid protecting groups are removed. The filtrate was filtered, and the filtrate was concentrated by nitrogen bubbling, followed by precipitation of the polypeptide in the concentrated filtrate with glacial ethyl ether, centrifugation at 4000rpm and decanting of the supernatant. And repeatedly washing with ice ether, centrifuging twice, and airing the polypeptide to obtain a crude peptide product.

2. Semi-preparative liquid phase separation and lyophilization

Reverse phase-high performance liquid chromatography (RP-HPLC) semipreparative separations were performed using LC-20At from Shimadzu. Mobile phase a was 0.1% TFA in acetonitrile and mobile phase B was 0.1% TFA in pure water. Ma-1-40-NHNH₂The half-preparative separations of (1) selected a gradient of 20% -50% -30min (acetonitrile/water) with a flow rate of 4 mL. Ma-41-57-NH₂The semi-preparative separation selection gradient of (1) was 10% -60% -30min (acetonitrile/water) with a flow rate of 4 mL. The single crude peptide sample was 25 mg. According to the condition of an ultraviolet absorption peak at 214nm, an EP tube is used for collecting the elution solution at the corresponding position, and LC-MS 2020 of Shimadzu is used for mass spectrum identification. And pre-freezing the collected solution with the correct molecular weight by using liquid nitrogen, and freeze-drying the solution in a freeze dryer to obtain the freeze-dried pure peptide of the corresponding fragment.

3. Chemical ligation by hydrazide method

56mg of 1.2eq equivalent of Ma-1-40-NHNH₂Dissolved in 6M guanidine hydrochloride solution (6M guanidine hydrochloride, 0.2M phosphate, pH 3) and cooled to about-10 ℃ in a chilled salt bath. Under magneton stirring, 7eq NaNO were added₂After 30min, the C-terminal hydrazide is oxidized to azide. The polypeptide is then removed from the ice salt bath and placed inAt room temperature, 40eq of 4-mercaptophenylacetic acid was added, the pH was adjusted to about 5, and the azide was converted to thioester. Then 19.5mg of 1-fold equivalent Ma-41-57-NH is added₂And (4) carrying out natural chemical ligation reaction by adjusting the pH value to about 6.8. The course of the reaction was monitored and characterized by analytical RP-HPLC and electrospray mass spectrometry. After completion of the reaction, TCEP (tris (2-carboxyethyl) phosphine) was added in a total amount of 0.1M to reduce the disulfide bonds in the system, and finally separated using semi-preparative chromatography and lyophilized.

The amino acid sequence of the obtained natural polypeptide analogue is shown as SEQ ID NO: 1, wherein the amino acids at positions 3 and 19 are linked by an S-S bond, the amino acids at positions 12 and 36 are linked by an S-S bond, the amino acids at positions 41 and 49 are linked by an S-S bond, and the amino acids at positions 50 and 55 are linked by a C-S bond.

LKCYQHGKVVTCHRDMKFCYHNTGMPFRNLKLILQGCSSSCSETENNKCCSTDRCNK(SEQ ID NO：1)

4. Natural polypeptide Mambalgin-1 renaturation

The lyophilized full-length native polypeptide, mambalagin-1, was dissolved in purified water containing 1mM glutathione (reduced form) and 0.1mM glutathione (oxidized form) to a final concentration of 0.01 mM. Adjusting the pH value to 7.8-8.0. The mixture was placed in a shaker at room temperature and shaken slowly at 60 rpm. Renaturation process monitoring was performed using analytical RP-HPLC and electrospray. After the renaturation was completed, the pH was adjusted to 2.0-3.0 using TFA. After most of water in the renatured solution is removed by using a freeze dryer, semi-preparative separation is carried out by using a gradient of 5-50-30 min (acetonitrile/water), and finally, the renatured and folded natural polypeptide Mambalgin-1 is obtained after freeze drying. The analysis chromatogram and mass spectrum identification chart of the natural polypeptide Mambalgin-1 after renaturation refer to the figure 1 and the figure 2.

The amino acid sequence of the natural polypeptide analogue is shown as SEQ ID NO: 1, wherein the amino acids at positions 3 and 19 are linked by an S-S bond, the amino acids at positions 12 and 36 are linked by an S-S bond, the amino acids at positions 41 and 49 are linked by an S-S bond, and the amino acids at positions 50 and 55 are linked by an S-S bond.

Second, electrophysiological experiments

The function experiment of the acid sensitive ion channel is mainly realized by the patch clamp electrophysiological technology, and the common extracellular fluid used in the experimentThe formula comprises 150mM NaCl, 4mM KCl and 2mM CaCl₂、1mM MgCl₂10mM HEPES, pH7.4 with NaOH solution, and osmolality around 310mOsm with water or sucrose. The electrode solution formula is 140mM KCl, 10mM NaCl, 5mM EGTA, 10mM HEPES, 1mM MgCl₂The pH is adjusted to 7.3 with KOH solution, and the osmolality is usually around 295 mOsm. When preparing extracellular fluid with different pH, the pH value can be adjusted to different pH values by NaOH solution, but it should be noted that when the pH is lower than 6, the buffering agent HEPES should be changed into MES, and then the pH value is adjusted to the desired pH value by NaOH. Mambalgin-1 solutions of different concentrations and pH were prepared with the corresponding extracellular fluid. Each solution was filtered through a 0.22mm filter and used.

In the experimental operation, the electrode was first drawn using a P-1000 horizontal electrode drawing machine from SUTTER and a borosilicate glass electrode with an inner core, with an outer diameter of 1.5mm and an inner diameter of 0.86 mm. Before the electrodes are formally drawn, a proper electrode drawing program needs to be set, and a 4-step drawing method is adopted. Firstly, after an electrode is installed, a program is started to find out safe heating temperature, generally the temperature capable of pulling a glass electrode without breaking the electrode under certain tension, then a proper electrode drawing program is found out by adjusting the temperature below the safe temperature, the resistance of the single-cell patch clamp experimental glass electrode is generally controlled to be 2-5M omega, the higher the temperature is, the thinner the tip of the drawn electrode is, the larger the resistance is, and otherwise, the lower the temperature is, the smaller the resistance is, and generally the temperature adjustment is changed by multiples of 5. After the program is set, an electrode is arranged in the glass tube clamp on one side (the middle part of the electrode is not touched), the elastic sheets of the glass tube clamps fixed at two ends are loosened after the glass tube clamps are fixed slightly, the two glass tube clamps are clamped in the middle, the electrode is slowly pushed to the middle after the platinum sheet is not touched, the two sides are screwed, and the program is executed by clicking pull. After the electrode is broken, the glass tube clamp is slightly unscrewed, the tip end of the electrode is obliquely and downwards placed into the electrode box, the electrode tip end does not need to be touched, and the cleaning of the tip end of the electrode needs to be paid attention to in the whole process. And taking an electrode, pouring the electrode liquid from the tail part, and only submerging the tip of the electrode, so that the electrode flicks to ensure that no air bubbles exist. And unscrewing the electrode holder, screwing the electrode, observing whether the silver wire touches the internal liquid or not, resetting the electrode holder, and screwing. Taking the cells after 24h of transfection, taking out the cell slide, firstly rinsing the cell slide with physiological external liquid, then transferring the cell slide into another dish filled with the extracellular liquid, placing the dish on a microscope objective table for fixing, connecting a ground wire, erecting an administration tube, moving the administration tube to a proper position, and preferably viewing the clear cells and the height of the tip of the administration tube under a 10-fold microscope at the same time. Before the electrode enters the liquid, positive pressure is firstly applied to the tip of the electrode, so that the electrode is prevented from being blocked, and the subsequent sealing is facilitated. Then finding the electrode tip under the microscope, adjusting to the cell focal plane, placing the electrode above the cell, and adjusting the micromanipulation to slowly lower the electrode. When the resistance changes by 0.1 MOmega, the tee joint is opened, and sealing is carried out by giving light, slow and continuous negative pressure, and the sealing is successful when the resistance reaches GO. Then gradually adjusting the voltage to the clamping voltage to compensate the fast capacitor. And then, applying short and powerful negative pressure to suck and break the cells, taking the slow capacitance appearing at the base line as the standard, compensating the slow capacitance and recording the slow capacitance and the series resistance, wherein the series resistance is below 20M omega. At this point, the recording experiment can begin.

The patch clamp amplifier used in this experiment was EPC10(HEKA corporation) and its data acquisition software PatchMaster. When recording the current of the acid-sensitive ion channel ASICs, the voltage is clamped at the resting potential of the cell of-70 mV, the protocol records 20s each time, the sampling frequency is 2KHz, 4s of acid stimulation is given once to induce current generation during recording, the acid stimulation is generated by quickly replacing the extracellular fluid with pH of 7.4 to the extracellular fluid with certain pH through a Y tube, and the extracellular fluid with the pH of 7.4 is quickly replaced after 4 s. The interval is 2min before the next acid stimulation is given, since the acid sensitive ion channel requires time to fully return from the desensitized state to the closed state. Record H⁺The pH current value when the current is maximum is required to be recorded once every time when the concentration effect curve is used for normalizing data, and other pH current records can be stimulated randomly without a fixed sequence. The pharmaceutical effect of Mambalgin-1 adopts a pre-administration mode, and the literature reports that Mambalgin-1 can possibly combine with an ion channel in a closed state, and a good inhibition effect can be achieved only by pre-administration, so that the administration mode of pre-administration for 30s is selected. According to the pH concentration response curve of hASIC1a WT, the channel is almost completely opened at pH 6.0, so that the pH is selected for drug inhibition test. The administration is repeated several timesAcid stimulation at pH 6.0 gave a relatively stable control current value, which was then rapidly transferred to pH 6.0 for 4s after 30s of extracellular fluid containing a certain concentration of Mambalgin-1 at pH7.4 to give a current value after drug action, which was then likewise rapidly exchanged back to normal extracellular fluid at pH 7.4. The recording of drug concentration effect curve should be from low concentration to high concentration in case of accumulation effect affecting the accuracy of experimental result. The series resistance change of the cells should be kept within 10% in the whole recording process, and the experimental data exceeding 10% cannot be used for statistical analysis.

Referring to FIG. 3, it can be seen that the natural polypeptide Mambalgin-1 analog (VAD002-1) has better activity than the natural polypeptide (VAD-WT), so that ASIC1a channel can be better inhibited to exert analgesic effect.

In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above are not necessarily intended to refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples and features of different embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction.

Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.

SEQUENCE LISTING

<110> Suzhou Star continent Biotechnology Ltd

<120> polypeptide and use thereof in analgesia

<130> PIDC3204575

<160> 1

<170> PatentIn version 3.3

<210> 1

<211> 57

<212> PRT

<213> Artificial

<220>

<223> 1

<220>

<221> MISC_FEATURE

<222> (3)..(19)

<223> amino acids at positions 3 and 19 are linked by an S-S bond

<220>

<221> MISC_FEATURE

<222> (12)..(36)

<223> amino acids at positions 12 and 36 are linked by an S-S bond

<220>

<221> MISC_FEATURE

<222> (41)..(49)

<223> amino acids at positions 41 and 49 are linked by an S-S bond

<220>

<221> MISC_FEATURE

<222> (50)..(55)

<223> amino acids at positions 50 and 55 are linked by a C-S bond

<400> 1

Leu Lys Cys Tyr Gln His Gly Lys Val Val Thr Cys His Arg Asp Met

1 5 10 15

Lys Phe Cys Tyr His Asn Thr Gly Met Pro Phe Arg Asn Leu Lys Leu

20 25 30

Ile Leu Gln Gly Cys Ser Ser Ser Cys Ser Glu Thr Glu Asn Asn Lys

35 40 45

Cys Cys Ser Thr Asp Arg Cys Asn Lys

50 55

Claims (8)

1. A polypeptide having the sequence of SEQ ID NO: 1, wherein the amino acids at positions 50 and 55 are connected by a C-S bond.

2. The polypeptide of claim 1, wherein the amino acids at positions 3 and 19 are linked by an S-S bond, the amino acids at positions 12 and 36 are linked by an S-S bond, and the amino acids at positions 41 and 49 are linked by an S-S bond.

3. A medicament, comprising: the polypeptide of claim 1 or 2.

4. The medicament of claim 3, further comprising: pharmaceutically acceptable adjuvants.

5. The medicament of claim 3, wherein the medicament is in a form for oral administration or injection.

6. Use of the polypeptide of claim 1 in the manufacture of a medicament for the treatment of pain.

7. The use according to claim 1, wherein the medicament inhibits the acid sensitive ion channel subtype 1a of the central and peripheral nervous system.

8. A method of increasing the stability of a native polypeptide having the amino acid sequence of SEQ ID NO: 1, wherein the amino acids at positions 3 and 19 are linked by an S-S bond, the amino acids at positions 12 and 36 are linked by an S-S bond, the amino acids at positions 41 and 49 are linked by an S-S bond, and the amino acids at positions 50 and 55 are linked by an S-S bond, comprising: replacing S-S bonds of amino acids at positions 50 and 55 in the amino acid sequence with C-S bonds.

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