CN114874292A

CN114874292A - Multi-target chimeric peptide compound, preparation method and application thereof

Info

Publication number: CN114874292A
Application number: CN202210680593.5A
Authority: CN
Inventors: 陈罡; 赵龙; 马钰; 吴泳江; 朱圣泽
Original assignee: Nantong University
Current assignee: Nantong University
Priority date: 2022-06-16
Filing date: 2022-06-16
Publication date: 2022-08-09

Abstract

The invention belongs to the technical field of biological medical treatment, and discloses a multi-target chimeric peptide compound which can act on an opioid receptor and a PD-1 receptor simultaneously, wherein the amino acid sequence of the multi-target chimeric peptide is as follows: Tyr-Pro-Xaa 3-Phe-Ile-Ser-Tyr-Gly-Gly-Ala-Asp-Tyr-Lys-R; wherein Xaa3 is Trp with side chain being indole ring or Phe with benzene ring; r is-NH 2 group or-OH group. The invention also discloses a preparation method of the multi-target compound of the opium and PD-1 receptor, which comprises the following steps: (1) by utilizing a solid-phase synthesis method, the first lysine at the carboxyl terminal of a peptide chain is coupled on resin in sequence, and (2) the target peptide is obtained by cracking a lysate. The invention provides a new thought and theoretical basis for the later work of designing a novel opioid/PD-1 receptor multi-target chimeric peptide.

Description

Multi-target chimeric peptide compound, preparation method and application thereof

Technical Field

The invention belongs to the technical field of biological medicines, and relates to multi-target chimeric peptide compounds of opioid/PD-1 receptors, a preparation method of the compounds and application of the compounds in preparation of analgesic drugs.

Background

Pain affects the major global health problem for the world population. Opioids such as morphine, oxycodone and fentanyl are the main drugs for the treatment of moderate-severe pain. However, taking opioid drugs usually causes adverse reactions such as constipation, respiratory depression, sedation, nausea and the like, reduces the medication experience of patients, and has negative effects on the lives of patients. Extended administration of opioid analgesics also results in analgesic tolerance, which means that patients require higher doses of the analgesic to maintain analgesic efficacy, which makes opioid drugs a significant challenge in the area of treating chronic pain (ann. lnn. med, 2015, 162, 276-. In addition, overdose and increased mortality due to opioid abuse and addiction are also current public health concerns that are not negligible.

In order to overcome the above-mentioned drawbacks of opioid drugs and the crisis they face, a trend has arisen in the past decades towards the development of analgesic drugs targeting non-opioid receptors such as neurotensin, neuropeptides FF, cannabinoids, melanocortins and substance P analogues. In recent years, research on multi-target drugs shows that the multi-target drugs can act on multiple targets of a disease network, and the drug effect of the drugs can be improved and the side effect can be reduced through the synergistic effect among the targets. Coupling of non-opioid pharmacophores to opioid pharmacophores, which are involved in pain modulation/signaling, is one of the mainstream directions in current multi-target drug design.

In 1997, the Zadina project group isolated an endogenous neurotransmitter endomorphin from the frontal cortex of cattle and the cerebral cortex of humans (EMs, Nature, 1997, 386, 499-. EMs are EM-1(Tyr-Pro-Trp-Phe-NH2) and EM-2(Tyr-Pro-Trp-Phe-NH2), respectively, which exhibit extremely high affinity and selectivity for mu-opioid receptors. After central administration, EMs showed analgesic activity comparable to morphine, and had lower side effects than morphine.

The inventor previously found that an artificially synthesized PD-1 targeting peptide TP-01(Ile-Ser-Tyr-Gly-Gly-Ala-Asp-Tyr-Lys-OH, patent application number: 202110451639.1) can remarkably relieve acute pain and chronic pain by activating a downstream SHP-1 pathway, and in later researches, the inventor also found that TP-01 cannot generate analgesic tolerance after long-term administration and has a certain positive effect on negative emotions (anxiety and depression) and sleep of mice. Therefore, the inventors conducted further research based on the previous studies.

Disclosure of Invention

The purpose of the invention is as follows: aiming at the problems or the defects in the prior art, the invention provides a multi-target chimeric peptide, a preparation method of a series of compounds and application thereof in preparing analgesic drugs, so as to solve the problems in the background art.

In order to achieve the above object, the embodiments of the present invention provide a multi-target chimeric peptide compound, wherein the multi-target chimeric peptide compound can act on an opioid receptor and a PD-1 receptor simultaneously, and the amino acid sequences of the multi-target chimeric peptides are as follows:

Tyr-Pro-Xaa3-Phe-Ile-Ser-Tyr-Gly-Gly-Ala-Asp-Tyr-Lys-R

wherein Xaa3 is Trp with side chain being indole ring or Phe with benzene ring;

r is-NH ₂ A group or an-OH group.

Further, the multi-target chimeric peptide compounds are compound 1, compound 2, compound 3 and compound 4, wherein the amino acid sequences of the compounds 1-4 are shown in SEQ ID NO: 1-4, SEQ ID NO: 1-4 are respectively:

SEQ ID NO：1：Tyr-Pro-Trp-Phe-Ile-Ser-Tyr-Gly-Gly-Ala-Asp-Tyr-Lys-OH；

SEQ ID NO：2：Tyr-Pro-Trp-Phe-Ile-Ser-Tyr-Gly-Gly-Ala-Asp-Tyr-Lys-NH ₂ ；

SEQ ID NO：3：Tyr-Pro-Phe-Phe-Ile-Ser-Tyr-Gly-Gly-Ala-Asp-Tyr-Lys-OH；

SEQ ID NO：4：Tyr-Pro-Trp-Phe-Ile-Ser-Tyr-Gly-Gly-Ala-Asp-Tyr-Lys-NH ₂ 。

the embodiment of the invention also provides a preparation method of the multi-target chimeric peptide compound, which is characterized by comprising the following steps of: (1) sequentially coupling the first lysine at the carboxyl terminal of a peptide chain on resin by using a solid-phase synthesis method; (2) obtaining the target peptide by cracking the lysate.

Further, the step (1) specifically includes the following steps:

(1-1) resin pretreatment: adding a calculated amount of corresponding resin into a synthesizer, adding anhydrous dichloromethane for swelling for 30 minutes, draining, adding N, N-dimethylformamide for washing for 3 times, wherein the indene detection resin is colorless, and the solution is light yellow; adding 20% hexahydropyridine N, N-dimethylformamide solution to elute the amino protecting group 9-fluorenylmethyloxycarbonyl on the resin for 3 times and 5 minutes/time; subsequently, N-dimethylformamide was washed 3 times, indene checked, resin blue, solution dark blue;

(1-2) amino acid condensation: adding 3 times of Fmoc-AA-OH, 6 times of N, N-diisopropylethylamine, 3 times of 1-hydroxybenzotriazole and O-benzotriazol-tetramethylurea hexafluorophosphate into a synthesizer, and reacting for 1h under the protection of argon; adding N, N-dimethylformamide to wash for 3 times, wherein the indene detection resin is colorless, and the solution is light yellow; adding 20% N, N-dimethylformamide solution of hexahydropyridine to elute Fmoc-group on the peptide resin for 3 times and 5 min/time; subsequently, N-dimethylformamide was washed 3 times, the resin was indenylated, the solution was dark blue;

(1-3) peptide chain extension: repeating the operation of step (1-2) starting from the amino acid at the C-terminal end of the polypeptide sequence to extend the polypeptide chain.

Further, the step (2) specifically includes the following steps:

(2-1) carrying out swelling-compressing operation on the peptide resin obtained in the step (1-3) by using anhydrous dichloromethane/anhydrous methanol for 3 times, and draining;

(2-2) cleavage of peptide resin: using the cleavage reagent trifluoroacetic acid triisopropylsilane: separating the crude peptide from the resin by double distilled water (95: 2.5: 2.5), extracting, and freeze-drying to obtain solid powder of the crude peptide;

(2-3) polypeptide purification: and (3) purifying the crude peptide obtained in the step (2-2) by using a reversed phase high performance liquid chromatography column (RP-HPLC preparative column) to obtain the opioid/PD-1 receptor multi-target chimeric peptide, and then performing purity analysis by using mass spectrometry and an HPLC analytical column.

The embodiment of the invention also provides application of the multi-target chimeric peptide compound in preparation of analgesic drugs.

Further, the drug includes one of compound 1, compound 2, compound 3, and compound 4.

Furthermore, the therapeutic targets of the drug are opioid receptors and PD-1 receptors.

Further, the analgesic drug is used for treating acute pain and inflammatory pain.

Further, the medicament adopts any one mode of intrathecal administration, subcutaneous administration, abdominal administration and oral administration.

The technical scheme of the invention has the following beneficial effects:

(1) the compounds disclosed herein are capable of acting on opioid and PD-1 receptors; centrally administered, the compounds have significant inhibitory effects on acute pain; in addition, the compound also shows a strong and lasting analgesic effect in inflammatory pain, has an effective analgesic time of 3 hours, and has the potential of clinical application. The compound structure disclosed by the invention provides a new thought and theoretical basis for later work of designing a novel opioid/PD-1 receptor multi-target chimeric peptide.

(2) The invention relates to a novel multi-target analgesic peptide structure obtained by embedding an opioid receptor pharmacophore EMs and a non-opioid receptor pharmacophore TP-01, which has the advantages of being capable of combining opioid receptors and activating PD-1 receptors simultaneously, and aims to improve the pharmacological activity and the possibility of drug formation of the analgesic peptide.

Drawings

FIG. 1 ESI-MS spectra of Compound 1 in an example of the invention;

FIG. 2 ESI-MS spectra of Compound 2 in an example of the invention;

FIG. 3 ESI-MS spectra of Compound 3 in an example of the invention;

FIG. 4 ESI-MS spectrum of Compound 4 in an example of the invention;

FIG. 5 is a graph of the analgesic effect of intrathecal injection of 20nmol of compounds 1-4 on capsaicin-induced acute pain in an example of the invention;

FIG. 6 is a graph of the analgesic effect of intrathecal injection of various doses of Compound 1 on capsaicin-induced acute pain in an embodiment of the invention;

FIG. 7 is a graph of the analgesic effect of intrathecal injection of various doses of Compound 2 on capsaicin-induced acute pain in an embodiment of the invention;

FIG. 8 is a graph of the effect of intrathecal injection of Compound 1 on carrageenan-induced inflammatory pain in accordance with embodiments of the invention;

figure 9 is a graph of the effect of intrathecal injection of compound 2 on carrageenan-induced inflammatory pain in accordance with embodiments of the invention.

Detailed Description

In order to make the technical problems, technical solutions and advantages to be solved by the present invention clearer, the following detailed description is given with reference to specific embodiments.

Example 1, solid phase synthesis of compound 1:

(1) swelling of the resin: weighing 500mg of wang-resin in a synthesizer, adding a proper amount of anhydrous dichloromethane for swelling for 30 minutes, draining, adding N, N-dimethylformamide for washing for three times, wherein the resin is colorless, and the solution is light yellow. Subsequently, the Fmoc-protecting group on the resin was eluted 3 times for 5 minutes each time by adding a suitable amount of a mixture of 20% piperidine in N, N-dimethylformamide. After elution, a proper amount of N, N-dimethylformamide was added for washing, and the indene resin was blue and the solution was blue after 3 washes.

(2) Condensed amino acids: 3 times of Fmoc-Lys (Boc) -OH, 3 times of 1-hydroxybenzotriazole and 3 times of O-benzotriazol-tetramethylurea hexafluorophosphate are sequentially added into a synthesizer, and finally 6 times of N, N-diisopropylethylamine is added for reaction for 1 hour under the protection of argon. After condensation, adding a proper amount of N, N-dimethylformamide to wash for 3 times, wherein the indene detection resin is colorless, and the solution is light yellow. The Fmoc-group on the peptide resin was eluted 3 times for 5 minutes each time by adding 20% piperidine in N, N-dimethylformamide. Subsequently, the indene resin was washed 3 times with N, N-dimethylformamide and the solution was dark blue.

(3) Peptide chain extension: condensing Fmoc-Tyr (tBu) -OH, Fmoc-Asp (OtBu) -OH, Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Tyr (tBu) -OH, Fmoc-Ser (tBu) -OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Trp (tBu) -OH, Fmoc-Pro-OH, and Tyr (tBu) -OH on the peptide resin in sequence according to the method of step (2). Boc-Tyr (tBu) -Pro-Trp (tBu) -Phe-Ile-Ser (tBu) -Tyr (tBu) -Gly-Gly-Ala-Asp (OtBu) -Tyr (tBu) -Lys (Boc) -Resin was obtained.

(4) Peptide resin cleavage and purification: and (3) adding a proper amount of anhydrous dichloromethane into the peptide resin obtained in the step (3) for swelling, compressing with anhydrous methanol, and repeating the swelling and compressing process for three times. After vacuum pumping, adding a proper amount of cutting reagent trifluoroacetic acid: the reaction was carried out at room temperature for 3 hours with 95:2.5:2.5 double distilled water to cleave the polypeptide from the resin. After freezing and drying, crude peptide is obtained and purified by using a reversed phase high performance liquid chromatography column (RP-HPLC preparative column) to obtain a compound 1: H-Tyr-Pro-Trp-Phe-Ile-Ser-Tyr-Gly-Gly-Ala-Asp-Tyr-Lys-OH, purification yield: 25%, and the results of mass spectrometry and chromatography are shown in Table 3. The mass spectrum is shown in figure 1.

Example 2 solid phase synthesis of compound 2:

(1) swelling of the resin: weighing 500mg of MBHA-resin in a synthesizer, adding a proper amount of anhydrous dichloromethane for swelling for 30 minutes, draining, adding N, N-dimethylformamide for washing for three times, wherein the resin is colorless, and the solution is light yellow. Subsequently, the Fmoc-protecting group on the resin was eluted 3 times for 5 minutes each time by adding a suitable amount of a mixture of 20% piperidine in N, N-dimethylformamide. After the elution is finished, a proper amount of N, N-dimethylformamide is added for washing, the washing is carried out for 3 times, the indene detection resin is blue, and the solution is blue.

(2) Condensed amino acids: the same procedure as in step (2) of the synthesis of Compound 1 in example 1;

(3) peptide chain extension: the same procedure as in step (3) of the synthesis of Compound 1 in example 1 was carried out;

(4) peptide resin cleavage and purification: crude peptide obtained from step (4) in the synthesis of compound 1 was purified by RP-HPLC preparative column to give compound 2: H-Tyr-Pro-Trp-Phe-Ile-Ser-Tyr-Gly-Gly-Ala-Asp-Tyr-Lys-NH2, purification yield: 29%, and the results of mass spectrometry and chromatography are shown in Table 3. The mass spectrum is shown in figure 2.

Example 3, solid phase synthesis of compound 3:

(1) swelling of the resin: the procedure was as in step (1) of the synthesis of Compound 1 in example 1;

(2) condensed amino acid: the same procedure as in step (2) of the synthesis of Compound 1 in example 1;

(3) peptide chain extension: condensing Fmoc-Tyr (tBu) -OH, Fmoc-Asp (OtBu) -OH, Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Tyr (tBu) -OH, Fmoc-Ser (tBu) -OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Phe-OH, Fmoc-Pro-OH and Boc-Tyr (tBu) -OH on the peptide resin in sequence according to the method of step (2). Boc-Tyr (tBu) -Pro-Phe-Phe-Ile-Ser (tBu) -Tyr (tBu) -Gly-Gly-Ala-Asp (OtBu) -Tyr (tBu) -Lys (Boc) -Resin was obtained.

(4) Peptide resin cleavage and purification: crude peptide obtained from step (4) in the synthesis of compound 1 was purified by RP-HPLC preparative column to give compound 3: Tyr-Pro-Phe-Phe-Ile-Ser-Tyr-Gly-Gly-Ala-Asp-Tyr-Lys-OH, purification yield: 35% and the results of mass spectrometry and chromatography are shown in Table 3. The mass spectrum is shown in figure 3.

Example 4, solid phase synthesis of compound 4:

(1) swelling of the resin: the step (1) in the process of synthesizing the compound 2;

(2) condensed amino acids: the step (2) in the process of synthesizing the compound 1;

(3) peptide chain extension: the step (3) in the process of synthesizing the compound 3;

(4) peptide resin cleavage and purification: crude peptide obtained from step (4) in the synthesis of compound 1 was purified by RP-HPLC preparative column to give compound 4: Tyr-Pro-Phe-Phe-Ile-Ser-Tyr-Gly-Gly-Ala-Asp-Tyr-Lys-NH2, purification yield: 34%, and the results of mass spectrometry and chromatography are shown in Table 3. The mass spectrum is shown in figure 4.

The amino acid abbreviations of the present invention are shown in table 1 below:

table 1 table for amino acid abbreviations in the present invention

Full scale	English abbreviation
		Tyrosine	Tyr
Proline	Pro
		Tryptophan	Trp
Phenylalanine	Phe
		Isoleucine	Ile
Serine	Ser
		Glycine	Gly
Alanine	Ala
		Aspartic acid	Asp
Lysine	Lys

The amino acid protecting groups in the present invention are abbreviated as shown in the following Table 2:

TABLE 2 abbreviated table of amino acid protecting groups in the present invention

Name (R)	Abbreviations
		9-fluorenylmethoxycarbonyl group	Fmoc
Tert-butyl radical	tBu
		Oxy tert-butyl	OtBu
Tert-butyloxycarbonyl radical	Boc

Physicochemical Properties of the Compounds of Table 3

EXAMPLE 5 evaluation of analgesic Activity of Compounds in capsaicin-induced acute pain model

ICR mice with the weight range of 25-30g are selected for experiments.

Mice were acclimated to the test environment 30 minutes earlier.

Saline/compound 1-4 was injected intrathecally. After 5 minutes, the left hind paw of the mouse was injected subcutaneously with capsaicin (1.6 ug/paw) and immediately placed in the observation room, and the cumulative time of painful behavior of the mouse within 5 minutes after capsaicin injection was recorded.

As shown in figures 5-7, the injection of capsaicin to the sole of the foot can cause the pain behaviors of the mouse in licking, biting and throwing the injected foot. However, the accumulation time of the pain behavior is obviously reduced after 20nmol of the compound 1-4 is injected in the intrathecal cavity, and the inhibition rate of the pain behavior is 48-60%. In addition, compounds 1 and 2 were able to dose-dependently inhibit capsaicin-induced acute pain behavior. At doses of 0.4, 4 and 20nmol, the inhibition rates of compound 1 were 24%, 32% and 58%, respectively; the inhibition rates of compound 2 were 35%, 42% and 60%, respectively. The results of the ability of compound 3 and compound 4 to dose-dependently inhibit capsaicin-induced acute pain behavior are similar to those of compound 1 and compound 2, and are not repeated herein.

EXAMPLE 6 evaluation of analgesic Activity of preferred Compounds 1 and 2 in Carrageenan-induced inflammatory pain

The invention selects 25-30g ICR mouse, and adapts to the mouse on the wire net rack for measuring mechanical pain for 2 hours each time.

And after the adaptation is finished on the third day, screening out mice with the mechanical pain threshold value within the range of 0.75-1.3 for modeling. The left hind paw of the mouse was injected underfoot with 20ul of 1% lambda-carrageenan. After 1 day, mice developed mechanorelgesia, different doses of compound or saline were injected intrathecally, and the mechanical pain threshold of mice was determined 30 minutes, 45 minutes, 1 hour, 1.5 hours, 2 hours and 3 hours at each time point after administration.

The experimental results are shown in fig. 8 and 9, and the mean value of the mechanical pain threshold of the mice is reduced from 1 to about 0.2 after injecting carrageenan subcutaneously on the soles for 1 day, which indicates that the mice generate hyperalgesia. After intrathecal injection of compound 1 or 2, the mechanical hyperalgesia of mice was significantly restored and this analgesic effect of the compound showed dose-dependence. The compound 1 can restore the mechanical pain threshold of the mice to the normal mouse level under the dosage of 20nmol or the compound 2 under the dosage of 40nmol, and the analgesic effect lasts for 3 hours.

The results in the assessment of analgesic activity of compounds 3 and 4 in carrageenan-induced inflammatory pain were similar to those of compounds 1 and 2 and are not repeated here.

In conclusion, the chimeric peptide can generate strong and durable analgesic effect through opium and PD-1 receptor. The chimeric peptide can obviously inhibit acute pain and inflammatory pain, and has potential application value in the aspect of preparing analgesic drugs.

While the foregoing is directed to the preferred embodiment of the present invention, it will be appreciated by those skilled in the art that various changes and modifications may be made therein without departing from the principles of the invention as set forth in the appended claims.

Claims

1. A multi-target compound chimeric peptide compound is characterized in that the multi-target compound chimeric peptide can act on an opioid receptor and a PD-1 receptor simultaneously, wherein the amino acid sequence of the multi-target compound chimeric peptide compound is as follows:

Tyr-Pro-Xaa3-Phe- Ile-Ser-Tyr-Gly-Gly-Ala-Asp-Tyr-Lys-R；

wherein Xaa3 is Trp with side chain being indole ring or Phe with benzene ring;

r is-NH ₂ A group or an-OH group.

2. The class of multi-target chimeric peptide compounds of claim 1, wherein the class of multi-target chimeric peptide compounds are compound 1, compound 2, compound 3, and compound 4, wherein the amino acid sequences of compounds 1-4 are as set forth in SEQ ID NO: 1-4, SEQ ID NO: 1-4 are respectively:

SEQ ID NO：1：Tyr-Pro-Trp-Phe- Ile-Ser-Tyr-Gly-Gly-Ala-Asp-Tyr-Lys-OH；

SEQ ID NO：2：Tyr-Pro-Trp-Phe- Ile-Ser-Tyr-Gly-Gly-Ala-Asp-Tyr-Lys-NH2；

SEQ ID NO：3：Tyr-Pro-Phe-Phe- Ile-Ser-Tyr-Gly-Gly-Ala-Asp-Tyr-Lys-OH；

SEQ ID NO：4：Tyr-Pro-Trp-Phe- Ile-Ser-Tyr-Gly-Gly-Ala-Asp-Tyr-Lys-NH2。

3. the method for preparing the multi-target chimeric peptide compound according to claim 1, which comprises the following steps: (1) sequentially coupling the first lysine at the carboxyl terminal of a peptide chain on resin by using a solid-phase synthesis method; (2) obtaining the target peptide by cracking the lysate.

4. The method for preparing a multi-target chimeric peptide compound of claim 3, wherein the step (1) comprises the following steps:

(1-1) resin pretreatment: adding a calculated amount of corresponding resin into a synthesizer, adding anhydrous dichloromethane for swelling for 30 minutes, draining, adding N, N-dimethylformamide for washing for 3 times, wherein the indene detection resin is colorless, and the solution is light yellow; adding 20% N, N-dimethylformamide solution of hexahydropyridine to elute the amino protecting group 9-fluorenylmethyloxycarbonyl on the resin for 3 times and 5 minutes/time; subsequently, N-dimethylformamide was washed 3 times, indene checked, resin blue, solution dark blue;

5. The method for preparing multi-target chimeric peptide compounds according to claim 4, wherein the step (2) comprises the following steps:

(2-2) cleavage of peptide resin: using the cleavage reagent trifluoroacetic acid triisopropylsilane: double distilled water =95:2.5:2.5, separating the crude peptide from the resin, extracting, and freeze-drying to obtain solid powder of the crude peptide;

6. The use of a class of multi-target chimeric peptide compounds according to claim 1 in the preparation of analgesic drugs.

7. The use of claim 6, wherein the medicament comprises one of compound 1, compound 2, compound 3, and compound 4.

8. The use according to claim 6, wherein the therapeutic targets of the drug are opioid and PD-1 receptors.

9. Use according to claim 6, wherein the analgesic medicament is for the treatment of acute pain and inflammatory pain.

10. The use of claim 6, wherein the medicament is administered intrathecally, subcutaneously, intraperitoneally, or orally.