CN111097037A

CN111097037A - PSD95-PDZ1/2 structural domain inhibitor and application thereof

Info

Publication number: CN111097037A
Application number: CN202010051511.1A
Authority: CN
Inventors: 刘成龙
Original assignee: Individual
Current assignee: Individual
Priority date: 2020-01-16
Filing date: 2020-01-16
Publication date: 2020-05-05

Abstract

The invention discloses a PSD95-PDZ1/2 structural domain inhibitor and application thereof, belonging to the technical field of biomedicine. The inhibitor is a small molecule peptide containing 9 amino acids, and the amino acid sequence of the small molecule peptide is from N terminal to C terminal: Lys-Leu-Ser-Ser-Ile-Glu-Ser-Asp-Val. The small molecule peptide is synthesized by a solid phase peptide synthesis technology based on fluorenylmethyloxycarbonyl acyl, and the myristic acid acylation is carried out at the nitrogen end to increase the membrane permeability of the small molecule peptide. The PSD95-PDZ1/2 domain inhibitor can be applied to the preparation of drugs for treating cancer pain. The invention treats pain by using the method of inhibiting the adaptor protein structural domain by the inhibitor, provides a theoretical basis for clinically preventing cancer pain, and provides a new idea and a new method.

Description

PSD95-PDZ1/2 structural domain inhibitor and application thereof

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to a PSD95-PDZ1/2 domain inhibitor and application thereof.

Background

Cancer pain seriously affects the life quality of cancer patients and causes huge economic burden to the society. According to the international association for pain research (IASP)2009 there are over 1000 million new patients with cancer worldwide each year. Cancer pain is the most common symptom in cancer patients, with pain associated with over 51% of cancer hospitalized patients and over 53% of cancer outpatients. Studies have shown that central sensitization of pain is mainly caused by excitatory amino acids and their receptors. Among them, ion channel type receptors play a major role. At present, the research on ion channel type receptors related to central sensitization of pain at home and abroad is mainly focused on NMDA receptors and AMPA receptors, and due to the lack of selective inhibitors on KA receptors (the KA receptors are one type of glutamate ion channel type receptors and consist of five subunits of GluR5, GluR6, GluR7, KA1 and KA2, each subunit contains four hydrophobic transmembrane domains of M1-4, one extracellular N-terminal and one intracellular C-terminal), the research on pathways of the NMDA receptors and the AMPA receptors is rarely carried out, but the drugs are difficult to be clinically used due to serious side effects such as memory impairment, hallucinogenic effect, ataxia and the like. Therefore, new targets for treating pain by targeting glutamate ion channel signaling pathways are sought.

Disclosure of Invention

The purpose of the invention is as follows: aiming at the problems in the prior art, the invention aims to provide a PSD95-PDZ1/2 structural domain inhibitor which is a small molecular peptide inhibitor, and compared with most common organic small molecular drugs, the peptide drug has the advantages of high activity, small dosage, low toxic and side effects, amino acid as a metabolic end product and the like. The invention also aims to provide application of the PSD95-PDZ1/2 domain inhibitor in preparation of a medicine for treating cancer pain, provide a theoretical basis for clinical preparation of the medicine for treating cancer pain, and provide a new idea and a new method.

The technical scheme is as follows: in order to solve the problems, the technical scheme adopted by the invention is as follows:

a PSD95-PDZ1/2 structural domain inhibitor is a small molecule peptide containing 9 amino acids.

The amino acid sequence of the PSD95-PDZ1/2 structural domain inhibitor is shown in SEQ ID NO. 1. The amino acid sequence of the polypeptide is from N terminal to C terminal: Lys-Leu-Ser-Ser-Ile-Glu-Ser-Asp-Val (K-L-S-S-I-E-S-D-V); wherein Lys (K) represents lysine, Leu (L) represents leucine, Ser (S) represents serine, Ile (I) represents isoleucine, Glu (E) represents glutamic acid, Asp (D) represents aspartic acid, and Val (V) represents valine.

The PSD95-PDZ1/2 domain inhibitor carries out myristic acid acylation on the nitrogen end of the small molecule peptide.

The PSD95-PDZ1/2 structural domain inhibitor is prepared by adopting a solid phase peptide synthesis technology based on fluorenylmethoxycarbonyl.

An application of PSD95-PDZ1/2 domain inhibitor in preparing medicine for treating cancer pain is disclosed.

The application of the PSD95-PDZ1/2 domain inhibitor in preparing a medicine for treating cancer pain is bone cancer pain.

Has the advantages that: compared with the prior art, the invention has the advantages that:

(1) the invention breaks through the angle of treating pain by various kinase or receptor inhibitors in the past, treats pain by using a method of inhibiting a linker protein structural domain by using a small peptide inhibitor, provides a theoretical basis for clinically preventing cancer pain, and provides a new thought and a new method.

(2) The small molecule peptide drug has clear structure and definite action mechanism; compared with most common organic small molecular drugs, the peptide drug has the outstanding characteristics of high activity, small dosage, low toxic and side effects, amino acid as a final metabolic product and the like; small molecule peptides are hardly immunogenic compared to proteins; can be chemically synthesized, and has high product purity and controllable quality.

Detailed Description

In order to make the aforementioned objects, features and advantages of the present invention comprehensible, embodiments accompanied with examples are described in detail below.

Example 1

1. Material

(1) Reagents and instruments: von Frey fiber wool (stockling corporation, usa); ME-410C type full automatic thermal radiometer (UGO BASILE 7370 plant Test Apparatus, Italy); NCTC2472 fibrosarcoma cells (batch No. 2087787, American model cell species Collection center).

Synthesizing small-molecule peptide Lys-Leu-Ser-Ser-Ile-Glu-Ser-Asp-Val, synthesizing the small-molecule peptide of 9 amino acids by using a solid phase peptide synthesis technology (standard Fmoc-based solid-phase peptide synthesis) based on fluorenylmethyloxycarbonyl acyl, and performing myristic acid acylation (Myristolation) at a nitrogen end to improve the membrane permeability of the small-molecule peptide. Wherein Lys (K) represents lysine, Leu (L) represents leucine, Ser (S) represents serine, Ile (I) represents isoleucine, Glu (E) represents glutamic acid, Asp (D) represents aspartic acid, and Val (V) represents valine.

(2) Animals and groups

SPF grade C3H/HeJ male mice, offered by beijing weitongli laboratory animal technology ltd, 4-6 weeks old, weighing 20-25g, 40 mice were randomly assigned to 5 groups using a random number table (n-8): sham group (group S), cancer pain model control group (group c), PSD95-PDZ1/2 domain inhibitor 2.5 μ g group (group K1), 5 μ g group (group K2) and 10 μ g group (group K3). And after the preparation of the intrathecal catheter and the model is finished, corresponding intervention is given, and corresponding indexes are observed.

2. Method of producing a composite material

(1) Cell culture: NCTC2472 fibrosarcoma cells (American Type Culture Collection, ATCC, 2087787) containing 5% CO at 37 ℃₂The culture box is cultured in 135 medium containing 10% horse serum, and the culture is passaged 1-2 times per week.

(2) Building a bone cancer pain model: after the mice are anesthetized, left knee osteotomy is performed, a puncture hole is drilled between the inner and outer ankles of the femur, NCTC2472 cells are inoculated into the marrow cavity at the far end of the femur, and bone wax seals the puncture hole.

(3) Administration: groups K1, K2, K3 were intrathecally injected with 2.5, 5, 10 μ g of inhibitor (dissolved in 20% DMSO, 5 μ L total volume), groups S and C were injected with the same volume of 20% DMSO solvent, and the drug was injected over 10S.

(4) And (3) measuring the blood of the pain behavior: the mechanical foot-shrinking reflection threshold and the heat-shrinking foot-emitting latency are measured in each group before and after the molding at 5, 7, 10 and 14 d. 1) Mechanical hyperalgesia assay: the mice were placed in separate plexiglas boxes with a metal mesh on the bottom. Mechanical thresholds were tested with a set of Von Frey fibers. 2) Thermal hyperalgesia assay: the mice are placed in mutually independent organic glass boxes with a bottom layer of a 3mm thick glass plate. The thermal pain threshold was tested with a thermal radiation stimulator.

(5) Statistical analysis: data analysis was performed using SPSS 16.0 statistical software. All data are expressed as mean soil standard deviation. The comparison between groups was analyzed by one-way anova, and the difference was considered statistically significant when P was less than 0.05.

3. Results

(1) Effect of mouse PWMT following intrathecal injection of PSD95-PDZ1/2 Domain inhibitor

As can be seen from Table 1, PWMT was significantly increased (P < 0.05) 14d after vaccination, 2, 6, 12h after intrathecal injection of KLSSIESDV, in the K2 and K3 groups compared to the pre-dose levels in the C and corresponding groups, and the level in the K3 group was higher than that in the K2 group at the same time point (P < 0.05). The K3 group had no statistical significance (P > 0.05) compared with the S group at time points 2 and 6h after administration, which reached the level of the grafting group, while the K1 group had no difference compared with the C group.

TABLE 1 Effect of mouse PWMT after intrathecal injection of KLSSIESDV

(2) Effect of intrathecal injection of PSD95-PDZ1/2 Domain inhibitor mouse PWTL

As can be seen from Table 2, PwTL levels were significantly increased (P < 0.05) 14d after vaccination, 2, 6, 12h after intrathecal KLSSIESDV injection, in the K2 and K3 groups compared to the pre-dose levels in the C and corresponding groups, and the K3 group was higher than the K2 group at the same time point (P < 0.05). The K3 group had no statistical difference (P > 0.05) from the S group at time points 2, 6 and 12h after administration, which reached the level of the sham operation group.

TABLE 2 Effect of mouse PWTL following intrathecal injection of inhibitors

Intrathecal administration of 5. mu.g, 10. mu.g of PSD95-PDZ1/2 domain inhibitor improves pain behavior in mice dose-dependently. During the generation and development of bone cancer pain, a presynaptic membrane depolarizes and releases excitatory neurotransmitters such as glutamic acid, a postsynaptic membrane receives an injurious signal and activates PSD95 and recruits GluR6 and nNOS, glutamic acid acts on GluR6 receptors on the postsynaptic membrane to cause calcium ion inflow, local high-solubility calcium ions and nNOS are beneficial to the activation of nNOS, a PSD95-PDZ1/2 structural domain is inhibited, and PSD95 cannot recruit GluR6 and nNOS, so that the pain transmission path is blocked to play an analgesic role.

Through the experimental research, the PSD95-PDZ1/2 domain small peptide inhibitor KLSSIESDV can be used for preparing small molecule peptide drugs for treating cancer pain, and the angle of pain treatment through various kinases or receptor inhibitors in the past is broken through.

Sequence listing

<110> Liu Cheng Long

<120> PSD95-PDZ1/2 structural domain inhibitor and application thereof

<130>0

<160>1

<170>SIPOSequenceListing 1.0

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<211>9

<212>PRT

<213> Artificial Synthesis (small peptide)

<400>1

Lys Leu Ser Ser Ile Glu Ser Asp Val

15

Claims

1. A PSD95-PDZ1/2 domain inhibitor, characterized in that it is a small molecule peptide.

2. The PSD95-PDZ1/2 domain inhibitor as claimed in claim 1, wherein the amino acid sequence of the small molecule peptide is represented by SEQ ID NO. 1.

3. The PSD95-PDZ1/2 domain inhibitor of claim 1, wherein a nitrogen terminus of the small molecule peptide is myristoylated.

4. The PSD95-PDZ1/2 domain inhibitor as claimed in claim 1, wherein the inhibitor is prepared by solid phase peptide synthesis technique based on fluorenylmethyloxycarbonyl acyl.

5. Use of a PSD95-PDZ1/2 domain inhibitor as defined in claim 1 in the manufacture of a medicament for the treatment of cancer pain.

6. The use of the PSD95-PDZ1/2 domain inhibitor for the manufacture of a medicament for the treatment of cancer pain, wherein said cancer pain is bone cancer pain.