Polypeptide with PresoCT domain as target and use thereof in preparing neuroprotective drugs

Abstract

The present invention relates to the field of biomedicine, and specifically to a polypeptide with a Preso CT domain as a target and its use in the preparation of a neuroprotective drug. The polypeptide has an amino acid sequence as shown below: PKIKETTV. The polypeptide has a good binding ability with the Preso CT domain and can effectively inhibit the apoptosis of primary neuronal cells in the mouse cortex. In addition, the polypeptide can effectively alleviate the anxiety-depressive behavior of chronic traumatic encephalopathy model mice after tail vein injection, proving that it has a neuroprotective effect. After determining that the Preso CT domain can be used as a target for neuroprotection, it can also be used to develop other neuroprotective drugs with the Preso CT domain as a target, which is of great value for medical research and drug development.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a polypeptide with a Preso CT domain as a target and use thereof in preparing a neuroprotective drug. Background Art

[0002] Traumatic brain injury (TBI) is an acquired injury to the brain caused by external mechanical force, which may cause temporary or permanent damage, with a high incidence and disability rate, and is the main cause of death in young and middle-aged people. Although a large number of basic and clinical studies have been carried out on TBI, the molecular pathological mechanism after TBI has not been fully elucidated, and there is a lack of effective clinical drug intervention measures. Chronic traumatic encephalopathy (CTE) is the most typical neurodegeneration after TBI, manifested as cognitive dysfunction such as dementia, which seriously affects the quality of life of the middle-aged and elderly population and brings great burden to society. At present, Tau protein pathology, as a hallmark pathological change of traumatic brain injury, remains unclear in its postsynaptic mechanism. Previous studies have confirmed that the postsynaptic scaffold protein PSD-95 forms a complex with Tau protein, which is the basis for Tau protein to localize and exert its effects in the postsynapse. Previous studies have shown that the postsynaptic scaffold protein Preso directly regulates the structure and function of the PSD-95-related complex after TBI, which is a key mechanism affecting neuronal outcome. However, there are currently no truly safe and effective neuroprotective drugs or measures. Exploring safe and effective anti-neural injury measures is an important issue currently facing the field of traumatic encephalopathy research.

[0003] Preso (FERM and PDZ domain-containing protein 4) is mainly expressed in the brain and contains WW (domain with two conserved Trp residues), PDZ (PSD-95 / Dlg / ZO-1), FERM (4.1, ezrin, radixin and moesin) and CT (C-terminal PDZ binding domain). It can participate in the regulation of the function of the postsynaptic scaffold protein complex and play a role in regulating synaptic function in neurological diseases such as neuropathic pain, schizophrenia, and mental retardation. Studies have shown that the CT binding domain at the carboxyl terminus of Preso can directly connect to the PDZ domain of PSD-95, thereby providing a platform for PSD-95 and its connecting molecules to play a role in regulating synaptic structure and function. Regulate excitotoxic damage of the NMDAR signaling pathway after TBI.

[0004] After craniocerebral injury, glutamate accumulates in large quantities, inducing overactivation of NMDA receptors, increasing Preso expression, and binding of Preso to PSD-95, triggering the formation of the Preso-PSD-95-Tau protein machine. This protein machine is formed by the interaction between the Preso CT domain and the PSD-95 PDZ domain, and through the interaction between the PSD-95 SH3 domain and Tau. The formation of this protein machine further positions the Tau protein in the postsynaptic region and close to the NMDAR, which is conducive to the effector molecules that bind to the Tau protein to participate in the regulation of NMDAR function, strengthen the regulatory effect of the postsynaptic Tau protein on excitotoxic injury, and ultimately cause neurodegeneration after CTE.

[0005] The above studies suggest that during craniocerebral injury, Preso recruits related molecules to form protein machines, which ultimately lead to cell death. Many compounds formed by protein machines during craniocerebral injury have been shown to have certain excitotoxic damage effects. In particular, the peptide NA-1 that destroys the interaction between NMDAR and PSD-95 has obvious neuroprotective effects and extremely strong drugability, and is considered to have opened the "spring" of neuroprotective agents. However, there are currently no truly safe and effective neuroprotective drugs or measures. As an important component of Preso, the CT domain is considered to be an important binding site for interacting with other proteins. However, its functions under physiological and pathological conditions are not yet clear. Summary of the invention

[0006] The present invention provides a polypeptide with a Preso CT domain as a target and use of the polypeptide in preparing a neuroprotective drug.

[0007] In a first aspect of the present invention, a polypeptide with the Preso CT domain as a target is provided, wherein the polypeptide has the following amino acid sequence: PKIKETTV.

[0008] In some embodiments, the polypeptide further comprises a cell-penetrating peptide, wherein the cell-penetrating peptide is a TAT tag, and the TAT tag can better penetrate the membrane, and the amino acid sequence of the cell-penetrating peptide is YGRKKRRQRRR.

[0009] In some embodiments, the polypeptide may be connected to a poly-His tag at the amino terminus for purification. The polypeptide targeting the Preso CT domain may be directly synthesized artificially, or its encoding gene may be synthesized first and then expressed biologically.

[0010] The second aspect of the present invention provides use of the polypeptide in preparing a neuroprotective drug.

[0011] In some embodiments, the present invention provides use of the polypeptide in preparing a drug for inhibiting the binding between PSD-95 protein and Tau protein.

[0012] In some embodiments, the present invention provides use of the polypeptide in preparing a drug for treating craniocerebral injury.

[0013] In some embodiments, the medicament is capable of reducing neuronal death following traumatic brain injury.

[0014] In some embodiments, the present invention provides use of the polypeptide in preparing a medicament for treating chronic traumatic encephalopathy.

[0015] In some embodiments, the medicament is used to alleviate anxiety-depression-like behaviors caused by chronic traumatic encephalopathy.

[0016] The third aspect of the present invention provides a neuroprotective drug, which uses the polypeptide as an active ingredient.

[0017] In some embodiments, the drug further comprises a pharmaceutically acceptable excipient, which is selected from one or more of a buffer, a solvent (such as water for injection, physiological saline), a solubilizer, a preservative, a stabilizer, a surfactant, and an antioxidant.

[0018] The present invention has the following beneficial effects:

[0019] The present invention discovered for the first time that the Preso CT domain is associated with excitotoxic injury in craniocerebral injury and can be used as a key target for neuroprotection. The polypeptide targeting the Preso CT domain can effectively inhibit apoptosis of primary cortical neurons in mice. In addition, tail vein injection of the polypeptide can effectively alleviate the anxiety-depressive behavior of CTE model mice, proving that it has a neuroprotective effect. The results of in vitro experiments showed that the Preso CT domain, as a key target, significantly inhibited the binding of PSD-95 and Tau protein. Therefore, the Preso CT domain is expected to be developed as an effective means to treat craniocerebral injury. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 These are the results of neuronal activity after TNI modeling and peptide administration under in vitro conditions.

[0021] Figure 2 The results and statistical analysis of the open field test of mice given the peptide. In the figure, A is the total distance statistics of OFT, B is the inner zone distance statistics, and C is the inner zone time statistics.

[0022] Figure 3 The results and statistical analysis of the elevated plus maze experiment of mice given the polypeptide. In the figure, A is the total distance statistics of EPM, B is the distance statistics of open arms, and C is the statistics of time of open arms.

[0023] Figure 4 Co IP experimental results of the effect of peptide on the interaction between Preso and PSD-95 on PSD-95 / Tau (A) and statistical analysis of the IP group results (B).

[0024] Figure 5 Co-IP experimental results of the effect of Preso CT domain mutants on the interaction between Preso and PSD-95 and PSD-95 / Tau (A) and statistical analysis of the IP group results (B). DETAILED DESCRIPTION

[0025] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments, but they should not be construed as limitations of the present invention.

[0026] In some embodiments, the term "neuroprotection" involved in the present invention may refer to measures such as restoring blood flow after craniocerebral injury, reducing infarct volume, preventing penumbra expansion, and saving reversibly damaged nerve cells, so as to restore or reshape nerve tissue function as much as possible. In some embodiments, the neuroprotection includes at least one of affecting the binding of PSD-95 to Tau, treating craniocerebral injury, and treating chronic traumatic encephalopathy, but is not limited thereto. In some embodiments, the affecting the binding of PSD-95 to Tau includes reducing the binding ability of PSD-95 to Tau, or reducing the binding ratio of PSD-95 to Tau.

[0027] In some embodiments, the polypeptide targeting the Preso CT domain has the following amino acid sequence: PKIKETTV. The polypeptide targeting the Preso CT domain can be connected to a poly-His tag at the amino terminus for purification. The polypeptide targeting the Preso CT domain can be directly synthesized artificially, or its encoding gene can be synthesized first and then obtained by biological expression. The encoding gene of the polypeptide targeting the Preso CT domain can also be connected to the coding sequence of the poly-His tag at its 5′ end.

[0028] In some embodiments, the drug of the present invention targeting the Preso CT domain, in particular the Preso CT domain inhibitor, comprises a polypeptide. In some embodiments, the sequence of the polypeptide comprises the protein sequence of the carboxyl terminal domain of Preso. In some embodiments, the sequence of the polypeptide comprises the protein sequence of the carboxyl terminal domain of Preso and a transmembrane peptide sequence. In some embodiments, the amino acid sequence of the polypeptide is as shown in SEQ ID NO.1, wherein positions 1-11 of SEQ ID NO.1 are transmembrane peptide sequences, and positions 12-19 of SEQ ID NO.1 are protein sequences of the carboxyl terminal domain of Preso.

[0029] In some embodiments, the Preso CT domain is associated with excitotoxic damage in craniocerebral injury and can be used as a target for neuroprotection for screening or developing neuroprotective drugs; in some embodiments, drugs targeting the Preso CT domain, particularly Preso CT domain inhibitors, can be used for neuroprotection against excitotoxic damage after craniocerebral injury and can be used to prepare neuroprotective products.

[0030] The following examples are only used to illustrate the present invention for better understanding, and are not to be considered as limiting the scope of the present invention. The reagents or instruments involved in the examples without indicating the manufacturer are all products that can be purchased from conventional biochemical reagent suppliers.

[0031] Example 1: Protective effect of peptides targeting the Preso CT domain on primary cortical neurons in TNI model mice

[0032] E15-17 C57BL / 6 pregnant mice were selected, and the fetal brains were dissected on ice, and the meninges were peeled off and the cortex was separated. After 5 minutes of 0.25% trypsin digestion, the cells were blown off and filtered through a 0.7μM cell strainer before being seeded in a 96-well plate. The seeding medium was a high-glucose DMEM medium containing 20% ​​fetal bovine serum and 1% double antibody, and the seeding density was 12.5×10 4 Pieces / cm 2 After culturing for 4-6 hours at 37°C and 5% CO2, the cells were replaced with a maintenance medium containing 2% GibcoTM B-27TM supplement, 1% glutamine, and 1% double antibody. After culturing for 4-5 days, half of the medium was replaced and the cells were cultured for 7-9 days before use.

[0033] 1.2 Peptides

[0034] The amino acid sequence of the polypeptide used in this example is shown in SEQ ID NO.1, where positions 1-11 of SEQ ID NO.1 are the cell-penetrating peptide sequence, and positions 12-19 of SEQ ID NO.1 are the protein sequence of the carboxyl terminal domain of Preso. The polypeptide was constructed by Shanghai GenScript Biotechnology Co., Ltd. with a purity greater than 98%. During the experiment, ultrapure water was used to dissolve the polypeptide lyophilized powder to prepare a mother solution with a concentration of 10 mM, and then diluted to the desired concentration using culture medium.

[0035] 1.3 Effects of peptides targeting the Preso CT domain on the viability of primary cortical neurons in mice after TNI modeling

[0036] The CCK8 method was used to observe the effect of polypeptides on the viability of primary cortical neurons in mice after TNI modeling. TNI modeling was performed after primary neurons were cultured to the 7th to 9th day: half of the medium was replaced within 24 hours before modeling; a 10μl micropipette tip was used to injure the cultured neurons according to the scratch line drawn in advance. The scratch path was 1mm wide, 4mm apart, and distributed in a grid-like manner. After the TNI modeling was completed, the solvent group and 400μg / ml polypeptide solution were added in the form of liquid replacement to treat the cells for 24 hours; then cell culture medium containing 10% CCK8 reagent was added to each well, and culture was continued for 1 hour at 37°C and 5% CO2. The absorbance of each well at 450nm was measured using a multifunctional microplate reader (Molecular Devices).

[0037] Figure 1 The results of neuronal activity after TNI modeling and peptide administration in vitro showed that the addition of peptide treatment reduced TNI-induced cell death to a certain extent, suggesting that the peptide has a neuroprotective effect.

[0038] Example 2: Peptides targeting the Preso CT domain can effectively reduce anxiety-like depressive behaviors in CTE model mice

[0039] In order to demonstrate the in vivo neuroprotective effect of the peptide, a mouse chronic traumatic encephalopathy (CTE) model was used. The beneficial effects of the peptide on anxiety and depression-like symptoms in CTE mice were detected to clarify the in vivo protective effect of the peptide.

[0040] 2.1 Experimental animals and groups

[0041] In this example, SPF male C57BL / 6 mice weighing 20-24 g were selected and, after one week of adaptive feeding, were randomly divided into a model group (control group) and an experimental group (15 mg / kg group).

[0042] 2.2 Peptides

[0043] The amino acid sequence of the polypeptide used in this example is shown in SEQ ID NO.1, where positions 1-11 of SEQ ID NO.1 are the cell-penetrating peptide sequence, and positions 12-19 of SEQ ID NO.1 are the protein sequence of the carboxyl terminal domain of Preso. The polypeptide was constructed by Shanghai GenScript Biotechnology Co., Ltd. The polypeptide lyophilized powder was prepared with physiological saline to the desired concentration for tail vein injection.

[0044] 2.3 Establishment of mouse CTE model and peptide treatment

[0045] In this embodiment, a cortical impactor was used to model CTE in mice. The steps are as follows: In the early stage of modeling, the mice were placed in a Reward anesthesia machine and anesthetized with isoflurane. After anesthesia, ear tags were added to the mice, and the hair between the two ears was shaved. At the beginning of modeling, we first placed the mice on a 4 cm thick sponge after anesthesia. After that, we used a CCI impactor with parameters of 1.5 mm depth, 3 m / s speed, and 200 ms residence time to directly strike the bregma of the unopened mice. The impact was 5 times at an interval of 24 hours to simulate the CTE model caused by repeated mild craniocerebral injury; behavioral tests were performed after 4 weeks of treatment. After the model was constructed, the peptide solution (15 mg / kg) or an equal amount of solvent without peptide was injected through the tail vein according to the grouping, and the mice were returned to the cage for monitoring after completion.

[0046] 2.4 Behavioral testing and analysis

[0047] The mice after CTE modeling were subjected to behavioral testing and analysis in the open field test (OFT) and elevated-plus maze test (EPM). The experimenter needs to record the number, date, status and other information of the mice; all behavioral tests are carried out in a quiet, soundproof and dimly lit environment, and the curtains need to be drawn to prevent affecting the test results; the mice to be tested need to be taken to the test room to adapt to the environment about 30 minutes in advance before the test. The behavioral test was monitored and recorded by a digital camera connected to a computer, and then analyzed and plotted using Smart 3.0 behavioral software and GraphpadPrism 9.0 software.

[0048] Figure 2 The results and statistical analysis of the open field test in mice given the peptide. Figure 3 The results and statistical analysis of the elevated plus maze test on mice given the peptide. Figure 2 , Figure 3 As shown, it can be seen that the peptide can significantly reduce the anxiety-depression-like behaviors of CTE model mice and has a neuroprotective effect.

[0049] Example 3: Peptide inhibits the binding of PSD-95 to Tau through the Preso CT domain

[0050] Since the Preso CT domain interacts with its PSD-95 PDZ domain, it is speculated that the peptide may play a neuroprotective role by disrupting the binding of PSD-95 and Tau during craniocerebral injury. Therefore, the immunoprecipitation method was used for verification.

[0051] 3.1 Experimental Grouping

[0052] PSD-95 was co-transfected with Tau; PSD-95 was co-transfected with Tau and treated with peptides. Preso, PSD-95, and Tau were co-transfected; Preso CT domain mutation overexpression plasmid, PSD-95, and Tau were co-transfected.

[0053] 3.2 Overexpression plasmid construction

[0054] The construction of Preso CT domain mutant overexpression plasmid was completed by Shanghai GeneCare Gene Medicine Technology Co., Ltd. The Preso CT domain mutant was constructed by knocking out the 1312-1320 amino acids of Preso (NCBI ACCESSION: A2AFR3), thereby destroying the interaction between Preso and PSD-95.

[0055] 3.3 Peptides

[0056] The amino acid sequence of the polypeptide used in this example is shown in SEQ ID NO.1, where positions 1-11 of SEQ ID NO.1 are the cell-penetrating peptide sequence, and positions 12-19 of SEQ ID NO.1 are the protein sequence of the carboxyl terminal domain of Preso. The polypeptide was constructed by Shanghai GenScript Biotechnology Co., Ltd. During the experiment, ultrapure water was used to dissolve the polypeptide lyophilized powder to prepare a mother solution with a concentration of 10mM, and then diluted to the desired concentration using culture medium.

[0057] 3.4 Cell culture and treatment

[0058] HEK-293T cells were cultured in 10 cm culture dishes for 24 hours, and then co-transfected with 3 μg of PSD-95 and Tau overexpression plasmids, respectively. After 24 hours of treatment, the culture medium was replaced with a medium containing polypeptides or a medium containing an equal amount of solvent according to the grouping. Continue to culture for 24 hours, harvest the cells, lyse on ice for half an hour, and collect the supernatant. Co-transfect Preso, PSD-95, Tau overexpression plasmids, Preso CT domain mutation overexpression plasmids, PSD-95, and Tau co-transfection, harvest the cells 48 hours after transfection, lyse on ice for half an hour, and collect the supernatant.

[0059] 3.5 Co-immunoprecipitation (Co-IP) experiment

[0060] An equal amount of cell lysate was taken and incubated with agarose beads linked to anti-PSD-95 antibody on a shaker at 4°C overnight for 16 hours for immunoprecipitation. The agarose beads were then washed to remove nonspecifically bound proteins. After the agarose beads were collected, Western blot analysis was performed using anti-PSD-95 antibody.

[0061] Figure 4 The Co-IP experimental results of the effect of peptides on the interaction between Preso and PSD-95 on PSD-95 / Tau and the statistical analysis of the IP group results are shown. Figure 5 The results of the Co-IP experiment of the effect of Preso CT domain mutants on the interaction between Preso and PSD-95 and PSD-95 / Tau and the statistical analysis of the IP group results. Figure 4 , Figure 5 It can be seen that peptides and Preso CT domain mutants can effectively reduce the binding of PSD-95 to Tau, and this effect is achieved through the Preso CT domain.

[0062] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0063] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A polypeptide targeting the Preso CT domain, characterized in that: The amino acid sequence of the polypeptide is shown in SEQ ID NO.

1.

2. Use of the polypeptide according to claim 1 in the preparation of a neuroprotective drug, characterized in that: The neuroprotective drug is a drug for treating chronic traumatic encephalopathy and alleviating anxiety-depression-like behaviors caused by chronic traumatic encephalopathy.

3. Use of the polypeptide according to claim 2 in the preparation of a neuroprotective drug, characterized in that: The use is the use of the polypeptide in preparing a drug for inhibiting the binding between PSD-95 protein and Tau protein.

4. Use of the polypeptide according to claim 2 in preparing a neuroprotective drug, characterized in that: The use is the use of the polypeptide in preparing a medicine for treating craniocerebral injury.

5. Use of the polypeptide according to claim 4 in the preparation of a neuroprotective drug, characterized in that: The drug is used to reduce neuronal death after craniocerebral injury.

6. A neuroprotective drug, characterized in that: The invention comprises the polypeptide according to claim 1 and auxiliary materials.

7. The drug according to claim 6, characterized in that The auxiliary material is selected from one or more of a buffer, a solvent, a solubilizer, a preservative, a stabilizer, a surfactant, and an antioxidant.

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