CN114533874B - Application of PSD-95GK domain as neuroprotective target - Google Patents

Application of PSD-95GK domain as neuroprotective target Download PDF

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CN114533874B
CN114533874B CN202210102478.XA CN202210102478A CN114533874B CN 114533874 B CN114533874 B CN 114533874B CN 202210102478 A CN202210102478 A CN 202210102478A CN 114533874 B CN114533874 B CN 114533874B
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王玺玫
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Beijing Heyu Medicine Technology Co ltd
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Abstract

Provides the application of the PSD-95GK domain as a neuroprotective target and provides the application of the drug taking the PSD-95GK domain as an action target in the preparation of neuroprotective drugs. Drugs that target the PSD-95GK domain include polypeptides, the sequence of which includes a sequence that targets the PSD-95GK domain, and may include a transmembrane sequence. Polypeptides targeting the PSD-95GK domain are provided. The compound has better binding capacity with PSD-95GK structural domain, can be used as a tool for exploring the physiological function of the PSD-95GK structural domain, can be used as a lead compound of a neuroprotective medicament acting on the PSD-95GK structural domain, and has great value for medical research and medicament development.

Description

Application of PSD-95GK domain as neuroprotective target
Technical Field
The application relates to the field of biological medicine, in particular to application of a PSD-95GK domain as a neuroprotective target.
Background
Cerebral apoplexy is the second leading cause of death in the world and is also the leading cause of death in China. Ischemic cerebral apoplexy accounts for 60-70% of cerebral apoplexy, has the characteristics of high morbidity, high mortality and high disability rate, and is a major public health problem. At present, vascular recanalization is usually realized in a mode of rt-PA venous thrombolysis combined with mechanical thrombolysis, but the existence of ischemia reperfusion injury can not effectively relieve the injury of nerve function even if vascular recanalization is performed within a time window.
Postsynaptic compact 95 (postsynaptic density-95, PSD-95), belonging to the superfamily of membrane-associated guanylate kinases (membrane associated guanylate kinase, MAGUK), is primarily localized to postsynaptic compact (postsynaptic density, PSD) of excitatory synapses. PSD-95 consists of 3 PDZ (PSD-95/disks large/zona occomprising-1) domains, one SH3 (Ser homolog 3) domain and one Guanylate Kinase (GK) domain that loses catalytic function. Studies have shown that PSD-95 mediated protein machinery plays an important role in ischemia reperfusion injury. On the one hand, the accumulation of glutamate in large amounts after cerebral ischemia induces the excessive activation of NMDA receptors, promoting the formation of NMDAR-PSD-95-nNOS protein machinery. In this process PSD-95 interacts with the NR2B subunit of the NMDA receptor and with neuronal nitric oxide synthase (nNOS) via its PDZ1 and PDZ2 domains, respectively. The formation of this protein machinery results in the activation of nNOS to produce large amounts of NO, which combines with large amounts of oxygen radicals in the cell after stroke to produce oxynitrite (ONOO-) which ultimately leads to cell death. On the other hand, glutamate activates KA receptors and triggers the formation of KAR-PSD95-MLK3 protein machinery. This protein machinery is formed by the interaction of the PSD-95 PDZ domain with the GluR6 subunit of the KA receptor and through the interaction of the SH3 domain with MLK 3. The formation of this protein machinery in turn leads to increased levels of phosphorylation of MLK3 and JNK, ultimately activating Caspase 3 to trigger apoptosis. The above studies suggest that PSD-95 ultimately leads to cell death during cerebral ischemia reperfusion injury by recruiting related molecules to form protein machinery. Many compounds that disrupt protein machinery formation during cerebral ischemia reperfusion injury have been shown to have some neuroprotective effect. In particular, polypeptide NA-1, which disrupts NMDAR interactions with PSD-95, has a pronounced neuroprotective effect and extremely strong drug-forming properties, which are believed to turn on the "spring" of the neuroprotective agent. However, clinical studies have found that NA-1 is cleaved by rt-PA to inactivate it to some extent, which limits its use.
The GK domain, as an important component of PSD-95, is considered an important binding site for interaction with other proteins. However, the functions under physiological and pathological conditions are not clear at present.
Disclosure of Invention
In some embodiments, the application provides the use of a PSD-95GK domain as a neuroprotective target. In some embodiments, the PSD-95GK domain of the present application is a neuroprotective target.
In some embodiments, the application provides the use of a PSD-95GK domain in neuroprotection. In some embodiments, the application provides for the use of a drug targeting the PSD-95GK domain in neuroprotection. In some embodiments, the application provides the use of a PSD-95GK domain in the preparation of a neuroprotective medicament. In some embodiments, the application provides the use of a drug targeting the PSD-95GK domain in the preparation of a neuroprotective drug. In some embodiments, the application provides the use of a PSD-95GK domain to affect PSD-95 binding to nNOS. In some embodiments, the present application provides the use of a drug targeting the PSD-95GK domain to affect PSD-95 binding to nNOS. In some embodiments, the application provides the use of a PSD-95GK domain in the preparation of a medicament for affecting PSD-95 binding to nNOS. In some embodiments, the application provides the use of a drug targeting the PSD-95GK domain for the preparation of a drug that affects PSD-95 binding to nNOS. In some embodiments, the application provides the use of a PSD-95GK domain in the treatment of cerebral ischemia reperfusion injury. In some embodiments, the application provides the use of a drug targeting the PSD-95GK domain in the treatment of cerebral ischemia reperfusion injury. In some embodiments, the application provides the use of a PSD-95GK domain in the preparation of a medicament for treating cerebral ischemia reperfusion injury. In some embodiments, the application provides the use of a medicament targeting the PSD-95GK domain in the manufacture of a medicament for treating cerebral ischemia reperfusion injury. In some embodiments, the application provides the use of a PSD-95GK domain in the treatment of ischemic stroke. In some embodiments, the application provides for the use of a medicament targeting the PSD-95GK domain in the treatment of ischemic stroke. In some embodiments, the application provides the use of a PSD-95GK domain in the manufacture of a medicament for treating ischemic stroke. In some embodiments, the application provides the use of a drug with a PSD-95GK domain as an action target in the preparation of a drug for treating ischemic stroke.
In some embodiments, the present application provides a neuroprotective method comprising administering a drug targeted to the PSD-95GK domain. In some embodiments, the present application provides a method of affecting binding of PSD-95 to nNOS comprising administering a drug targeted to the PSD-95GK domain. In some embodiments, the present application provides a method of treating cerebral ischemia reperfusion injury comprising administering a drug targeting the PSD-95GK domain. In some embodiments, the present application provides a method for treating ischemic stroke comprising administering a drug targeted to the PSD-95GK domain.
In some embodiments, the present application provides agents targeting the PSD-95GK domain for neuroprotection. In some embodiments, the present application provides agents targeting the PSD-95GK domain for affecting binding of PSD-95 to nNOS. In some embodiments, the present application provides agents targeting the PSD-95GK domain for use in treating cerebral ischemia reperfusion injury. In some embodiments, the present application provides agents targeting the PSD-95GK domain for use in the treatment of ischemic stroke. In some embodiments, the present application provides a neuroprotective product comprising a drug targeting the PSD-95GK domain. In some embodiments, the present application provides a product that affects binding of PSD-95 to nNOS, including a drug targeted to the PSD-95GK domain. In some embodiments, the present application provides a product for treating cerebral ischemia reperfusion injury comprising a drug targeting the PSD-95GK domain. In some embodiments, the present application provides a product for treating ischemic stroke comprising a drug targeting the PSD-95GK domain.
In some embodiments, the agent for which the PSD-95GK domain of the present application is a target for action comprises a PSD-95GK domain inhibitor. In some embodiments, the PSD-95GK domain of the present application is a drug that targets the effect, particularly a PSD-95GK domain inhibitor.
In some embodiments, polypeptides targeting the PSD-95GK domain are provided. In some embodiments, the application provides a polypeptide whose sequence includes a sequence that targets a PSD-95GK domain. In some embodiments, the present application provides a polypeptide whose sequence includes a sequence that targets the PSD-95GK domain and a transmembrane sequence. In some embodiments, the application provides a polypeptide whose sequence consists of a sequence targeting the PSD-95GK domain. In some embodiments, the present application provides a polypeptide whose sequence consists of a sequence targeting the PSD-95GK domain and a transmembrane sequence. In some embodiments, the application provides the use of the polypeptide in neuroprotection. In some embodiments, the application provides the use of the polypeptide in the preparation of a neuroprotective medicament.
In some embodiments, the agent targeting the PSD-95GK domain, particularly the PSD-95GK domain inhibitor, may increase neuronal survival and reduce infarct size after stroke. In some embodiments, the PSD-95GK domain can serve as a target for neuroprotection; in some embodiments, polypeptides targeting the PSD-95GK domain are provided, which have better binding capacity to the PSD-95GK domain, can be used as tools for exploring the physiological functions of the PSD-95GK domain, and can be used as lead compounds of neuroprotective drugs acting on the PSD-95GK domain, and have great value for medical research and drug development.
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FIG. 1 shows the results of neuronal viability following administration of a polypeptide NDLS, a polypeptide CDLS under physiological conditions.
FIG. 2 shows the results of neuronal viability following OGD model treatment and administration of polypeptide CDLS.
FIG. 3 shows the results of brain TTC staining and statistical analysis of mice given the polypeptide CDLS after MCAO molding and reperfusion.
FIG. 4 is a statistical analysis of Co-IP experimental results and IP group results of the effect of polypeptide CDLS on PSD-95 binding to nNOS.
FIG. 5 is a statistical analysis of Co-IP experimental results of the effect of polypeptide CDLS on binding of PSD-95GK domain mutants to nNOS and results from the IP group.
FIG. 6 is a graph showing the effect of polypeptide CDLS on neuronal NO production following OGD and statistical analysis.
Detailed Description
In order to further describe the technical means and effects adopted by the present application for achieving the preset purpose, the following detailed description is given below of the specific implementation, structure, characteristics and effects according to the present application with reference to the accompanying drawings and preferred embodiments.
In some embodiments, the term "neuroprotection" as referred to herein may refer to restoring or remodeling neural tissue function as much as possible by, for example, restoring blood flow after an ischemic stroke has occurred, reducing infarct volume, preventing expansion of the penumbra, and rescuing neural cells that have been reversibly damaged. In some embodiments, the neuroprotection includes at least one of affecting PSD-95 binding to nNOS, treating ischemic reperfusion injury, and treating ischemic stroke, but is not limited thereto. In some embodiments, the affecting binding of PSD-95 to nNOS comprises decreasing the binding capacity of PSD-95 to nNOS or decreasing the ratio of PSD-95 to nNOS binding.
In some embodiments, the amino acid sequence of the PSD-95GK domain is set forth in SEQ ID NO. 1. The amino acid sequence of the PSD-95GK domain may be amino-terminally linked to a poly-His tag (SEQ ID NO: 22) for ease of purification. The PSD-95GK structural domain can be directly synthesized by manpower or can be obtained by synthesizing the encoding gene and then biologically expressing. The coding gene of the PSD-95GK domain may also be linked at its 5' end to the coding sequence of a poly-His tag (SEQ ID NO: 22). In some embodiments, the amino acid sequence of the PSD-95 PDZ2 domain is shown in SEQ ID NO. 2. In some embodiments, the amino acid sequence of nNOS is shown in SEQ ID NO. 3.
In some embodiments, the agents of the present application that target the PSD-95GK domain, particularly PSD-95GK domain inhibitors, include polypeptides. In some embodiments, the sequence of the polypeptide comprises a sequence that targets a PSD-95GK domain. In some embodiments, the sequence of the polypeptide includes a sequence that targets the PSD-95GK domain and a transmembrane sequence. In some embodiments, the polypeptide sequence consists of a sequence targeting the PSD-95GK domain. In some embodiments, the sequence of the polypeptide consists of a sequence that targets the PSD-95GK domain, and a transmembrane sequence.
In some embodiments, the sequence targeting the PSD-95GK domain in the polypeptides of the present application comprises a sequence having at least 93.75% sequence identity to the sequence set forth in SEQ ID NO. 8. In some embodiments, the sequence targeting the PSD-95GK domain in the polypeptide of the present application has at least 84.21% sequence identity to the sequence set forth in SEQ ID NO. 8. In some embodiments, the sequence targeting the PSD-95GK domain in the polypeptide of the present application has at least 93.75% sequence identity to the sequence set forth in SEQ ID NO. 8. In some embodiments, the sequence targeting the PSD-95GK domain in the polypeptide of the present application has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO. 8.
In some embodiments, the polypeptide of the present application, the sequence targeting the PSD-95GK domain comprises or consists of the sequence set forth in SEQ ID NO. 8 or a mutant thereof, wherein the mutant is an amino acid substitution in the sequence set forth in SEQ ID NO. 8. In some embodiments, the mutant is one in which arginine in the sequence shown in SEQ ID NO. 8, particularly arginine at position-5 in the sequence, is replaced, particularly with phenylalanine, glycine, alanine, or valine. In some embodiments, the mutant is a substitution of phenylalanine in the sequence shown in SEQ ID NO. 8, particularly a substitution of phenylalanine at position 10 in the sequence, particularly a substitution of tryptophan. In some embodiments, the mutant is one in which the asparagine in the sequence shown in SEQ ID NO. 8 is replaced, particularly the asparagine at position 6 in the sequence is replaced, particularly with cysteine, glutamic acid, or histidine. In some embodiments, the mutant is one in which tyrosine in the sequence shown in SEQ ID NO. 8 is replaced, particularly tyrosine at position 1 in the sequence, particularly arginine.
In some embodiments, the sequence targeting the PSD-95GK domain in the polypeptides of the present application comprises or consists of at least one of the sequences set forth in any one of SEQ ID NOs 4, 8-19. In some embodiments, the sequence of the targeting PSD-95GK domain of the polypeptides of the present application comprises or consists of at least one of the sequences set forth in any one of SEQ ID NOs 4, 8-13. In some embodiments, a polypeptide DLS targeting the PSD-95GK domain is provided, the sequence of which is shown in SEQ ID NO. 4.
In some embodiments, the polypeptide of the present application has a transmembrane sequence shown in SEQ ID NO. 5. In some embodiments, polypeptide NDLS (SEQ ID NO: 6) and polypeptide CDLS (SEQ ID NO: 7) can be obtained by combining the sequence of polypeptide DLS (SEQ ID NO: 4) with the transmembrane sequence TAT (SEQ ID NO: 5). Polypeptide NDLS and polypeptide CDLS are described in the examples below as PSD-95GK domain inhibitors.
However, the present application is not limited thereto; in some embodiments, the polypeptide of the present application may be a polypeptide comprising or consisting of at least one of SEQ ID NOS: 4, 8-19, in particular comprising or consisting of at least one of SEQ ID NOS: 4, 8-13, in combination with a transmembrane sequence (e.g., SEQ ID NO: 5), or a polypeptide comprising or consisting of at least one of SEQ ID NOS: 4, 8-19, in particular comprising or consisting of at least one of SEQ ID NOS: 4, 8-13, and not in combination with a transmembrane sequence (e.g., SEQ ID NO: 5). In some embodiments, the polypeptides of the present application may be a polypeptide consisting of at least one of SEQ ID NOS: 4, 8-19, in particular a sequence consisting of at least one of SEQ ID NOS: 4, 8-13, in combination with a transmembrane sequence (e.g., SEQ ID NO: 5), or a polypeptide consisting of at least one of SEQ ID NOS: 4, 8-19, in particular a sequence consisting of at least one of SEQ ID NOS: 4, 8-13.
In some embodiments, a polypeptide QSF targeting the PSD-95GK domain is provided, the sequence of which is shown in SEQ ID NO. 8. In some embodiments, a polypeptide R-5F targeting the PSD-95GK domain is provided, the sequence of which is shown in SEQ ID NO 9. In some embodiments, a polypeptide R-5G targeting the PSD-95GK domain is provided, the sequence of which is shown in SEQ ID NO. 10. In some embodiments, a polypeptide R-5A targeting the PSD-95GK domain is provided, the sequence of which is shown in SEQ ID NO. 11. In some embodiments, a PSD-95GK domain targeting polypeptide F10W is provided, the sequence of which is shown in SEQ ID NO. 12. In some embodiments, a polypeptide R-5V targeting the PSD-95GK domain is provided, the sequence of which is shown in SEQ ID NO. 13. In some embodiments, a polypeptide R-5T targeting the PSD-95GK domain is provided, the sequence of which is shown in SEQ ID NO. 14. In some embodiments, a PSD-95GK domain targeting polypeptide N6H is provided, the sequence of which is shown in SEQ ID NO. 15. In some embodiments, a PSD-95GK domain targeting polypeptide Y1R is provided, the sequence of which is shown in SEQ ID NO. 16. In some embodiments, a polypeptide R-5S targeting the PSD-95GK domain is provided, the sequence of which is shown in SEQ ID NO. 17. In some embodiments, a polypeptide N6E targeting the PSD-95GK domain is provided, the sequence of which is shown in SEQ ID NO. 18. In some embodiments, a polypeptide N6C targeting the PSD-95GK domain is provided, the sequence of which is shown in SEQ ID NO. 19.
Wherein, the polypeptide QSF with the sequence shown in SEQ ID NO. 8, wherein arginine at the-5 position in the sequence is mutated into phenylalanine, glycine, alanine and valine, so that the polypeptide R-5F with the sequence shown in SEQ ID NO. 9, the polypeptide R-5G with the sequence shown in SEQ ID NO. 10, the polypeptide R-5A with the sequence shown in SEQ ID NO. 11 and the polypeptide R-5V with the sequence shown in SEQ ID NO. 13 can be obtained respectively. The polypeptide QSF shown in SEQ ID NO. 8 has phenylalanine at position 10 mutated into tryptophan, so that the polypeptide F10W shown in SEQ ID NO. 12 can be obtained.
In some embodiments, polypeptide NF10W of SEQ ID NO. 20 and polypeptide CF10W of SEQ ID NO. 21 may be obtained as PSD-95GK domain inhibitors by combining the sequence of polypeptide F10W (SEQ ID NO: 12) with the transmembrane sequence TAT (SEQ ID NO: 5).
In some embodiments, the polypeptides of the present application include or consist of the sequence set forth in SEQ ID NO. 4 and optionally a transmembrane sequence, particularly a transmembrane sequence comprising or consisting of SEQ ID NO. 5. In some embodiments, the polypeptides of the present application include or consist of the sequence set forth in SEQ ID NO. 8 and optionally a transmembrane sequence, particularly a transmembrane sequence comprising or consisting of SEQ ID NO. 5. In some embodiments, the polypeptides of the present application include or consist of the sequence set forth in SEQ ID NO. 9 and optionally a transmembrane sequence, particularly a transmembrane sequence comprising or consisting of SEQ ID NO. 5. In some embodiments, the polypeptides of the present application include or consist of the sequence set forth in SEQ ID NO. 10 and optionally a transmembrane sequence, particularly a transmembrane sequence comprising or consisting of SEQ ID NO. 5. In some embodiments, the polypeptides of the present application include or consist of the sequence set forth in SEQ ID NO. 11 and optionally a transmembrane sequence, particularly a transmembrane sequence comprising or consisting of SEQ ID NO. 5. In some embodiments, the polypeptides of the present application include or consist of the sequence set forth in SEQ ID NO. 12 and optionally a transmembrane sequence, particularly a transmembrane sequence comprising or consisting of SEQ ID NO. 5. In some embodiments, the polypeptides of the present application include or consist of the sequence set forth in SEQ ID NO. 13 and optionally a transmembrane sequence, particularly a transmembrane sequence comprising or consisting of SEQ ID NO. 5. In some embodiments, the polypeptides of the present application include or consist of the sequence set forth in SEQ ID NO. 14 and optionally a transmembrane sequence, particularly a transmembrane sequence comprising or consisting of SEQ ID NO. 5. In some embodiments, the polypeptides of the present application include or consist of the sequence set forth in SEQ ID NO. 15 and optionally a transmembrane sequence, particularly a transmembrane sequence comprising or consisting of SEQ ID NO. 5. In some embodiments, the polypeptides of the present application include or consist of the sequence set forth in SEQ ID NO. 16 and optionally a transmembrane sequence, particularly a transmembrane sequence comprising or consisting of SEQ ID NO. 5. In some embodiments, the polypeptides of the present application include or consist of the sequence set forth in SEQ ID NO. 17 and optionally a transmembrane sequence, particularly a transmembrane sequence comprising or consisting of SEQ ID NO. 5. In some embodiments, the polypeptides of the present application include or consist of the sequence set forth in SEQ ID NO. 18 and optionally a transmembrane sequence, particularly a transmembrane sequence comprising or consisting of SEQ ID NO. 5. In some embodiments, the polypeptides of the present application include or consist of the sequence set forth in SEQ ID NO. 19 and optionally a transmembrane sequence, particularly a transmembrane sequence comprising or consisting of SEQ ID NO. 5. In some embodiments, the agent targeting the PSD-95GK domain comprises or consists of a polypeptide having the sequence shown in SEQ ID NO. 6. In some embodiments, the agent targeting the PSD-95GK domain comprises or consists of a polypeptide having the sequence shown in SEQ ID NO. 7. In some embodiments, the agent targeting the PSD-95GK domain comprises or consists of a polypeptide having the sequence shown in SEQ ID NO. 20. In some embodiments, the agent targeting the PSD-95GK domain comprises or consists of a polypeptide having the sequence shown in SEQ ID NO. 21.
In some embodiments, the PSD-95GK domain is associated with ischemic reperfusion injury in ischemic stroke and can be used as a target for neuroprotection for screening or developing neuroprotective agents; in some embodiments, drugs targeting the PSD-95GK domain, particularly PSD-95GK domain inhibitors, may be used for neuroprotection of ischemic reperfusion injury following ischemic stroke, and may be used for the preparation of neuroprotection products.
The following examples are merely illustrative of the present application for better understanding and are not to be construed as limiting the scope of the present application. The reagents or apparatus referred to in the examples are available commercially from conventional biochemical reagent suppliers without the manufacturer's knowledge.
Example 1: protection of primary neurons in murine cortex of OGD model by polypeptide DLS targeting PSD-95GK domain
A mouse primary neuron oxygen-sugar deprivation (oxygen glucose deprivation, OGD) model is selected, and the in vitro neuroprotection effect of the polypeptide is confirmed by detecting the change of the cell viability after the polypeptide is given.
1.1 extraction and culture of murine cortical primary neuronal cells
C57BL/6 pregnant mice of E15-17 are selected, brains of the pregnant mice are dissected on ice, and meninges are stripped, and cortex is separated. Cells were blown off after 5 min of 0.25% pancreatin digestion, and after filtration on a 0.7 μm cell screen, the cells were seeded in 96-well plates. The seed solution is high sugar DMEM medium containing 20% fetal calf serum and 1% double antibody, and has a seed density of 12.5X10 4 Individual/cm 2 . At 37℃with 5% CO 2 After 4-6 hours of cultivation under the condition, the medium is changed into a maintenance medium which contains 2% Gibco TM B-27 TM Additive+1% glutamine+1% diad Neurobasal medium. After culturing for 4-5 days, the liquid is changed for half, and culturing is continued until 7-9 days later.
1.2 Polypeptides
This example relates to the synthesis of polypeptides NDLS (SEQ ID NO: 6) and CDLS (SEQ ID NO: 7) by the blaze Biotechnology Co., ltd. With a purity of more than 98%. In the experimental process, ultrapure water is used for dissolving polypeptide freeze-dried powder to prepare mother solution with the concentration of 10mM, and then culture medium is used for dilution to the required concentration.
1.3 Effect of polypeptide DLS targeting PSD-95GK Domain on Primary neuronal cell viability in Normal murine cortex
The effect of the polypeptide on the viability of normal murine cortical primary neuronal cells was observed using the MTS method. After the primary neurons are cultured to the 7 th to 9 th days, adding a polypeptide solution with a certain concentration in a liquid change mode to treat the cells for 24 hours; then 20. Mu.L MTS was added to each well at 37℃with 5% CO 2 The culture was continued for 2 hours under the conditions. The absorbance of each well at 490nm was measured using a multifunctional microplate reader (Molecular Devices).
FIG. 1 shows the results of neuronal viability following administration of polypeptide DLS under physiological conditions. The results show that the polypeptide CDLS (SEQ ID NO: 7) was non-toxic to normal murine primary cortical neuronal cells within 32. Mu.M; however, the polypeptide NDLS (SEQ ID NO: 6) at high concentrations may cause damage to cells.
1.4 Effect of polypeptide CDLS targeting PSD-95GK Domain on cell viability of murine cortical primary neurons after OGD
The effect of the polypeptide on the activity of murine cortical primary neuronal cells after OGD modeling was observed using the MTS method. OG was performed after the primary neurons were cultured to day 7-9D, molding: the maintenance medium was replaced with sugarless DMEM and the cells were placed in an anoxic environment (5% co) 2 、95％NO 2 ) Culturing for 2 hours. After the end of OGD, the polypeptide CDLS (SEQ ID NO: 7) dissolved at different concentrations or the maintenance medium dissolved in the same solvent was added in the form of a liquid change. Then at 37 ℃ and 5% CO 2 The culture was continued for 22 hours under the conditions. MTS was then added to determine cell viability per well.
FIG. 2 shows the results of neuronal viability following OGD modeling treatment and administration of polypeptide CDLS. The result shows that the polypeptide CDLS can reduce cell death caused by OGD within a certain concentration range, and the polypeptide CDLS has neuroprotection.
Example 2: polypeptide CDLS targeting PSD-95GK domain reduces cerebral infarction volume of MCAO model mice
To demonstrate the in vivo neuroprotective effect of the polypeptide, a mouse middle cerebral artery occlusion (middle cerebral artery occlusion, MCAO) model is adopted, and the in vivo protective effect of the polypeptide is clarified by detecting the beneficial effect of the polypeptide on cerebral infarction of ischemia reperfusion injury mice.
2.1 laboratory animals and groups
In this example, SPF-grade male C57BL/6 mice weighing 20-24g were selected, and after one week of adaptive feeding, they were randomly divided into 3 groups, namely, a model group (control group), a low dose group (3 nmol/g group) and a high dose group (10 nmol/g group).
2.2 Polypeptides
This example relates to the synthesis of polypeptide CDLS (SEQ ID NO: 7) with a purity of greater than 98% by blaze Biotechnology Co., ltd. The lyophilized powder of polypeptide is formulated with physiological saline to a desired concentration for intravenous injection.
2.3 establishment of mouse MCAO model and polypeptide treatment
In this example, the wire bolt method was used for the molding of mouse MCAO. The method comprises the following steps: first pentobarbital sodium anesthetized mice shave neck hair. The left Common Carotid Artery (CCA), internal Carotid Artery (ICA), and External Carotid Artery (ECA) were exposed and isolated, the CCA was ligated near the tail, the ECA was ligated and the proximal trunk was prepared at both ligatures. A plug is inserted into the ECA incision, and the plug is adjusted to enter ICA 9-10mm so as to realize the occlusion and ischemia of the middle cerebral artery. The cord is then secured with the previously prepared cord and the slip knot at the CCA is untied. After 90 minutes of occlusion, the plug was slowly removed and the ECA was ligated at the end of the vessel and cerebral perfusion was resumed. The skin is then sutured and sterilized. After reperfusion was initiated, mice were returned to the cage for monitoring after completion, depending on the grouping by tail vein injection of different concentrations of polypeptide CDLS solution (3 nmol/g, 10 nmol/g) or equivalent amounts of solvent without polypeptide.
2.4 TTC staining and analysis
Mice after MCAO molding described above were sacrificed at 24 hours of reperfusion for brain removal. After freezing at-80℃the coronal position was serially sliced backwards with a slice thickness of 2mm. The sections were then immersed in a 2% ttc solution and incubated at 37 ℃ for 15 minutes in the dark, during which time the brain pieces were turned over at 5 minute intervals to homogenize the staining. TTC solution was discarded after incubation was completed, and after fixation with 4% paraformaldehyde for 24 hours, photographs were taken and ischemia was counted using ImageJ software.
FIG. 3 shows the results of TTC staining of the brains of mice given the polypeptide CDLS and statistical analysis. As shown in FIG. 3, the polypeptide CDLS (SEQ ID NO: 7) can obviously reduce the infarct volume of MCAO model mice, obviously reduce the infarct rate, and has neuroprotection effect.
Example 3: polypeptide CDLS inhibits binding of PSD-95 to nNOS via the PSD-95GK domain
Because of the interaction of the PSD-95GK domain with its PDZ2 domain, it is speculated that the polypeptide may exert neuroprotective effects by disrupting PSD-95 binding to nNOS during cerebral ischemia and reperfusion. Therefore, the method of co-immunoprecipitation was adopted for verification.
3.1 Experimental grouping
PSD-95 co-transfection with nNOS: solvent group (0. Mu.M group), low concentration polypeptide group (5. Mu.M group), medium concentration polypeptide group (10. Mu.M group), high concentration polypeptide group (20. Mu.M group), positive control group (ZL 006 group)
PSD-95GK mutant cotransfection of nNOS: solvent group (0. Mu.M group), low concentration polypeptide group (5. Mu.M group), medium concentration polypeptide group (10. Mu.M group), high concentration polypeptide group (20. Mu.M group), positive control group (ZL 006 group)
3.2 construction of the overexpression plasmid
Construction of HA-tagged PSD-95 overexpression plasmid, HA-tagged PSD-95GK domain mutation overexpression plasmid and Flag-tagged nNOS overexpression plasmid was completed by Shanghai Ji Kai Gene technologies and technologies Co.
3.3 Polypeptides
The polypeptide CDLS (SEQ ID NO: 7) was synthesized by the blaze Biotechnology Co., ltd and had a purity of more than 98%. In the experimental process, ultrapure water is used for dissolving polypeptide freeze-dried powder to prepare mother solution with the concentration of 10mM, and then culture medium is used for dilution to the required concentration.
3.4 cell culture and treatment
HEK-293T cells were incubated in 10cm dishes for 24 hours and then co-transfected with 3. Mu.g each of the HA-tagged PSD-95 over-expression plasmid and the Flag-tagged nNOS over-expression plasmid, or 3. Mu.g each of the HA-tagged PSD-95GK domain mutant over-expression plasmid and the Flag-tagged nNOS over-expression plasmid, respectively. After 24 hours of treatment, the medium was changed to the medium containing the polypeptide or the medium containing the same amount of solvent in a liquid-changing manner according to the group. Culture was continued for 24 hours, and after harvesting cells were lysed on ice for half an hour, the supernatant was collected.
3.5 Co-immunoprecipitation (Co-IP) experiments
Equal amount of cell lysate was incubated with agarose beads attached to anti-HA antibody for 3 hours in a shaker at 4deg.C for co-immunoprecipitation, after which the agarose beads were washed to remove non-specifically bound proteins, and after agarose beads were collected, western blot analysis was performed using anti-HA antibody and anti-Flag antibody.
FIG. 4 is a statistical analysis of Co-IP experimental results and IP group results of the effect of CDLS on PSD-95 and nNOS binding, and FIG. 5 is a statistical analysis of Co-IP experimental results and IP group results of CDLS on PSD-95GK domain mutants and nNOS binding. From FIGS. 4 and 5, it can be seen that polypeptide CDLS reduces binding of PSD-95 to nNOS in a dose dependent manner and this is accomplished by the PSD-95GK domain.
Example 4: effect of polypeptide CDLS on NO production in mouse cortical neurons after OGD
Detection of the effect product NO of mouse cortical neurons nNOS using DAF-FM DA further demonstrates that polypeptide targeting the PSD-95 domain affects neuroprotective effects through the interaction of PSD-95 with nNOS.
4.1 Experimental grouping set-up
In this example, a physiological group (control group), a solvent group (carrier group) and a 10. Mu.M polypeptide group (CDLS group) were set.
4.2 Polypeptides
The polypeptide CDLS (SEQ ID NO: 7) was synthesized by the blaze Biotechnology Co., ltd and had a purity of more than 98%. In the experimental process, ultrapure water is used for dissolving polypeptide freeze-dried powder to prepare mother solution with the concentration of 10mM, and then culture medium is used for dilution to the required concentration.
4.3 detection of the Effect of polypeptide CDLS on NO production in mouse cortical neurons after OGD
The extraction and culture methods of mouse cortical neurons are as described above. OGD molding is carried out when the culture is carried out until D7. After reoxygenation, DAF-FM DA working solution was prepared at a concentration of 5. Mu.M using HBSS buffer without phenol red. DAF FM-DA was washed off after incubation at 37℃for 30 minutes. Fluorescence pictures were taken with laser confocal and analyzed for fluorescence intensity with ImageJ software.
FIG. 6 is a graph showing the effect of polypeptide CDLS on neuronal NO production following OGD and statistical analysis. It can be seen that OGD modeling significantly increased NO production in neuronal cells, while CDLS (SEQ ID NO: 7) targeting the PSD-95GK domain reduced OGD-induced NO production.
Example 5: polarized fluorescence competitive binding assay to determine binding capacity of polypeptide to PSD-95GK Domain
In order to obtain the polypeptide with better neuroprotective effect and targeting PDS-95 GK structural domain, a series of mutants (SEQ ID NO: 9-19) are designed based on the polypeptide QSF (SEQ ID NO: 8), and the polypeptide with better molecular dynamics simulation result is synthesized. Its binding capacity to the PSD-95GK domain was determined by polarized fluorescence competitive binding experiments.
5.1 Prokaryotic expression and purification of PSD-95GK structural domain
The PSD-95GK structural domain (SEQ ID NO: 1) gene sequence is cloned into a Pet-15b vector containing a His6 tag at the N end and transformed into BL21 (DE 3) escherichia coli for expression. Prokaryotic expression of the protein was performed using 0.2M isopropyl-beta-D-thiogalactoside (IPTG) induced at 16℃for 18 hours. The His 6-tagged protein was then purified for the first time using Ni2+ -NTA agarose affinity chromatography and further purified using size exclusion chromatography, eluting with protein eluent (50 mM Tris-HCl pH 8.0, 100mM NaCl,1mM EDTA,1mM DTT).
5.2 Polypeptides
QSF (SEQ ID NO: 8) and polypeptides having lower free energy of binding than it: R-5F (SEQ ID NO: 9), R-5G (SEQ ID NO: 10), R-5A (SEQ ID NO: 11), F10W (SEQ ID NO: 12), R-5V (SEQ ID NO: 13) were synthesized by the blaze Biotechnology Co., ltd, and the purity was more than 90%. The FITC-SAPAP (SEQ ID NO: 24) and the unlabeled control polypeptide SAPAP (SEQ ID NO: 23) were also synthesized by the blaze Biotechnology Co.
5.3 polarized fluorescence competitive binding experiments
In this example, the binding capacity of the polypeptide to the PSD-95GK domain was determined by detecting the change in the system polarization value resulting from the design of the polypeptide to compete with the FITC-tagged fluorescent peptide FITC-SAPAP (SEQ ID NO: 24) for binding to the PSD-95GK domain protein. Specifically, a black 384-well plate was used, and 20. Mu.L of each well of the reaction system was determined; the buffer system was ph=7.5 Tris-HCl buffer (50mM Tris,100mM NaCl, pH adjusted to 7.5 with HCl). The final concentration of FITC-SAPAP in the system is determined to be 10nM through a pre-experiment; the final concentration of PSD-95GK protein was 2. Mu.M. The polypeptides to be tested were set up at a total concentration gradient of eight from 0.06. Mu.M to 125. Mu.M. In addition to the experimental group, a protein control group, a FITC-SAPAP control group, a negative control group and a positive control group were additionally provided.
Firstly adding PSD-95GK protein and FITC-SAPAP into a system, fully mixing, then adding a polypeptide solution to be detected, shaking, uniformly mixing, incubating for 1h at room temperature in a dark place, and then using a multifunctional enzyme-labeled instrument (Molecular Devices), setting the fluorescence excitation wavelength of FITC to 485nm and the emission wavelength to 530nm to read the polarization value. The determined polarization values were used for calculation of the dissociation constant Kd, affinity constant Ki, and half inhibition concentration IC50 of the polypeptide with PSD-95 GK. The results show that the designed polypeptide can be combined with PSD-95GK domain, and the mutant has improved binding capacity (Ki=5.64+ -0.51 μm) compared with QSF, especially F10W binding capacity (Ki=0.75+ -0.25 μm).
The above embodiments are only preferred embodiments of the present application, and the scope of the present application is not limited thereto, but any insubstantial changes and substitutions made by those skilled in the art on the basis of the present application are intended to be within the scope of the present application.
Sequence listing
<110> Beijing and public medical science and technology Co., ltd
Use of <120> PSD-95GK domain as neuroprotective target
<130> PT2022000158-DD-P
<160> 24
<170> PatentIn version 3.5
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Phe Val Ser Ser Arg Glu Lys Met Glu Lys Asp Ile Gln Ala His Lys
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Leu Ala Val Asn Gly Arg Pro Leu Val Asp Leu Ser Tyr Asp Ser Ala
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Leu Glu Val Leu Arg Gly Ile Ala Ser Glu Thr His Val Val Leu Ile
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Leu Arg Gly Pro Glu Gly Phe Thr Thr His Leu Glu Thr Thr Phe Thr
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Gly Asp Gly Thr Pro Lys Thr Ile Arg Val Thr Gln Pro Leu Gly Pro
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Pro Thr Lys Ala Val Asp Leu Ser His Gln Pro Pro Ala Gly Lys Glu
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Gln Pro Leu Ala Val Asp Gly Ala Ser Gly Pro Gly Asn Gly Pro Gln
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His Ala Tyr Asp Asp Gly Gln Glu Ala Gly Ser Leu Pro His Ala Asn
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Gly Leu Ala Pro Arg Pro Pro Gly Gln Asp Pro Ala Lys Lys Ala Thr
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Arg Val Ser Leu Gln Gly Arg Gly Glu Asn Asn Glu Leu Leu Lys Glu
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Ile Glu Pro Val Leu Ser Leu Leu Thr Ser Gly Ser Arg Gly Val Lys
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Gly Gly Ala Pro Ala Lys Ala Glu Met Lys Asp Met Gly Ile Gln Val
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Asp Arg Asp Leu Asp Gly Lys Ser His Lys Pro Leu Pro Leu Gly Val
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Glu Asn Asp Arg Val Phe Asn Asp Leu Trp Gly Lys Gly Asn Val Pro
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Val Val Leu Asn Asn Pro Tyr Ser Glu Lys Glu Gln Pro Pro Thr Ser
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Gly Lys Gln Ser Pro Thr Lys Asn Gly Ser Pro Ser Lys Cys Pro Arg
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Phe Leu Lys Val Lys Asn Trp Glu Thr Glu Val Val Leu Thr Asp Thr
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Leu His Leu Lys Ser Thr Leu Glu Thr Gly Cys Thr Glu Tyr Ile Cys
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Met Gly Ser Ile Met His Pro Ser Gln His Ala Arg Arg Pro Glu Asp
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Val Arg Thr Lys Gly Gln Leu Phe Pro Leu Ala Lys Glu Phe Ile Asp
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Gln Tyr Tyr Ser Ser Ile Lys Arg Phe Gly Ser Lys Ala His Met Glu
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Arg Leu Glu Glu Val Asn Lys Glu Ile Asp Thr Thr Ser Thr Tyr Gln
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Leu Lys Asp Thr Glu Leu Ile Tyr Gly Ala Lys His Ala Trp Arg Asn
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Ala Ser Arg Cys Val Gly Arg Ile Gln Trp Ser Lys Leu Gln Val Phe
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Leu Gln Ala Asn Gly Asn Asp Pro Glu Leu Phe Gln Ile Pro Pro Glu
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Leu Val Leu Glu Val Pro Ile Arg His Pro Lys Phe Glu Trp Phe Lys
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Asp Leu Gly Leu Lys Trp Tyr Gly Leu Pro Ala Val Ser Asn Met Leu
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Leu Glu Ile Gly Gly Leu Glu Phe Ser Ala Cys Pro Phe Ser Gly Trp
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Tyr Met Gly Thr Glu Ile Gly Val Arg Asp Tyr Cys Asp Asn Ser Arg
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Tyr Asn Ile Leu Glu Glu Val Ala Lys Lys Met Asn Leu Asp Met Arg
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His Ser Ala Thr Glu Ser Phe Ile Lys His Met Glu Asn Glu Tyr Arg
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Ser Gly Ser Ile Thr Pro Val Phe His Gln Glu Met Leu Asn Tyr Arg
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Trp Lys Gly Thr Asn Gly Thr Pro Thr Lys Arg Arg Ala Ile Gly Phe
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Lys Lys Leu Ala Glu Ala Val Lys Phe Ser Ala Lys Leu Met Gly Gln
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Ala Met Ala Lys Arg Val Lys Ala Thr Ile Leu Tyr Ala Thr Glu Thr
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Ala Phe Asp Ala Lys Val Met Ser Met Glu Glu Tyr Asp Ile Val His
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Gly Asp Pro Pro Glu Asn Gly Glu Lys Phe Gly Cys Ala Leu Met Glu
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Asn Val Arg Phe Ser Val Phe Gly Leu Gly Ser Arg Ala Tyr Pro His
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Phe Cys Ala Phe Gly His Ala Val Asp Thr Leu Leu Glu Glu Leu Gly
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Ser Leu Ile Ser Asn Asp Arg Ser Trp Lys Arg Asn Lys Phe Arg Leu
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Thr Phe Val Ala Glu Ala Pro Glu Leu Thr Gln Gly Leu Ser Asn Val
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His Lys Lys Arg Val Ser Ala Ala Arg Leu Leu Ser Arg Gln Asn Leu
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Gln Ser Pro Lys Ser Ser Arg Ser Thr Ile Phe Val Arg Leu His
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Glu Arg Leu Glu Asp Ala Pro Pro Val Asn Gln Met Val Lys Val
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Glu Leu Leu Glu Glu Arg Asn Thr Ala Leu Gly Val Ile Ser Asn
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Trp Thr Asp Glu Leu Arg Leu Pro Pro Cys Thr Ile Phe Gln Ala
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Leu Leu Gln Pro Arg Tyr Tyr Ser Ile Ser Ser Ser Pro Asp Met
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Thr Arg Asp Gly Glu Gly Pro Ile His His Gly Val Cys Ser Ser
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Cys Ile Leu Val Gly Pro Gly Thr Gly Ile Ala Pro Phe Arg Ser
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Phe Trp Gln Gln Arg Gln Phe Asp Ile Gln His Lys Gly Met Asn
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Pro Cys Pro Met Val Leu Val Phe Gly Cys Arg Gln Ser Lys Ile
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Asp His Ile Tyr Arg Glu Glu Thr Leu Gln Ala Lys Asn Lys Gly
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Val Phe Arg Glu Leu Tyr Thr Ala Tyr Ser Arg Glu Pro Asp Lys
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Pro Lys Lys Tyr Val Gln Asp Ile Leu Gln Glu Gln Leu Ala Glu
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Ser Val Tyr Arg Ala Leu Lys Glu Gln Gly Gly His Ile Tyr Val
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Cys Gly Asp Val Thr Met Ala Ala Asp Val Leu Lys Ala Ile Gln
1355 1360 1365
Arg Ile Met Thr Gln Gln Gly Lys Leu Ser Ala Glu Asp Ala Gly
1370 1375 1380
Val Phe Ile Ser Arg Met Arg Asp Asp Asn Arg Tyr His Glu Asp
1385 1390 1395
Ile Phe Gly Val Thr Leu Arg Thr Tyr Glu Val Thr Asn Arg Leu
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Arg Ser Glu Ser Ile Ala Phe Ile Glu Glu Ser Lys Lys Asp Thr
1415 1420 1425
Asp Glu Val Phe Ser Ser
1430
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Arg Ile Arg Arg Glu Glu Tyr Arg Arg Ala Ile Asn Gly Gln Ser Phe
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Asp Leu Ser
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Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Pro Gln
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Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Pro Gln Arg Ile Arg Arg
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Glu Glu Tyr Arg Arg Ala Ile Asn Gly Gln Ser Phe Asp Leu Ser
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<400> 7
Arg Ile Arg Arg Glu Glu Tyr Arg Arg Ala Ile Asn Gly Gln Ser Phe
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Asp Leu Ser Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Pro Gln
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Arg Ile Arg Arg Glu Glu Tyr Arg Arg Ala Ile Asn Gly Gln Ser Phe
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Phe Ile Arg Arg Glu Glu Tyr Arg Arg Ala Ile Asn Gly Gln Ser Phe
1 5 10 15
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Gly Ile Arg Arg Glu Glu Tyr Arg Arg Ala Ile Asn Gly Gln Ser Phe
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Ala Ile Arg Arg Glu Glu Tyr Arg Arg Ala Ile Asn Gly Gln Ser Phe
1 5 10 15
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Arg Ile Arg Arg Glu Glu Tyr Arg Arg Ala Ile Asn Gly Gln Ser Trp
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Val Ile Arg Arg Glu Glu Tyr Arg Arg Ala Ile Asn Gly Gln Ser Phe
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Thr Ile Arg Arg Glu Glu Tyr Arg Arg Ala Ile Asn Gly Gln Ser Phe
1 5 10 15
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<223> N6H
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Arg Ile Arg Arg Glu Glu Tyr Arg Arg Ala Ile His Gly Gln Ser Phe
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Arg Ile Arg Arg Glu Glu Arg Arg Arg Ala Ile Asn Gly Gln Ser Phe
1 5 10 15
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Ser Ile Arg Arg Glu Glu Tyr Arg Arg Ala Ile Asn Gly Gln Ser Phe
1 5 10 15
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Arg Ile Arg Arg Glu Glu Tyr Arg Arg Ala Ile Glu Gly Gln Ser Phe
1 5 10 15
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Arg Ile Arg Arg Glu Glu Tyr Arg Arg Ala Ile Cys Gly Gln Ser Phe
1 5 10 15
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<223> NF10W
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Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Pro Gln Arg Ile Arg Arg
1 5 10 15
Glu Glu Tyr Arg Arg Ala Ile Asn Gly Gln Ser Trp
20 25
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<220>
<223> CF10W
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Arg Ile Arg Arg Glu Glu Tyr Arg Arg Ala Ile Asn Gly Gln Ser Trp
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Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Pro Gln
20 25
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His His His His His His
1 5
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<212> PRT
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<220>
<221> PHOSPHORYLATION
<222> (6)
<223>
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Ala Ala Arg Arg Glu Ser Tyr Leu Lys Ala Thr Gln
1 5 10
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<221> BINDING
<222> (1)
<223> FITC
<220>
<221> PHOSPHORYLATION
<222> (6)
<223>
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Ala Ala Arg Arg Glu Ser Tyr Leu Lys Ala Thr Gln
1 5 10

Claims (9)

1. The application of the medicine taking the PSD-95GK structural domain as an action target point in preparing the neuroprotective medicine is characterized in that,
the medicine taking the PSD-95GK domain as an action target point comprises a polypeptide, and the sequence of the polypeptide is SEQ ID NO. 7.
2. The use of claim 1, wherein the neuroprotection comprises at least one of affecting PSD-95 binding to nNOS, treating ischemic reperfusion injury, and treating ischemic stroke.
3. The use of claim 1, wherein the sequence of the polypeptide comprises a sequence that targets the PSD-95GK domain.
4. The use of claim 1, wherein said sequence of said polypeptide comprises a transmembrane sequence.
5. The method of claim 4, wherein the transmembrane sequence consists of SEQ ID NO. 5.
6. The use of claim 1, wherein said neuroprotection comprises affecting PSD-95 binding to nNOS.
7. The use of claim 1, wherein the neuroprotection comprises treating cerebral ischemia reperfusion injury.
8. The use of claim 1, wherein said neuroprotection comprises treating ischemic stroke.
9. A polypeptide, characterized in that the sequence of the polypeptide is SEQ ID NO. 7.
CN202210102478.XA 2022-01-27 2022-01-27 Application of PSD-95GK domain as neuroprotective target Active CN114533874B (en)

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