CN113880923B - A small molecule polypeptide that interferes with the binding of CDK5 and TRPV1 and its application - Google Patents

Abstract

The invention discloses a small molecular polypeptide interfering with CDK5 and TRPV1 combination and application thereof, wherein the amino acid sequence of the small molecular polypeptide is shown as SEQ ID NO. 1. The present invention is designed based on the interaction between interfering proteins, refers to the binding sequence on TRPV1 and CDK5 by literature, and determines the final small molecule polypeptide sequence based on the binding sequence, so that it can bind to CDK 5. The finally designed small molecule polypeptide can enter the cell interior through one end of the penetrating peptide sequence, and the other part can compete with TRPV1 for binding to CDK5, so that the binding between TRPV1 and CDK5 is reduced. The small molecule polypeptide provided by the invention can specifically interfere the interaction between CDK5 and TRPV1, thereby preventing the TRPV1 in cytoplasm from transferring to a cell membrane and relieving cerebral ischemia injury.

Description

A small molecule polypeptide that interferes with the binding of CDK5 and TRPV1 and its application

Technical field

The invention belongs to the field of biomedicine technology, and specifically relates to a small molecule polypeptide that interferes with the binding of CDK5 and TRPV1 and its application.

Background technique

Stroke is a common cerebrovascular disease and the leading cause of death and disability in adults. Stroke is divided into hemorrhagic stroke and ischemic stroke, among which ischemic stroke patients account for more than 80% of stroke patients. After a stroke, a variety of cells in the brain, including neurons, glial cells, and cerebrovascular endothelial cells, will be damaged, thus leading to dysfunction in the corresponding brain areas. Currently, tissue plasminogen activator (tPA) is still the only therapeutic drug approved by the U.S. Food and Drug Administration. However, the time window during which patients can use tPA is very narrow. It must be used within 4.5 hours of stroke onset, and there are to the risk of hemorrhagic transformation (Fukuta T, Asai T, Yanagida Y, et al. Combinationtherapy with liposomal neuroprotectants and tissue plasminogen activator for treatment of ischemic stroke. The FASEB Journal. 2017; 31(5):1879-1890.). Therefore, the development of new drugs to reduce the damage caused by stroke attacks and extend the patient's treatment time window is of great significance for the treatment of stroke.

After a stroke, multiple signaling pathways are often activated, leading to brain damage through signaling cascades. In the transmission process of the signal cascade, the interaction between upstream and downstream proteins plays an extremely critical role. More and more studies are turning their attention to the interactions between proteins. The LiXing team inhibited neuronal apoptosis induced by cerebral ischemia by designing a cell-penetrating peptide that interferes with the binding of annexin A1 (ANXA1) and importin β (Li X, Zheng L, Xia Q, et al .A novel cell-penetrating peptide protects against neuronapoptosis after cerebral ischemia by inhibiting the nuclear translocation ofannexin A1. Cell Death Differ. 2019; 26(2):260-275.). Research by Anthony J. Schulien's team shows that designing small peptides to interfere with the connection between delayed rectifier voltage-gated potassium channel Kv2.1 and vesicle-associated membrane protein interacting protein A (VAPA) can provide neuroprotection after stroke (Schulien AJ, Yeh CY, Orange BN, et al. Targeted disruption of Kv2.1-VAPA association provides neuroprotection against ischemic stroke in mice by declustering Kv2.1 channels. Sci Adv. 2020; 6(27).). These studies demonstrate that protein-protein interactions are becoming increasingly popular in stroke research.

TRPV1 has many important physiological regulatory functions, including temperature regulation, pain and nociception, and heat conduction. However, in most basic studies on TRPV1, the effect of interfering with TRPV1 is mainly through gene knockout or its inhibitor Capsazepine, AMG9810 (Hakimizadeh E, Shamsizadeh A, Roohbakhsh A, et al. Inhibition of transient receptor potential vanilloid-1confersneuroprotection, reduces tumor necrosis factor-alpha, and increases IL-10in arat stroke model. Fundam Clin Pharmacol. 2017; 31(4):420-428.; Miyanohara J, Shirakawa H, Sanpei K, Nakagawa T, Kaneko S. A pathophysiological role of TRPV1in ischemic injury after transient focal cerebral ischemia in mice. BiochemBiophys Res Commun. 2015; 467(3):478-483.). This will completely block the effect of TRPV1, thereby affecting other physiological functions of TRPV1. There are currently no reliable methods for specifically inhibiting the interaction between TRPV1 and downstream signaling. And many proteins used as therapeutic tools are unable to cross cell membranes, limiting their usefulness.

Contents of the invention

In view of the shortcomings of the existing technology, the present invention provides a small molecule polypeptide that interferes with the binding of CDK5 and TRPV1 and its application, which can specifically interfere with the interaction between CDK5 and TRPV1, thereby preventing TRPV1 in the cytoplasm from moving to the cell membrane. transfer and reduce cerebral ischemic damage.

The present invention is achieved through the following technical solutions:

A small molecule polypeptide that interferes with the binding of CDK5 and TRPV1. The amino acid sequence of the small molecule polypeptide is shown in SEQ ID NO. 1.

Application of a small molecule polypeptide that interferes with the binding of CDK5 and TRPV1 in the preparation of drugs for the treatment of cerebrovascular diseases.

Preferably, the cerebrovascular disease is ischemic stroke.

A pharmaceutical composition for treating cerebrovascular disease, including the above-mentioned small molecule polypeptide and a pharmaceutically acceptable carrier.

Preferably, the cerebrovascular disease is ischemic stroke.

The beneficial effects of the present invention are as follows:

1. The small molecule polypeptide designed in the present invention can specifically interfere with the binding of TRPV1 and CDK5 without affecting other physiological functions of TRPV1 and CDK5.

2. The present invention adds a cell-penetrating peptide sequence to one end of the small molecule polypeptide and connects it through four glycines, so that it can enter cells and exert its effects.

3. The present invention uses the thread embolization method to construct a mouse cerebral ischemia model, and injects small molecule polypeptides into the mouse brain at an appropriate dose through mouse brain stereotaxy. The results show that the small molecule polypeptide of the present invention improves cerebral ischemia. Blood damage, reduced infarct volume after cerebral ischemia in mice and pathological brain damage in mice, providing experimental basis and direction for drug application.

Description of the drawings

Figure 1 is a quantitative analysis of the correction of cerebral infarct volume in each group of mice in Example 2 (n=6, I/R vs. sham, ^**** P<0.0001; I/R+TAT vs. I/R, ^*** P＜0.001); data are expressed as mean±SD;

Figure 2 shows the open/closed field experiment of each group of mice in Example 3: Figure 2A, Figure 2B, and Figure 2C are respectively the trajectory diagram of the mouse open field experiment and the horizontal movement distance, number of explorations and central zone time statistics, Figure 2D , Figure 2E and Figure 2F respectively show the trajectory diagram and horizontal movement distance, number of explorations and central zone time statistics of the mouse closed-field experiment (n=12; one-way ANOVA, Tukey's test; I/R vs. sham, ^{* **} P＜0.001, ^** P＜0.01; I/R+TAT-T407 vs. I/R, ^* P＜0.05); data are expressed as mean ± standard deviation (means ± SD);

Figure 3 shows the water maze experiment of each group of mice in Example 4: Figure 3A shows the platform search during the escape latency (learning) period 1-5 days after reperfusion (n=12; multi-factor repeated measurement analysis of variance, Tukey's test, ^* P<0.05, I/R+TAT-T407vs.I/R); Figure 3B shows the number of times the mice crossed the platform in the exploration test, Figure 3C shows the distance percentage of the mice in the platform quadrant in the exploration test, Figure 3D shows the mice in the exploration test Percentage of time spent in the platform quadrant (n=12; one-way ANOVA, Tukey's test; I/R vs. sham, ^*** P < 0.001, ^** P <0.01; I/R + TAT-T407 vs. I /R, ^* P<0.05); data are expressed as means±SD;

Figure 4 shows Bax (Figure 4A), Bcl-2 (Figure 4B), Caspase-3 (Figure 4C), Cleaved Caspase-3 (Figure 4D), Cytochrome c ( Figure 4E) and NOX2 (Figure 4F) quantitative analysis of protein levels (n=3, I/R vs. sham, ^* P < 0.05, ^**** P <0.0001; I/R + TAT-407 vs. I/R, ^* P<0.05, ^** P<0.01, ^*** P<0.001); samples were taken from the ischemic penumbra; data are expressed as means±SD.

Detailed ways

The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments.

Example 1

A small molecule polypeptide that interferes with the binding of CDK5 and TRPV1. Its amino acid sequence is shown in SEQ ID NO.1, specifically: YGRKKRRQRRR-GGGG-IAYSSSETPNRHDML.

The present invention is designed based on the interaction between interfering proteins. The binding sequence 400-IAYSSSETPNRHDML-414 on TRPV1 and CDK5 is determined by consulting the article, and the final small molecule polypeptide sequence is determined based on this binding sequence, so that it can be combined with CDK5 binding. Since the small molecule peptide designed based solely on this sequence cannot enter cells to function, we added a cell-penetrating peptide sequence to one end of the small molecule peptide, connected by four glycines in the middle. The final designed small molecule peptide can enter the cell interior with the help of the membrane-penetrating peptide sequence at one end, and the other part can compete with TRPV1 for binding to CDK5, thereby reducing the binding of TRPV1 to CDK5.

In Examples 2-5, we applied the designed small molecule polypeptide (hereinafter referred to as small peptide) in the mouse middle cerebral artery occlusion (MCAO) model at a dose of 1 mg/80 μL through targeted injection in the cortex and hippocampus. Inject the small peptide into the brain, and the volume of each spot is 1 μL. First, we observed through fluorescence microscopy that the diffusion effect of the small peptide was good. Secondly, compared with the model group, the model + small peptide group significantly reduced the cerebral infarction volume and neuropathological damage of mice.

The experimental groups and steps designed in Examples 2-5 are as follows: C57BL/6J wild-type mice were randomly divided into 4 groups: sham operation group (sham group), ischemia-reperfusion group (I/R group), small peptide intervention group (I/R+TAT-T407 group), small peptide control group (I/R+TAT-T407A group). The TAT-T407 sequence is shown in SEQ ID NO.1, specifically: YGRKKRRQRRR-GGGG-IAYSSSETPNRHDML, and the TAT-T407A sequence is shown in SEQ ID NO.2, specifically: YGRKKRRQRRR-GGGG-IAYSSSEAPNRHDML, both by Sangon Bioengineering (Shanghai) Co., Ltd. The small peptide was dissolved in PBS, and the injection dose was 1 mg/kg. It was injected into the cortex M1 area and the hippocampus CA1 area 2 hours before ischemia.

Example 2 TAT-T407 reduces infarct volume after cerebral stroke in mice

The experimental animals were C57BL/6J mice, weighing 22-25g, license number: SCXK (Lu) 2019-003, provided by Jinan Pengyue Experimental Animal Co., Ltd. All mice used were kept under a 12-h light/dark cycle, the temperature was controlled at about 23°C by adjusting equipment, and the humidity was maintained in the range of 60 to 65%.

A mouse stroke model was constructed using the suture method. After 1 hour of ischemia, the mouse was anesthetized after 24 hours of cerebral ischemia and reperfusion. The brain was removed by decapitation. After the dura mater was peeled off, the mouse brain was placed in a -20°C refrigerator. Freeze at medium speed for 20 minutes. When the mouse brain is moderately soft and hard, remove excess parts such as the olfactory bulb, cerebellum and lower brainstem, and cut into 6 slices evenly along the coronal plane. Then, the cut brain slices were soaked in 2% TTC dye solution and placed in a 37°C oven to avoid light for 15 min. The successfully stained brain slices were fixed in 4% paraformaldehyde overnight and then taken out. The tissues were arranged in order, photographed and saved, and the cerebral infarct volume of the mice was calculated.

The dosage in this experiment was 1 mg/80 μL, and the administration route was stereotaxic injection into the brain. The injection points were the cortex and hippocampus, with 1 μL injected at each point.

The control group in this experiment was the sham operation group (sham group).

Figure 1 shows the quantitative analysis of the correction of cerebral infarct volume of mice in each group. This experiment was repeated 6 times. The results showed that TAT-T407 reduced the infarct volume of mice after stroke.

Example 3 Open Field Experiment

Field experiments are a method of evaluating the autonomous behavior, exploratory behavior and tension of experimental animals in new environments. On the 7th day after MCAO surgery, an open field experiment was conducted using the animal behavior tracking analysis system. The system of this open field experiment consists of a cubic openable and closable box with a side length of 50cm and a corresponding camera acquisition system. The mice were placed from the central zone for a total duration of 5 min. During this period, the total distance traveled by the mice, the time in the central zone and the number of crossings were recorded. Pay attention to cleaning up feces and urine between experiments, and spray 75% alcohol. Figures 2A, 2B, and 2C are open-field experiments, and Figures 2D, 2E, and 2F are closed-field experiments.

As shown in Figure 2, the results were repeated 13 times. The results of the open field experiment showed that compared with the I/R group, the mice pretreated with the small peptide intervention group (I/R+TAT-T407 group) exercised better in the open field experiment. The total distance, the number of explorations in the central area and the time spent in the central area were significantly increased, while the small peptide control group (I/R+TAT-T407A group) had no such effect. It shows that TAT-T407 can improve the voluntary movement and exploratory movement of mice after cerebral ischemia.

Example 4 Water Maze Experiment

The Morris water maze experiment trains animals' spatial learning and memory abilities by forcing them to swim. The Morris water maze pool has a diameter of 1m and a height of 0.5m. The pool is divided into 4 quadrants. Four different reference objects are placed diagonally in the quadrants. The fourth quadrant is set as the target quadrant, and a circle with a diameter of 6cm and a height of 30cm is placed in the center. Shape platform, the platform is placed 1cm underwater in the target quadrant. The water temperature is kept constant at (21±1)℃. Experiment preparation: water maze, electric blanket, milk powder.

The water maze experiment was started on the 7th day after MCAO surgery, and the room temperature was adjusted to approximately 24°C. The entire training period lasted for 6 days.

Visual platform experiment: Place the mice in the water maze room one day in advance to adapt to the environment, put the mice in from the opposite quadrant of the platform, and guide the mice to find the platform and stay on it for 30 seconds.

Positioning and navigation experiment: Day 1 is adaptive training, and days 2 to 5 are positioning and navigation experiments. Training is performed at a fixed time every day. On 4 days, the mice enter the maze from fixed water entry points in the 4 quadrants, and the mice are recorded within 60 seconds. The time required from entering the water to finding the platform was used as the escape latency period. If the platform is not found within 60 s, the mouse needs to be guided to the platform and stay on it for 30 s to guide its learning and directional memory. At this time, the escape latency is 60 s.

Space exploration experiment: On the 6th day of the experiment, remove the platform, randomly select the water entry point, and record and calculate within 60 seconds: swimming trajectory, target quadrant residence time as a percentage of the total time, number of times crossing the platform, and target quadrant distance as a percentage of the total distance.

Note: Due to reduced grooming, experimental mice can be placed on an electric blanket to maintain body temperature and quickly recover their physical strength.

The results were repeated 13 times. The results showed that in the Morris water maze test, compared with the I/R group of mice, the long-term effects of the small peptide intervention group (I/R+TAT-T407 group) on spatial learning and memory could be revealed. Obvious beneficial effects. During the spatial learning phase, all mice from different groups benefited from 5 days of training and showed gradually decreasing latency (as shown in Figure 3A). Mice pretreated with the small peptide intervention group (I/R+TAT-T407 group) shortened the latency to find the hidden platform and the swimming path to the platform, thereby improving spatial memory, with significant differences appearing on the fifth day. In the spatial detection test, compared with the mice in the I/R group, the small peptide intervention group (I/R+TAT-T407 group) increased the number of times they crossed the platform, and increased the time and distance in the target quadrant ( As shown in Figure 3B, Figure 3C, and Figure 3D). These results indicate that TAT-T407 improves learning and memory impairment after ischemic stroke in mice.

Example 5 Western Blotting Method

1. Gel preparation

(1) Insert the thick and thin glass plates into the plate clamps, making sure that the clamping plates are clamped and the bottom edges are kept flush.

(2) Water sealing experiment: Fill the plate with deionized water and let it sit for about 20 minutes. If there is no leakage, proceed to the next step.

(3) Since the molecular weight of the target protein in this experiment is 17 to 110 kDa, the separation gels with concentrations of 12%, 10%, and 8% and the stacking gel with 5% concentration are configured according to Table 1 and Table 2.

Table 1 5% SDS-PAGE stacking gel formula (2 gels)

Table 2 SDS-PAGE separation gel formula (2 gels)

(4) When an obvious coagulation line appears between the upper edge of the separation gel and absolute ethanol, discard the absolute ethanol, rinse the coagulation line with deionized water, discard the absolute ethanol on the separation gel, and use filter paper to absorb the remaining liquid. Add 5% concentrated gel, insert the comb gently, and wait until the concentrated gel solidifies.

2. Protein sample preparation and loading

(1) Assembly: Align the gel plate with the bayonet of the electrophoresis tank and assemble it together. Fill the inner tank with newly prepared electrophoresis solution and carefully remove the comb.

(2) Loading: The sample to be tested is kept in 100°C water for 6 minutes. After cooling, load 30 μg of protein per lane. The loading amount will be further corrected based on the band results. The marker is generally 2 to 3 μL, and is filled with waste sample to keep the volume of all lanes consistent. (The samples are protein samples obtained from the brain tissue on the same side of the mouse model in each group, extracted, and tested for BCA)

3. Electrophoresis

(1) Connection: Fill the outer tank with the electrophoresis solution to the inner tank page, and confirm that the positive and negative electrodes of the outer tank, cover, and power supply are consistent.

(2) Electrophoresis: Adjust the parameters to a constant voltage of 80V on the stacking gel until all the samples enter the separation gel and the markers are dispersed, then change to a constant voltage of 110V. The appearance of bubbles on the metal wire under the inner tank indicates the start of electrophoresis.

(3) Termination: The electrophoresis can be terminated when the upper and lower markers of the target protein can be completely distinguished and can be used for transfer to the membrane. Wash away the residual gel and electrophoresis solution on the surface of the gel plate with clean water.

4. Transfer film

(1) Cut the film: Cut the NC film into a size that fits the glue. Cut the upper right corner to mark the front side of the film and number it with a waterproof marker.

(2) Cut the gel: refer to the pre-stained protein marker to cut the gel containing the target protein.

(3) "Sandwich" structure of the film transfer clamp: Place the pre-cooled sponge, thick filter paper and thin filter paper in the electrotransfer solution, and place the black plate, sponge, thick filter paper, thin filter paper, strips, etc. in order from bottom to top. NC membrane, thin filter paper, thick filter paper, sponge, clamp the transparent plate and put it into the transfer tank, with the large molecular weight facing downwards. Note that there should be no air bubbles in between. Insert the positive and negative electrodes into the inner tank of the transfer film according to the corresponding colors. Put it in an ice box and add pre-cooled electrotransfer fluid to cover the electrotransfer clamp. Cover the outside of the transfer tank with crushed ice.

(4) Transfer: After confirming that the positive and negative electrodes are correct (the glue is on the negative electrode and the membrane is on the positive electrode), a constant current of 300mA is generally used, and the transfer time is calculated based on the protein molecular weight plus 20. Cleaved Caspase-3 can adopt constant pressure switching mode.

(5) Blocking: After the transfer is completed, wash the NC membrane with TBST for 1 min × 3 times, 5 min each time, then put it into 5% skim milk powder and block it for 1 to 2 hours at room temperature on a horizontal shaker or overnight at 4°C on a shaker. If it is a phosphorylated protein, it needs to be blocked with 5% BSA. After completion, wash the membrane with TBST for 5 min × 3 times, 5 min each time.

(6) Antibody incubation: After blocking, wash the strip with 1×TBST for 5 min×3 times, place it in the primary antibody incubation box, shake at 4°C overnight, and wash with TBST for 5 min×3 times the next day. Place the secondary antibody incubation box corresponding to the primary antibody species, shake at room temperature for 1.5 to 2 hours, and wash with TBST for 5 minutes × 3 times. If the cleaning effect is not good, the cleaning time can be appropriately extended.

The results were repeated three times. The results showed that interference with TAT-T407 can reduce apoptosis induced by I/R in mice.

It has been well documented that Bax activation induces cytosolic release of cytochrome c from mitochondria after ischemia, and that the released cytochrome c ultimately leads to cleavage and activation of caspase-3, representing intrinsic caspases The final step in a pathway that has previously been shown to regulate ischemic cell death. To investigate whether Bax, Cleaved Caspase-3, Cytochrome c, Bcl-2, Caspase-3 and NOX2 are involved in the protective mechanism of TAT-T407 peptide, we performed Western blotting assay.

As shown in Figure 4, compared with the sham group, mice in the I/R group had significantly increased NOX2 (Figure 4F), Bax (Figure 4A), Cleaved Caspase-3 (Figure 4D), and Cytochrome c (Figure 4E), and Bcl- 2 (Fig. 4B) was also significantly reduced. TAT-T407 treatment significantly reduced MCAO-induced expression of NOX2, Bax, Cytochrome c, and Cleaved Caspase-3, and increased the expression of Bcl-2, but had little effect on the expression of Caspase-3 (Figure 4C). At the same time, TAT-T407A does not have this therapeutic effect. The results further showed that the small peptide TAT-T407 interfered with the binding of TRPV1 and CDK5 and significantly improved the oxidative stress and apoptosis response after cerebral ischemia.

sequence list

<110> Xuzhou Medical University

Kuoran Precision Medicine Technology (Xuzhou) Research Institute Co., Ltd.

<120> A small molecule polypeptide that interferes with the binding of CDK5 and TRPV1 and its application

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 30

<212> PRT

<213> Artificial Sequence

<400> 1

Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Gly Gly Gly Gly Ile

1 5 10 15

Ala Tyr Ser Ser Ser Glu Thr Pro Asn Arg His Asp Met Leu

20 25 30

Claims (1)

1. The application of a small molecular polypeptide interfering with the combination of CDK5 and TRPV1 in preparing medicaments for treating ischemic stroke is characterized in that the amino acid sequence of the small molecular polypeptide is shown as SEQ ID NO. 1.