A permeabilizing peptide targeting the functional domain of Migfilin and its applications

Abstract

This invention belongs to the field of biomedical technology, and particularly relates to a transmembrane peptide targeting the functional domain of Migfilin and its applications. The transmembrane peptide comprises a functional segment and a transmembrane segment, wherein the transmembrane segment is located at the N-terminus or C-terminus of the functional segment, and the amino acid sequence of the functional segment is shown in SEQ ID NO:1 or SEQ ID NO:2. The transmembrane peptide of this invention can selectively intervene in the Migfilin-mediated platelet integrin αIIbβ3 outside-in signaling pathway, thereby inhibiting excessive platelet activation, aggregation, particle release, and thrombus formation, while minimizing adverse effects on normal physiological hemostasis.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a transmembrane peptide targeting the functional domain of Migfilin, a pharmaceutical composition containing the transmembrane peptide, a method for preparing the same, and its application in regulating platelet adhesion, spreading, secretion, aggregation, thrombosis and prevention and treatment of related thrombotic diseases. Background Technology

[0002] Thrombotic cardiovascular and cerebrovascular diseases, such as cerebral infarction, myocardial infarction, and other arterial thrombotic events, are among the leading causes of clinical death and disability. Platelet activation, adhesion, aggregation, and thrombus stabilization play a crucial role in the development and progression of these diseases.

[0003] While existing antiplatelet and antithrombotic therapies can inhibit thrombus formation to some extent, most still carry varying degrees of bleeding risk. One reason for this is that many existing drugs directly inhibit the activation or ligand binding function of platelet integrin αIIbβ3, or interfere with upstream signaling processes equally important for normal physiological hemostasis. Thus, while inhibiting pathological thrombus formation, they also weaken normal hemostatic capacity. Previous studies have proposed that selectively intervening in integrin αIIbβ3-mediated outside-in signaling may, to some extent, achieve a balance between antithrombotic effects and preservation of basic hemostatic function. Furthermore, designing transmembrane short peptides around the interaction interface between the intracellular segment of integrin and its aptamer proteins has also proven to be a feasible technical approach. However, existing technologies mainly focus on the interaction interfaces such as Gα13, β3 tail ExE motif, and 14-3-3 / c-Src / β3 complex. For Migfilin, a key aptamer protein involved in the regulation of platelet integrin αIIbβ3 outside-in signaling, there is still a lack of mature technical solutions for selective intervention targeting its specific functional domains or key protein interaction sites.

[0004] Meanwhile, existing research has shown that Migfilin participates in platelet activation and thrombosis, and its role is more inclined towards the integrin-mediated outside-in signaling stage (Yangfan Zhou, Mengjiao Hu, Xiaoyan Chen, et al. Migfilin supports hemostasis and thrombosis through regulating platelet αIIbβ3 outside-in signaling. Haematologica 2020; 105(11):2608-2618.). However, on the other hand, although the overall absence of Migfilin can weaken thrombus stability, it may still lead to coagulation dysfunction. This indicates that not all functions of Migfilin can be broadly inhibited to achieve antithrombotic effects without increasing the risk of abnormal bleeding. The roles of different domains and binding interfaces of Migfilin in platelet activation, thrombosis, and physiological hemostasis may not be the same. Therefore, the key problem that the existing technology has not yet solved is: how to screen out specific targets that can preferentially regulate pathological thrombosis and have little impact on normal hemostasis from different functional regions of Migfilin and their interaction sites with effector molecules such as filamin A, kindlin-3, and Src, and further construct transmembrane short peptides that can enter the cell to exert their effects. Summary of the Invention

[0005] The technical problem this invention aims to solve is to provide a transmembrane peptide designed based on the functional domain of Migfilin or its key protein interaction sites, and its application scheme. This peptide can selectively intervene in the Migfilin-mediated platelet integrin αIIbβ3 outside-in signaling pathway, thereby inhibiting excessive platelet activation, aggregation, particle release, and thrombus formation, while minimizing adverse effects on normal physiological hemostasis. Furthermore, this invention also addresses how to screen and optimize truncated Migfilin sequences or candidate sites of action to obtain candidate transmembrane peptides with good antithrombotic activity while maintaining hemostatic safety, thus providing a technical basis for the development of new antithrombotic intervention molecules.

[0006] The present invention specifically adopts the following technical solution:

[0007] In a first aspect, the present invention provides a polypeptide that targets the functional domain of Migfilin, wherein the amino acid sequence of the polypeptide is GHAIPSEEELPPPPEEPVTL (SEQ ID NO:1) or KRVASS (SEQ ID NO:2).

[0008] Secondly, the present invention provides a transmembrane peptide that targets the functional domain of Migfilin, the transmembrane peptide comprising a functional segment and a transmembrane segment, the transmembrane segment being disposed at the C-terminus of the functional segment, and the amino acid sequence of the functional segment being as shown in SEQ ID NO:1 or SEQ ID NO:2.

[0009] In a further embodiment, the transmembrane segment is CCR7, and its amino acid sequence is shown as CCRRRRRRR (SEQ ID NO:3).

[0010] This invention, through experiments, has shown that the permeabilizing peptide of this invention has the ability to reduce platelet adhesion, spreading, and thrombus formation, and exhibits stable antithrombotic effects in in vivo thrombosis models without significantly increasing the risk of bleeding. Therefore, in a third aspect, this invention also provides the application of the polypeptide or the permeabilizing peptide in the preparation of drugs for the prevention and treatment of thrombotic diseases. The thrombotic diseases include: platelet adhesion, spreading, secretion, aggregation, and thrombus formation.

[0011] Fourthly, the present invention provides a drug for the prevention and treatment of thrombotic diseases, wherein the active ingredient of the drug is the polypeptide or the transmembrane peptide.

[0012] The beneficial effects of this invention are as follows:

[0013] The transmembrane peptides obtained by screening in this invention can selectively inhibit platelet outside-in signaling-related functions without affecting platelet inside-out signaling and basic hemostatic function, thereby reducing platelet adhesion, spreading and thrombus formation. They also exhibit stable antithrombotic effects in in vivo thrombosis models without significantly increasing bleeding risk, indicating that functional peptides designed based on the Migfilin domain have potential application value as a low-bleeding-risk antithrombotic intervention strategy.

[0014] This invention, for the first time, designs a transmembrane short peptide based on the key structural domain of Migfilin, achieving selective inhibition of platelet integrin outside-in signaling. This creates a balance between the antithrombotic and hemostatic functions of natural Migfilin, opening up a novel approach to antithrombotic drug design. It exerts its antithrombotic effect while preserving platelet inside-out signaling and basic hemostatic function. In vivo thrombosis models have verified that it does not significantly increase the risk of bleeding, achieving a balance between antithrombosis and hemostasis. This approach holds promise for addressing the clinical pain point of bleeding complications associated with existing antiplatelet drugs. Unlike previously reported RGT peptides and Myr-RKEFAK peptides that target the intracellular domain of integrin β3, this invention, based on the key structural domain of Migfilin, possesses a unique and irreplaceable amino acid sequence and target site. Attached Figure Description

[0015] Figure 1: Membrane permeation status of pep1 and pep2, which connect transmembrane sequences.

[0016] Figure 2 Effects of different concentrations of pep1 on platelet aggregation (Part 1) and effects of different concentrations of pep1 on agonist-induced ATP release levels (Part 2).

[0017] Figure 3 Effects of different concentrations of PEP2 on platelet aggregation (Part 1) and effects of different concentrations of PEP2 on agonist-induced ATP release levels (Part 2).

[0018] Figure 4 Pep1 and pep2 can significantly inhibit thrombus formation in mice.

[0019] Figure 5 Effects of Pep1 and pep2 on hemorrhage after tail amputation in mice.

[0020] Figure 6 Effects of Pep1 and pep2 on cerebral hemorrhage in mice.

[0021] In the figure, Con1 represents the negative control random sequence transmembrane peptide 1 in the pep1 group experiment, and Con2 represents the negative control random sequence transmembrane peptide 2 in the pep2 group experiment. Detailed Implementation

[0022] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions in the embodiments of this invention will be clearly described below. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0023] The experimental materials used in the examples are as follows:

[0024] 1. Candidate permeabilizing peptides

[0025] The candidate peptides described in this invention include pep1 and pep2, both peptides designed based on truncated Migfilin amino acid sequences and obtained through permeabilization modification. Specifically, the amino acid sequence of pep1 is GHAIPSEEELPPPPEEPVTL (SEQ ID NO:1), and the amino acid sequence of pep2 is KRVASS (SEQ ID NO:2). A CCR7 transmembrane sequence (CCRRRRRRR, SEQ ID NO:3) is linked to the C-terminus of both pep1 and pep2 via a disulfide bond, with a purity >95%. These peptides were designed by the inventors and synthesized by Zhongke Huayao (Jiangsu) Biotechnology Co., Ltd. FITC-labeled pep1 and pep2 for cell entry or localization experiments using fluorescently labeled peptides were also prepared by Zhongke Huayao (Jiangsu) Biotechnology Co., Ltd., with a purity >95% and C-terminal linkage. Since these candidate peptides are custom-synthesized peptides, they do not have publicly known CAS numbers. See Table 1 for details of the candidate peptides.

[0026] Table 1. Candidate peptides

[0027]

[0028] 2. Control peptide

[0029] To evaluate the specificity of candidate peptides and the direction of screening, a negative control was set up in this embodiment of the invention. The negative control was a random sequence transmembrane peptide 1 (Con1): EPHPEPGPLPIEASEVTPEL (SEQ ID NO:4), or a random sequence transmembrane peptide 2 (Con2)SVSKAR (SEQ ID NO:5).

[0030] 3. Animal materials

[0031] The background strain of experimental animals was C57BL / 6. The animals were male, 6-8 weeks old, and weighed 20-25g. They were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.

[0032] 4. Materials related to platelet preparation

[0033] Materials used for separating and detecting platelets include:

[0034] Sodium citrate anticoagulant (39 mM citrate powder, 75 mM trisodium citrate powder, 135 mM glucose powder, diluted with double-distilled water to volume, adjusted pH to 6.5, and stored at 4°C).

[0035] Tyrode's buffer (137 mM sodium chloride powder (NaCl), 20 mM HEPES powder, 2.5 mM potassium chloride powder (KCl), 13.8 mM sodium bicarbonate powder (NaHCO3), 0.36 mM sodium dihydrogen phosphate (NaH2PO4), 5.5 mM glucose powder, diluted to volume with double-distilled water, adjust pH to 7.4, and store at 4°C).

[0036] Collagen (P / N385 Chrono-log Corporation (USA));

[0037] apyrase (A6535-100UN, Sigma-Aldrich, USA);

[0038] BSA (V900933-100G Sigma-Aldrich (USA));

[0039] CaCl2 (10043-52-4 China National Pharmaceutical Chemical Reagent Co., Ltd.), etc.

[0040] 5. Platelet agonists and related reagents for functional testing

[0041] Agonists used in platelet aggregation and release assays include:

[0042] Collagen (P / N385 Chrono-log Corporation (USA));

[0043] Thrombin (P / N386 Chrono-log, USA), etc.

[0044] Reagents used for ATP release detection include:

[0045] Luciferase (P / N395 Chrono-log, USA)

[0046] 6. Reagents for in vivo thrombosis models

[0047] FeCl3-induced thrombosis model: ferric chloride (FeCl3) (157740, 97% purity, Sigma-Aldrich (USA)).

[0048] Example 1

[0049] 1. Design, construction, and preliminary screening of candidate permeabilizing peptides

[0050] Based on the role of migfilin in platelet integrin αIIbβ3-mediated outside-in signaling, the inventors truncated and combined amino acid sequences corresponding to different domains of migfilin, focusing on potential interaction regions between migfilin and aptamers such as filamin A, kindlin-3, and Src, to construct a transmembrane short peptide library based on the truncated migfilin. Through preliminary screening, pep1: GHAIPSEEELPPPPEEPVTL; and pep2: KRVASS were selected as candidate functional fragments. The preliminary experiments involved using an agglutinator to detect the effect of different short peptides on platelet aggregation rate.

[0051] Then, the transmembrane sequence CCR7 was ligated to the C-terminus of pep1 and pep2, respectively, to obtain candidate peptides capable of entering the cell interior. The peptides were synthesized by Zhongke Huayao (Jiangsu) Biotechnology Co., Ltd. using solid-phase synthesis and purified by HPLC with a purity >95%. Specifically, the migfilin fragment corresponding to pep1 is rich in the Pro intermediate domain 156-175, with the complete sequence GHAIPSEEELPPPPEEPVTL; the migfilin fragment corresponding to pep2 is the N-terminus 7-12, with the complete sequence KRVASS.

[0052] Additionally, negative controls were set up: random sequence transmembrane peptide 1: EPHPEPGPLPIEASEVTPEL, and random sequence transmembrane peptide 2: SVSKAR; positive control: a migfilin-derived peptide (KPEKRVASSVFITLAP, whose C-terminus is linked to the CCR7 transmembrane sequence via a disulfide bond, as shown in SEQ ID NO:6), which has been shown to have a proactivating effect (hereinafter referred to as the positive control peptide). The proactivating peptide mimics the binding site of the N-terminal region of migfilin to filamin A. In resting platelets, filamin A binds to the β3 tail, preventing the interaction between Talin, Kindlin, and integrins. When platelets are activated, the inside-out signal pulls down filamin A, enabling subsequent Talin and Kindlin to bind to β3, ultimately leading to a high-affinity conformational change of αIIbβ3. Once activated αIIbβ3 binds to fibrinogen, this activating peptide prevents Filamin A from rebinding to β3 and supports sustained outside-in signaling.

[0053] 2. Membrane permeation of pep1 and pep2, which connect transmembrane sequences.

[0054] Mice were intraperitoneally anestheticized according to body weight. The inferior vena cava was exposed via the peritoneum, and whole blood was collected using a 2 mL syringe pre-filled with 200 μL of anticoagulant. After removing the needle, the blood was transferred to a 10 mL centrifuge tube containing 300 μL of anticoagulant. Tyrode's buffer was then added to bring the volume to 5 mL, gently inverted to mix, and centrifuged horizontally at 180×g for 10 min. The supernatant was collected into a centrifuge tube containing 0.5 mL of ACD, avoiding aspiration of red blood cells and white blood cells. The resulting platelet-rich supernatant was mixed and centrifuged at 700×g for 10 min. The supernatant was discarded, and the tube walls were carefully wiped dry, avoiding disturbance of the platelet precipitate at the bottom. Finally, 0.5 mL of Tyrode's buffer was added, and the platelets were gently resuspended using a cut pipette tip. 10 μL of the suspension was collected for platelet counting, and the resuspended platelets were diluted to the appropriate concentration as needed for the experiment to obtain washed platelets.

[0055] FITC-labeled pep1 and pep2 were used to perform mouse platelet entry and localization experiments, with the transmembrane sequence CCR7 as a control.

[0056] like Figure 1 As shown, both pep1 and pep2, which connect transmembrane sequences, can successfully enter mouse platelets.

[0057] In summary, the above results indicate that pep1 and pep2 have the potential to inhibit platelet aggregation.

[0058] 3. Effects of candidate peptides on platelet aggregation and ATP release

[0059] Washed platelets obtained from the same source and using the same preparation method as in the experiment on "membrane permeability of pep1 and pep2 connecting transmembrane sequences" were used to prepare transmembrane peptide (pep1, pep2) solutions with physiological saline as the solvent. Mouse-derived washed platelets were used, and the platelet concentration was adjusted to 3 × 10⁻⁶. 8 The platelet aggregation rate was measured at 37°C and mixed separately with candidate permeabilizing peptides pep1 and pep2, and incubated for 5 min. Two final concentrations of the two permeabilizing peptides were set: 5 μM and 10 μM. A random sequence permeabilizing peptide was added to the negative control group, while a migfilin-derived peptide with proven activation-promoting activity was added to the positive control group. After incubation, 5 μL of luciferase was added, and the mixture was stirred in an aggregator in the dark. Then, platelet agonists collagen (0.4 μg, 0.8 μg) and thrombin (0.018 U / mL, 0.025 U / mL) were added to stimulate platelet activation. The maximum platelet aggregation rate and ATP release level were measured.

[0060] The results showed that, for the maximum aggregation rate, both PEP1 and PEP2 inhibited platelet aggregation under collagen or thrombin stimulation, with PEP2 showing a more pronounced effect. Figure 2 and Figure 3 As shown, the above-mentioned inhibitory effect exhibits a dose-dependent trend for different concentrations (final concentrations of 5 μM and 10 μM) of the transmembrane peptide.

[0061] In contrast, the positive control peptide derived from the filamin A binding site at the N-terminus of migfilin showed that it promoted aggregation or enhanced activation under the same experimental conditions. This indicates that different peptides designed using migfilin as a template have different functional outputs. The candidate peptides of this invention are not simply derived from migfilin and automatically possess antithrombotic effects, but are specific sequences obtained through screening.

[0062] Regarding ATP release levels: Compared with the negative control group, both candidate peptides pep1 and pep2 reduced agonist-induced ATP release levels, such as... Figure 2 and Figure 3 As shown in the figure. This result indicates that, in addition to inhibiting aggregation, the candidate peptide can also inhibit the release of platelet dense granules, suggesting that it has an inhibitory effect on the platelet activation amplification process.

[0063] In contrast, the positive control peptide derived from the N-terminal filamin A binding site of migfilin showed an enhanced release response under the same experimental conditions.

[0064] 4. Effects of candidate peptides on thrombosis in mice

[0065] Male C57BL / 6 mice, aged 6-8 weeks, were randomly divided into a solvent control group, a pep1 treatment group, a pep2 treatment group, and a positive control peptide treatment group. Mice in each group were administered the corresponding drugs via tail vein injection at the following dosages: control group: 1.5 μmol / kg pep1 random sequence transmembrane peptide; pep1 treatment group: 1.5 μmol / kg pep1; pep2 treatment group: 1.5 μmol / kg pep2; positive control peptide treatment group: 1.5 μmol / kg positive control peptide. A thrombosis model was established 5 min after drug administration. DIOC6 fluorescently labeled platelets were injected via tail vein. The thrombosis model was established using FeCl3-induced mesenteric artery injury model, with the blood vessels treated with 10% FeCl3 for 3 min. The occlusion time was observed under a microscope to evaluate thrombus formation.

[0066] The results showed that both pep1 and pep2 treatments could inhibit thrombus formation in mice to varying degrees. Figure 4 As shown in the figure. Compared with the control group, the pep2 treatment group showed prolonged vascular occlusion time, slowed thrombus formation rate, reduced thrombus volume, and decreased stability, with the occlusion time increasing from 17 min to 42 min; pep1 also showed a similar inhibitory effect, with the occlusion time increasing from 18 min to 48 min.

[0067] In contrast, the positive control peptide derived from the filamin A binding site at the N-terminus of migfilin showed an increased tendency for thrombosis in vivo under the same experimental conditions. This result indicates that peptides designed using migfilin as a template do not naturally possess antithrombotic effects, and different fragments may exhibit opposite biological functions.

[0068] 5. Verification of hemostatic safety

[0069] 5.1 Effects of Pep1 and pep2 on hemorrhage after tail amputation in mice

[0070] Male C57BL / 6 mice, aged 6-8 weeks, were randomly divided into a negative control group, a pep1 treatment group, and a pep2 treatment group. Mice in each group underwent standardized tail amputation after intravenous injection of the corresponding drug. Bleeding time was continuously recorded and statistically analyzed. The drug dosage was as follows: negative control group: 1.5 μmol / kg pep1 random sequence transmembrane peptide; pep1 treatment group: 1.5 μmol / kg pep1; pep2 treatment group: 1.5 μmol / kg pep2.

[0071] The results showed that there was no significant difference (ns) in bleeding time between the pep1 and pep2 treatment groups compared with the negative control group. Figure 5 As shown. The data were statistically analyzed using t-tests, and the results are expressed as mean ± SEM.

[0072] 5.2 Effects of Pep1 and pep2 on cerebral hemorrhage in mice

[0073] Male C57BL / 6 mice, aged 6-8 weeks, were randomly divided into a negative control group, a pep1 treatment group, a pep2 treatment group, and a positive control group. Brain injury models were established in each group by intravenous injection of the corresponding drugs via the tail vein. Brain tissue sections were then analyzed, and the area of ​​the cerebral hematoma was quantitatively measured. The drug dosages were as follows: negative control group: 1.5 μmol / kg pep1 random sequence transmembrane peptide; pep1 treatment group: 1.5 μmol / kg pep1; pep2 treatment group: 1.5 μmol / kg pep2; positive control group: tirofiban 0.75 mg / kg. The brain injury was established as follows: mice were anesthetized, and a 52 mm diameter hole was drilled lateral to the anterior fontanelle (the junction of the coronal and sagittal sutures) of the skull. A 25-G needle was inserted into the mouse brain to create a 4 mm lesion.

[0074] The results showed that, compared with the negative control group, there was no significant difference in the cerebral hematoma area between the pep1 and pep2 treatment groups; however, the cerebral hematoma area was significantly increased in the tirofiban treatment group (*P < 0.05), (**P < 0.01). Figure 6 As shown. Data are expressed as mean ± SEM, and statistical analysis was performed using t-tests.

[0075] In summary, while overall loss of Migfilin can inhibit outside-in signaling and affect thrombus stability, it may still lead to coagulation dysfunction. Therefore, the goal of this invention is not simply to block all functions of Migfilin, but to screen specific fragments as more refined intervention sites. Candidate peptides pep1 and pep2 effectively inhibited thrombus formation while showing no significant or only slight prolongation of bleeding time in mice.

[0076] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

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

2.

2. A membrane-permeable peptide targeting the functional domain of Migfilin, characterized in that, The permeabilizing peptide includes a functional segment and a transmembrane segment, wherein the transmembrane segment is located at the C-terminus of the functional segment, and the amino acid sequence of the functional segment is shown in SEQ ID NO:1 or SEQ ID NO:

2.

3. The membrane-permeable peptide targeting the functional domain of Migfilin according to claim 2, characterized in that, The transmembrane segment is CCR7.

4. The use of the polypeptide of claim 1 or the transmembrane peptide of claim 2 or 3 in the preparation of drugs for the prevention and treatment of thrombotic diseases.

5. The application according to claim 4, characterized in that, Thrombotic diseases include: platelet adhesion, spreading, secretion, aggregation, and thrombus formation.

6. A drug for the prevention and treatment of thrombotic diseases, characterized in that, The active ingredient of the drug is the polypeptide of claim 1 or the membrane-permeable peptide of claim 2 or 3.

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