Application of engineered PARP1 or engineered PARP1 polypeptide in preparation of product for treating aortic dissection
By engineering PARP1, engineered PARP1 or engineered PARP1 polypeptides are prepared to prepare products for treating aortic dissection, which solves the pathological progression and vascular lesions of aortic dissection, and achieves the effect of significantly reducing the incidence of AD and reducing vascular lesions.
Patent Information
- Application Number
- CN202510623128.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-15
- Publication Date
- 2025-08-08
AI Technical Summary
The prior art is difficult to effectively solve the pathological progression and vascular lesions of aortic dissection, and traditional treatment methods have serious complications and it is difficult to fundamentally solve the vascular problem.
By engineering specific sites of PARP1, engineered PARP1 or engineered PARP1 polypeptides are prepared to inhibit their lactation, and the therapeutic products of aortic dissections are prepared, including applications in drug form, using engineered PARP1 or engineered PARP1 polypeptides.
It significantly reduces the incidence of aortic dissection, reduces vascular lesions, reduces aortic diameter dilation, and provides therapeutic effects on aortic dissection.
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Abstract
Description
Technical Field
[0001] The present invention relates to the field of biotechnology, and in particular to an application of an engineered PARP1 or an engineered PARP1 polypeptide in preparing a product for treating aortic dissection. Background Art
[0002] Aortic dissection (AD) is a serious cardiovascular emergency. Its primary pathological feature is a tear in the aortic intima, through which blood enters the medial layer of the aortic wall, leading to delamination of the aortic wall and the formation of a false lumen. Furthermore, this false lumen may expand or rupture acutely, becoming life-threatening and carrying an extremely high mortality rate. Epidemiological studies have shown that the incidence rate is significantly higher in men than in women, with the average age of onset being between 60 and 70 years. Major risk factors for AD include hypertension, atherosclerosis, hereditary connective tissue diseases, chest trauma, and perinatal conditions. Currently, treatment options for aortic dissection include medication, interventional therapy, and surgery. Medication primarily involves beta-blockers combined with calcium channel blockers to control heart rate and blood pressure to reduce pressure on the aortic wall, while analgesics are used to relieve acute chest pain. Complex cases with Stanford A or associated organ dysfunction require open surgery or interventional therapy, using stent grafts to isolate the intimal rupture and restore aortic blood flow. Given that traditional treatment methods have serious complications such as aortic rupture, peripheral nerve damage and paraplegia, it is difficult to fundamentally solve the problem of vascular disease.
[0003] PARP1 (poly ADP-ribose polymerase 1) is a key regulatory protein for DNA damage repair. It participates in biological processes such as DNA repair, epigenetic regulation, and cell fate determination by catalyzing the ribosylation of substrate protein poly ADP (PARP). Under physiological conditions, PARP1 maintains genomic stability by recruiting repair factors, but when cells are exposed to oxidative stress or genotoxic damage, its abnormal activation will trigger NAD +Excessive depletion of PARP1 leads to ATP biosynthesis and mitochondrial metabolic disorders, ultimately causing cell apoptosis or necrosis. In addition, abnormal PARP1 function is associated with the pathological processes of many diseases. In cancer, PARP inhibitors target tumors with DNA repair defects through a "synergistic synthetic lethality" effect. In cardiovascular diseases, overactivation of PARP1 aggravates the progression of myocardial ischemia-reperfusion injury and atherosclerosis by inducing the production of reactive oxygen species (ROS), the release of inflammatory factors, and programmed necrosis pathways. In neurodegenerative diseases, abnormal PARP1 activation is associated with mitochondrial dysfunction, axonal transport disorders, and impaired synaptic plasticity. It is worth noting that in cardiovascular diseases or tumor microenvironments, the coordinated regulation of SIRT1 inactivation and PARP1 hyperfunction, metabolic reprogramming, and epigenetic mechanisms such as histone modification, accelerate the progression of the disease. At present, there are no studies on the effect of PARP1 modification on the occurrence of aortic dissection.
[0004] SIRT1, a well-known histone deacetylase, plays a crucial role in cancer, aging, and cardiovascular disease through epigenetic regulation and metabolic homeostasis. In vascular homeostasis, SIRT1 inhibits the NF-κB inflammatory pathway, activates the FOXO antioxidant pathway, and maintains the contractile phenotype of vascular smooth muscle cells (VSMCs), thereby delaying vascular remodeling and aortic medial degeneration. SIRT1 regulates gene transcription by targeting non-histone proteins such as p53, HIF-1α, and E2F1, as well as histone H3K9 for deacetylation. Furthermore, PARP1 and SIRT1 form a dynamic antagonistic relationship through NAD+ metabolism in the cardiovascular system. Under pathological conditions such as oxidative stress or high glucose, PARP1 is overactivated, consuming significant amounts of NAD+ and inhibiting NAD+-dependent SIRT1 activity, leading to insulin resistance, mitochondrial dysfunction, and cardiomyocyte senescence, potentially triggering diabetic cardiomyopathy or chemotherapeutic cardiotoxicity. SIRT1, through deacetylation, regulates antioxidant genes, improving energy metabolism and inhibiting fibrosis, exerting cardiovascular protective effects. PARP1 relies on NAD+ for DNA damage repair, and its overactivation consumes intracellular NAD+, thereby inhibiting the NAD+-dependent SIRT1 deacetylase activity, leading to mitochondrial dysfunction, autophagy disorders, and abnormal oxidative stress response. It has been reported that SIRT1 has a strong ability to delactate and modify, and can affect glycolysis and myocardial function by regulating the lactation levels of non-histone proteins such as MDH2 and α-MHC. However, the role of PARP1 and SIRT1 in AD is still unclear. Given the key role of SIRT1 delactate activity in cardiovascular disease, it is very important to determine whether SIRT1's delactate modification of PARP1 has a biological function in AD. Summary of the Invention
[0005] The object of the present invention is to provide an application of an engineered PARP1 in the preparation of a product for treating aortic dissection. The present invention can inhibit the lactation of PARP1 by engineering the lactation sites of K498 / 505 / 506 / 508 / 518 / 521 / 524 through engineered PARP1 or engineered PARP1 polypeptide, and can prevent the occurrence of AD and delay the progression of AD, thereby achieving a therapeutic effect on AD.
[0006] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are adopted:
[0007] In a first aspect, the present invention provides a use of an engineered PARP1 or an engineered PARP1 polypeptide in preparing a product for treating aortic dissection, wherein the amino acid sequence of the engineered PARP1 is shown in SEQ ID NO: 1; the amino acid sequence of the engineered PARP1 polypeptide is shown in SEQ ID NO: 2.
[0008] Preferably, the gene encoding the engineered PARP1 or engineered PARP1 polypeptide is used in the preparation of a product for treating aortic dissection.
[0009] Preferably, a recombinant plasmid containing a gene encoding an engineered PARP1 or an engineered PARP1 polypeptide is used in the preparation of a product for treating aortic dissection.
[0010] Preferably, the product comprises a medicament.
[0011] Preferably, the pharmaceutical dosage forms include tablets, capsules, granules, pills, syrups, oral solutions, oral suspensions, oral emulsions and injections.
[0012] A second aspect of the present invention provides a drug for treating aortic dissection, comprising a therapeutically effective amount of engineered PARP1, an engineered PARP1 polypeptide, a gene encoding an engineered PARP1, a gene encoding an engineered PARP1 polypeptide, a recombinant plasmid containing a gene encoding an engineered PARP1 polypeptide, or a recombinant plasmid containing a gene encoding an engineered PARP1, wherein the amino acid sequence of the engineered PARP1 is shown in SEQ ID NO: 1; the amino acid sequence of the engineered PARP1 polypeptide is shown in SEQ ID NO: 2.
[0013] Preferably, the drug for treating aortic dissection further comprises a pharmaceutically acceptable excipient.
[0014] Preferably, the dosage forms of the drug for treating aortic dissection include tablets, capsules, granules, pills, syrups, oral solutions, oral suspensions, oral emulsions and injections.
[0015] Compared with the prior art, the beneficial effects of the present invention include at least:
[0016] The engineered PARP1 or engineered PARP1 polypeptide of the present invention can inhibit the lactation of PARP1 by engineering the lactation sites at K498 / 505 / 506 / 508 / 518 / 521 / 524, and can significantly reduce the incidence of AD. At the same time, it can alleviate vascular lesions and reduce the expansion of aortic diameter, and has a certain therapeutic effect on AD. BRIEF DESCRIPTION OF THE DRAWINGS
[0017] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly describes the drawings required for the specific embodiments or the description of the prior art. Similar elements or parts are generally identified by similar reference numerals throughout the drawings. Elements or parts in the drawings are not necessarily drawn to scale.
[0018] Figure 1 This is the research result of the potential PARP1 modifying enzyme in Example 1 of the present invention;
[0019] Figure 2 This is the research result of the effect of engineered PARP1 on VSMCs in Example 2 of the present invention;
[0020] Figure 3 This is the research result on the therapeutic effect of the engineered PARP1 polypeptide on AD mice in Example 3 of the present invention. DETAILED DESCRIPTION
[0021] The following embodiments of the technical solution of the present invention are described in detail in conjunction with the embodiments. The following embodiments are only used to more clearly illustrate the technical solution of the present invention and are therefore only used as examples and cannot be used to limit the scope of protection of the present invention.
[0022] It should be noted that, unless otherwise specified, the technical or scientific terms used in this application should have the common meanings understood by those skilled in the art to which the present invention belongs.
[0023]
[0024] In the present invention, the engineered PARP1 is based on PARP1 WT (wild type), and the lysine with lactylation function at the K498 / 505 / 506 / 508 / 518 / 521 / 524 sites is replaced with arginine.
[0025] The engineered PARP1 or engineered PARP1 polypeptide of the present invention can inhibit the lactation of PARP1 by engineering the lactation sites at K498 / 505 / 506 / 508 / 518 / 521 / 524, and can significantly reduce the incidence of AD. At the same time, it can alleviate vascular lesions and reduce the expansion of aortic diameter, and has a certain therapeutic effect on AD.
[0026] In one embodiment, a gene encoding an engineered PARP1 or an engineered PARP1 polypeptide is used in the preparation of a product for treating aortic dissection.
[0027] In one embodiment, a recombinant plasmid containing a gene encoding an engineered PARP1 or an engineered PARP1 polypeptide is used in the preparation of a product for treating aortic dissection.
[0028] In one embodiment, the vector used in the recombinant plasmid is pcDNA3.1-CMV-MCS-3flag-EF1A-zsGreen-sv40-puromycin.
[0029] In one embodiment, the product comprises a pharmaceutical.
[0030] In one embodiment, the pharmaceutical dosage forms include tablets, capsules, granules, pills, syrups, oral solutions, oral suspensions, oral emulsions, and injections.
[0031] Another embodiment of the present invention provides a drug for treating aortic dissection, comprising a therapeutically effective amount of engineered PARP1, an engineered PARP1 polypeptide, a gene encoding an engineered PARP1, a gene encoding an engineered PARP1 polypeptide, a recombinant plasmid containing a gene encoding an engineered PARP1 polypeptide, or a recombinant plasmid containing a gene encoding an engineered PARP1, wherein the amino acid sequence of the engineered PARP1 is shown in SEQ ID NO: 1; the amino acid sequence of the engineered PARP1 polypeptide is shown in SEQ ID NO: 2.
[0032] The present invention does not impose any specific restrictions on the dosage form of the drug. Preferably, in one embodiment, the dosage form of the drug for treating aortic dissection includes tablets, capsules, granules, pills, syrups, oral solutions, oral suspensions, oral emulsions and injections.
[0033] In one embodiment, the drug for treating aortic dissection further comprises a pharmaceutically acceptable excipient.
[0034] In the present invention, there is no specific limitation on the types of the above-mentioned excipients. Those skilled in the art can routinely select conventional excipients in the art according to the specific type of pharmaceutical dosage form.
[0035] The technical solution of the present invention is further described in detail below through specific embodiments.
[0036] Example 1
[0037] This example is a study on potential modifying enzymes of PARP1:
[0038] This study focused on exploring downstream targets that directly bind to PARP1. By using immunoprecipitation combined with liquid chromatography-mass spectrometry technology for proteomic screening, it was predicted that PARP1 and SIRT1 interact with each other. Studies have shown that SIRT1 plays a key regulatory role in pathological processes such as vascular remodeling, atherosclerosis, and vascular restenosis in VSMCs through apoptosis, inflammatory response, and oxidative stress pathways. Therefore, after preliminary screening, SIRT1 was determined to be a potential binding target ( Figure 1 Middle A). Immunofluorescence double staining experiments revealed that SIRT1 and PARP1 co-localized in the cell nucleus, further confirming the interaction between the two ( Figure 1 Middle B). Immunoprecipitation was used to bidirectionally verify whether SIRT1 is a target gene directly bound by PARP1. Anti-IgG, anti-SIRT1, and anti-PARP1 antibodies were used to enrich the protein. The results showed that anti-SIRT1 antibodies could specifically enrich PARP1 protein. Anti-PARP1 antibodies could also effectively capture SIRT1 protein. No obvious binding signal was detected in the non-specific IgG control group ( Figure 1 CD), which suggests a strong interaction between SIRT1 and PARP1. Given the interaction between SIRT1 and PARP1, to clarify whether the lactylation level of PARP1 is regulated by delactylase activity, SIRT1 overexpression plasmid and SIRT1 small interfering RNA were transfected. Co-IP experiments showed that SIRT1 overexpression reduced the level of PARP1 lactylation modification, while siSIRT1 treatment increased the level of PARP1 lactylation ( Figure 1 These results indicate that SIRT1 is a PARP1 delactylating enzyme and regulates the PARP1 lactylation level.
[0039] Example 2
[0040] This example is a study on the effects of SIRT1 and engineered PARP1 (denoted as PARP1 KR) on the function of VSMCs:
[0041] PARP1 has 7 lactylation sites K498 / 505 / 506 / 508 / 518 / 521 / 524 (abbreviated as PARP1 7K), and these 7 sites are relatively conserved in humans and mice, such as Figure 2 As shown in A. In this study, a wild-type plasmid (PARP1 WT plasmid, constructed and synthesized by Ruipuyuan Company, encoding wild-type PARP1) and a mutant plasmid (PARP1KR plasmid, constructed and synthesized by Hangzhou Zhongpeptide Biochemical Co., Ltd., encoding engineered PARP1) were constructed through the PARP1 lactylation site to detect their effects on VSMCs function.
[0042] Based on previous studies, PARP1 has 7 lactylation sites K498 / 505 / 506 / 508 / 518 / 521 / 524 (abbreviated as PARP1 7K), such as Figure 2 As shown in A. Therefore, it is speculated whether the lactylation level of PARP1 site is regulated by SIRT1, thereby affecting the function of VSMCs. This study constructed a wild-type plasmid (PARP1 WT plasmid) and a mutant plasmid (PARP1 KR plasmid) through the PARP1 lactylation site to detect its effect on the function of VSMCs. First, under the stimulation of sodium lactate pathological conditions, PARP1 WT plasmid and PARP1 KR plasmid were transfected into VSMCs to detect their overexpression efficiency and changes in lactylation modification. Translation and transcription levels showed that PARP1 WT and PARP1 KR had a high overexpression efficiency ( Figure 2 Co-immunoprecipitation experiments revealed that PARP1 WT had a higher lactylation level than the negative control group, while the lactylation level of PARP1 KR was significantly lower than that of PARP1 WT, confirming the conclusion that lactylation levels tend to decrease after mutation of the lactylation site. Figure 2 Subsequently, to verify whether SIRT1 mediates the regulation of VSMCs function by the lactylation site of PARP1, PARP1 WT plasmid and PARP1 KR plasmid were double-transfected into VSMCs with a SIRT1 overexpression plasmid, and the changes in lactylation modification and VSMCs function were detected. The experimental results showed that overexpression of SIRT1 could reverse the increase in lactylation modification caused by PARP1 WT ( Figure 2 Next, we found that PARP1 KR showed higher expression of MYH11, CNN1, and α-SMA, and lower expression of MMP2 and MMP9 compared with PARP1 WT at the translation and transcription levels ( Figure 2In addition, the expression of MYH11, α-SMA and CNN1, the contractile markers of VSMCs, was significantly increased after overexpression of SIRT1 compared with that after overexpression of PARP1 WT. However, the expression of MMP2 and MMP9 was significantly decreased, which indicates that overexpression of SIRT1 after transfection of PARP1 WT plasmid can maintain cell functional homeostasis ( Figure 2 Meanwhile, overexpression of SIRT1 can also reverse the increase of inflammatory markers IL-1β and TNF-α caused by PARP1 WT ( Figure 2 DCFH-DA probe and flow cytometry showed that PARP1 KR reduced ROS production and cell apoptosis. After transfection of PARP1 WT plasmid, overexpression of SIRT1 reduced fluorescence intensity and cell apoptosis rate ( Figure 2 It is noteworthy that the expression of NF-κB, p53, STAT1 and Caspase3 in PARP1 KR showed a downward trend. Overexpression of SIRT1 reversed the abnormal increase caused by PARP1 WT, suggesting that PARP1 may regulate VSMCs function through the NF-κB, p53, STAT1 and Caspase3 pathways ( Figure 2 The above experimental data indicate that SIRT1 mediates the lactylation of PARP1 to regulate the function of VSMCs.
[0043] Example 3
[0044] This example is a study on the therapeutic effect of engineered PARP1 polypeptide on AD mice:
[0045] The wild-type PARP1 polypeptide (denoted as pe-PARP1 WT, the amino acid sequence is shown in SEQ ID NO: 3, specifically EVVAPRGKSGAALSKKSKGQVKEEGINKSEKRMKLTLKGG) and the engineered PARP1 polypeptide (denoted as pe-PARP1 KR) were designed and synthesized.
[0046] At the initial stage of modeling, mice were randomly divided into 5 groups, with 11 mice in each group. AD was established at the same time, and sodium lactate was used for intervention. Each group was intraperitoneally injected with pe-PARP1 WT, pe-PARP1 WT and SRT1720, pe-PARP1 KR, and pe-PARP1KR and SRT1720, respectively. The intervention was carried out by intraperitoneal injection (5 mg / kg / day) for 14 days. The experimental design was as follows. Figure 3 Middle A. The survival rate of mice was continuously monitored during this period. Survival curve analysis showed that after AD and sodium lactate treatment, the survival rate of pe-PARP1WT combined with SRT1720 and pe-PARP1 KR was significantly increased compared with pe-PARP1 WT ( Figure 3According to the statistics of AD incidence, pe-PARP1 WT combined with SRT1720 and pe-PARP1 KR can significantly inhibit the incidence of AD in mice ( Figure 3 We can observe that these five groups have different degrees of vascular lesions. The vascular lesions of pe-PARP1 WT are the most severe, while the vascular lesions can be alleviated by combining pe-PARP1 WT with SRT1720 and pe-PARP1 KR ( Figure 3 E), pe-PARP1 KR has a certain therapeutic effect. The results of small animal ultrasound showed that compared with the pe-PARP1 WT group, pe-PARP1 WT combined with SRT1720 and pe-PARP1KR significantly reduced the expansion of the aorta diameter ( Figure 3 FG), while there was no significant difference between pe-PARP1 KR and pe-PARP1 KR combined with SRT1720, indicating that SIRT1 can regulate the lactylation level of PARP1 sites. The above evidence suggests that pe-PARP1 KR can delay the progression of AD.
[0047] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or make equivalent replacements for some or all of the technical features therein. These modifications or replacements do not deviate the essence of the corresponding technical solutions from the scope of the technical solutions of the embodiments of the present invention, and they should all be included in the scope of the claims and description of the present invention.
Claims
1. Use of engineered PARP1 or engineered PARP1 polypeptide in the preparation of a product for treating aortic dissection, characterized in that: The amino acid sequence of the engineered PARP1 is shown in SEQ ID NO: 1; the amino acid sequence of the engineered PARP1 polypeptide is shown in SEQ ID NO:
2.
2. The use according to claim 1, characterized in that Use of a gene encoding engineered PARP1 or an engineered PARP1 polypeptide in the preparation of a product for treating aortic dissection.
3. The use according to claim 1, characterized in that Use of a recombinant plasmid containing a gene encoding engineered PARP1 or an engineered PARP1 polypeptide in the preparation of a product for treating aortic dissection.
4. The use according to any one of claims 1 to 3, characterized in that: The products include pharmaceuticals.
5. The use according to claim 4, characterized in that The pharmaceutical dosage forms include tablets, capsules, granules, pills, syrups, oral solutions, oral suspensions, oral emulsions and injections.
6. A drug for treating aortic dissection, characterized in that: The drug for treating aortic dissection includes a therapeutically effective amount of engineered PARP1, an engineered PARP1 polypeptide, a gene encoding an engineered PARP1, a gene encoding an engineered PARP1 polypeptide, a recombinant plasmid containing a gene encoding an engineered PARP1 polypeptide, or a recombinant plasmid containing a gene encoding an engineered PARP1, the amino acid sequence of the engineered PARP1 is shown in SEQ ID NO: 1; the amino acid sequence of the engineered PARP1 polypeptide is shown in SEQ ID NO:
2.
7. The medicament for treating aortic dissection according to claim 6, characterized in that The medicine for treating aortic dissection also includes pharmaceutically acceptable excipients.
8. The medicament for treating aortic dissection according to claim 6, characterized in that The dosage forms of the drug for treating aortic dissection include tablets, capsules, granules, pills, syrups, oral solutions, oral suspensions, oral emulsions and injections.