Polypeptide with ASIC1a selective inhibitory activity and application thereof

Abstract

The invention relates to the technical field of active polypeptides, and discloses a polypeptide with ASIC1a selective inhibitory activity and application thereof. The amino acid sequence of the polypeptide is EDCIPKWKGCVNRHGDCCEGLECYVRR KSFEVCIPKTPKT, and the amino acid sequence of the polypeptide is shown in the specification. According to the polypeptide with ASIC1a selective inhibitory activity and the application thereof, the polypeptide can specifically inhibit channel current of ASIC1a and can effectively inhibit channel current of ASIC1b, the molecular weight is 4629.40 Da, and compared with a monoclonal antibody with large molecular weight, the polypeptide has good blood-brain barrier penetration rate, does not have remarkable cytotoxicity after being tested by experiments, and can be used for preparing a monoclonal antibody with ASIC1a selective inhibitory activity. The possibility of causing human immune response is low, potential unsafe factors are relatively few, and the compound can be used for preparing neuroprotective drugs for treating ischemic brain injury caused by cerebral arterial thrombosis.

Description

Technical Field

[0001] The present invention relates to the technical field of active polypeptides, in particular to polypeptides with ASIC1a selective inhibitory activity and applications thereof. Background Art

[0002] Stroke is a devastating neurological disease with complex pathophysiology, characterized by high morbidity, recurrence, disability, and mortality. Ischemic stroke is the most common type, accounting for approximately 60% to 70% of all strokes. Therefore, the development of effective neuroprotective drugs for the treatment of ischemic brain damage caused by ischemic stroke is of paramount importance.

[0003] During a stroke, the infarcted area suffers from severe glucose and oxygen deprivation due to ischemia, which in turn causes excessive release and accumulation of glutamate, triggering excitotoxicity and leading to neuronal death. Recent studies have revealed that in ischemic brain injury, in addition to the well-known NMDARs (N-methyl-D-aspartate receptors), glutamate can also increase the sensitivity and openness of acid-sensing ion channels (ASICs) to acidic environments, promoting their activity and causing excitotoxicity. By comparing the effects of NMDAR antagonists and ASIC knockout on cerebral infarct volume in stroke-induced mice, the study showed that blocking ASICs can achieve a similar protective effect as blocking NMDARs in reducing ischemic brain damage. This finding suggests that ASICs may be as important as NMDARs in stroke treatment, indicating that ASICs are a new and underappreciated target for stroke treatment.

[0004] Selective inhibition of ASIC1a channel currents by inhibitors is considered a promising approach for the treatment of ischemic brain injury. In vitro studies have shown that in the simulation of cellular acidosis, the use of non-selective inhibitors (Amiloride) or selective inhibitors (PcTx1) of ASIC1a can effectively reduce intracellular Ca 2+ Concentrations of ASIC1a were significantly reduced, effectively reducing neuronal damage. Neurons lacking or knocked out of the ASIC1a gene were significantly resistant to acidic damage. In vivo studies further validated the role of ASIC1a in cerebral ischemia. In a model of focal cerebral ischemia, intraventricular administration of non-selective or selective ASIC1a inhibitors reduced infarct size by up to 60%.

[0005] At present, the reported inhibitors of ASIC1a channels mainly include two categories: small molecule organic compounds and polypeptide toxins. One category is small molecule organic compounds including amiloride and its derivatives, as well as non-steroidal anti-inflammatory drugs (such as efavirenz, ibuprofen, aspirin, etc.). Amiloride is the first reported ASICs inhibitor, but it is a weak non-selective inhibitor and has no significant difference in inhibitory effect on different ASIC subtypes within the micromolar concentration range (IC 50 :5-100μM). Nonsteroidal anti-inflammatory drugs have a higher inhibitory concentration (IC 50 :92-249μM) and have inhibitory effects on both ASIC1a and ASIC3. Although these small molecule compounds can reduce neuronal damage caused by acidosis, their poor selectivity and high inhibitory concentrations limit their potential as neuroprotective agents.

[0006] Another type of ASIC1a inhibitor is a polypeptide toxin extracted from animal venom glands. Compared with small molecule compounds, this type of polypeptide toxin shows higher selectivity and more significant inhibitory effects, and is therefore more suitable for development as a neuroprotective drug for the treatment of ischemic brain injury. At present, there are a total of four active polypeptides that have been discovered that can selectively inhibit ASIC1a channel currents. Among them, the polypeptide toxin PcTx1 isolated from the venom gland of the Trinidad Chevron wolf spider is the first discovered active polypeptide that can selectively inhibit homologous ASIC1a currents. It plays an important role in the treatment of ischemic stroke. PcTx1 can selectively inhibit homologous ASIC1a currents. PcTx1 has an effect on the IC of rat rASIC1a. 50 The IC value for human hASIC1a is 1 nM. 50 The concentration of PcTx1 in the ASIC1a channel was 3 nM (inhibits channel current at pH 7.2). However, it is worth noting that while PcTx1 selectively inhibits ASIC1a current, high concentrations of PcTx1 can enhance ASIC1b channel current (EC 50 ASIC1b is primarily expressed in peripheral sensory neurons and has been shown to play a role in the nociceptive system in rodents. Therefore, the development and application of PcTx1 as a drug molecule for the treatment of ischemic stroke may result in some predictable adverse reactions in the organism. Summary of the Invention

[0007] The purpose of the present invention is to provide a polypeptide with selective inhibitory activity of ASIC1a and its application. The polypeptide can specifically inhibit the channel current of ASIC1a and effectively inhibit the channel current of ASIC1b. The polypeptide has a molecular weight of 4629.40Da and has a better blood-brain barrier penetration rate than monoclonal antibodies with larger molecular weights. It has no significant cytotoxicity after experimental testing, is unlikely to cause an immune response in the human body, and has relatively few potential unsafe factors. It can be used to prepare a neuroprotective drug for treating ischemic brain damage caused by ischemic stroke.

[0008] To achieve the above objectives, the present invention provides a polypeptide having selective ASIC1a inhibitory activity, the amino acid sequence of which is EDCIPKWKGCVNRHGDCCEGLECYVRRKSFEVCIPKTPKT.

[0009] Furthermore, the present invention also provides the use of the above polypeptide in the preparation of an ASIC1a selective inhibitor.

[0010] Furthermore, the present invention also provides the use of the above polypeptide in the preparation of neuroprotective drugs for ischemic brain injury.

[0011] Furthermore, the present invention also provides the use of the above polypeptide in the preparation of a neuroprotective drug for treating ischemic brain damage caused by ischemic stroke.

[0012] Furthermore, the present invention also provides an ASIC1a selective inhibitor comprising the above-mentioned polypeptide.

[0013] Furthermore, the present invention also provides a neuroprotective drug comprising the above-mentioned polypeptide.

[0014] The polypeptide sequence of the present invention can inhibit the channel current of ASIC1a by 37.5% at a concentration of 10 nM. 50 The concentration of 10 nM was 37.829 nM, and it did not enhance the channel current of ASIC1b at high concentrations. It could inhibit the channel current of ASIC1b by 31.5% at a concentration of 10 nM. 50 It is 83.947nM.

[0015] The advantages and positive effects of the polypeptide having ASIC1a selective inhibitory activity and its application according to the present invention are:

[0016] The polypeptide described in the present invention can specifically inhibit the channel current of ASIC1a and can effectively inhibit the channel current of ASIC1b. It has a molecular weight of 4629.40Da and has a better blood-brain barrier penetration rate than monoclonal antibodies with larger molecular weights. It has no significant cytotoxicity after experimental testing, is unlikely to cause an immune response in the human body, and has relatively few potential unsafe factors. It can be used to prepare neuroprotective drugs for treating ischemic brain damage caused by ischemic stroke.

[0017] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Schematic diagram of the mutation sites of the polypeptides in the examples of the present invention;

[0019] Figure 2 This is a heat map of immunoadsorption between the mutant library-3rd selected monoclonal and HEK293T-hASIC1a in the embodiment of the present invention;

[0020] Figure 3 Schematic diagram of the inhibitory effect of the polypeptide on ASIC1a in an embodiment of the present invention;

[0021] Figure 4 Schematic diagram of the inhibitory effect of the polypeptide on ASIC1b in an embodiment of the present invention;

[0022] Figure 5 This is a schematic diagram of SDS-PAGE of heterologous expression of polypeptides in an embodiment of the present invention;

[0023] Figure 6 This is the cell activity detection result of the polypeptide in the embodiment of the present invention. DETAILED DESCRIPTION

[0024] The technical solution of the present invention is further described below with reference to the accompanying drawings and embodiments.

[0025] Unless otherwise defined, technical or scientific terms used in the present invention shall have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs.

[0026] All other embodiments obtained by persons of ordinary skill in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention. Experimental methods in the following examples where specific conditions are not specified are generally determined in accordance with national standards. Experimental instruments, equipment, and reagents in the following examples where the sources are not specified are all commercially available raw materials.

[0027] Unless otherwise defined or indicated, all technical and scientific terms used in this invention have the same meanings as those familiar to those skilled in the art. Furthermore, any methods and materials similar or equivalent to those described herein may be applied to the methods of the present invention. It should be noted that, unless there is a conflict, the embodiments and features of the embodiments of the present invention may be combined with each other.

[0028] Example 1

[0029] The present invention performs mutations on PcTx1 and first determines that four amino acid residues (Trp24, Lys25, Arg28, Thr37) play a key role in PcTx1's selection of ion channel subtypes. Therefore, these four active sites are used as randomized sites ( Figure 1 By saturating mutagenesis at key sites (amino acid residues marked in green), a random tetrapeptide library was constructed. The constructed random tetrapeptide library was then displayed on the phage surface using phage display technology.

[0030] In the present invention, the Sleeping Beauty technology was used to construct a stable cell line HEK293T-ASIC1a that stably expresses the voltage-gated ASIC1a ion channel, and the cell line was inoculated into a 96-well ELISA plate (1×10 4 cells / well), cultured in a 37°C, 5% CO2 incubator for 2-3 days, and can be used as a target antigen for detection.

[0031] 5×10 12 The random peptide library of CFU was incubated at room temperature for 2 hours. After washing the plate three times with PBS, the binding solution was added with Glycine-HCl (pH 7.4) for elution. The eluate was mixed with E. coli XL1-Blue and incubated at 37°C for 30 minutes, followed by shaking culture. The amplified antibody 1st library was obtained by shaking culture at 30°C overnight.

[0032] Repeat the above steps to obtain the 2nd and 3rd libraries respectively.

[0033] The second and third rounds of amplified libraries of the mutant library were used for ELISA detection (secondary antibody: M13-HRP). The immunoabsorption results of the mutant library-2nd and mutant library-3rd with the stable cell line HEK293T-ASIC1a are shown in Table 1

[0034] Table 1 Binding of mutant library-2nd and mutant library-3rd to the stable cell line HEK293T-ASIC1a

[0035]

[0036] Mutation library-3rd showed stronger immunoadsorption ability with the stable cell line HEK293T-ASIC1a, and further monoclonal screening was performed from mutation library-3rd to identify active peptide molecules with good selective inhibition effect on ASIC1a.

[0037] Based on the above test results, clones with significant absorbance differences relative to the negative control were selected for retesting and sequencing to obtain the polypeptide sequences, as shown in Table 2.

[0038] Table 2 Peptide sequences

[0039]

[0040] The active polypeptide sequence obtained by screening is as follows: EDCIPKWKGCVNRHGDCCEGLECYVR RKSFEVCIPKTPKT (SEQ ID NO. 1).

[0041] Example 2

[0042] Inhibition analysis:

[0043] This peptide sequence can inhibit 37.5% of ASIC1a channel current at a concentration of 10 nM. 50 37.829nM( Figure 3 It does not enhance the channel current of ASIC1b at high concentrations, but can inhibit the channel current of ASIC1b by 31.5% at a concentration of 10 nM, IC 50 83.947nM( Figure 4 shown).

[0044] The active polypeptides of the present invention can be obtained by chemical synthesis or various acquisition methods such as prokaryotic and eukaryotic methods.

[0045] The amino acid composition and molecular weight of the peptides were predicted using the Expasy ProtParam tool. The peptides were obtained by chemical synthesis or prokaryotic expression, and their molecular weights were identified by SDS-PAGE. Figure 5 As shown), the peptide molecular weight is 4629.40Da, which has a better blood-brain barrier penetration rate than monoclonal antibodies with larger molecular weight. And the peptide has no significant cytotoxicity ( Figure 6 As shown in Figure 2, the likelihood of triggering an immune response in the human body is low, and potential safety risks are relatively few. Therefore, active peptides can be used to develop neuroprotective drugs for the treatment of ischemic brain damage caused by ischemic stroke and are widely used in biotechnology, biomedicine and other fields.

[0046] Therefore, the present invention adopts the above-mentioned polypeptide with selective ASIC1a inhibitory activity and its application. The polypeptide can specifically inhibit the channel current of ASIC1a and effectively inhibit the channel current of ASIC1b. It has a molecular weight of 4629.40 Da and has a better blood-brain barrier penetration rate than monoclonal antibodies with larger molecular weight. It has no significant cytotoxicity after experimental testing, is unlikely to cause an immune response in the human body, and has relatively few potential unsafe factors. It can be used to prepare neuroprotective drugs for treating ischemic brain damage caused by ischemic stroke.

[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 the same. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solutions of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A polypeptide having selective ASIC1a inhibitory activity, characterized in that: Its amino acid sequence is EDCIPKWKGCVNRHGDCCEGLECYVRRKSFEVCIPKTPKT.

2. Use of the polypeptide according to claim 1 in the preparation of an ASIC1a selective inhibitor.

3. Use of the polypeptide according to claim 1 in the preparation of a neuroprotective drug for ischemic brain injury.

4. Use of the polypeptide according to claim 1 in the preparation of a neuroprotective drug for treating ischemic brain damage caused by ischemic stroke.

5. An ASIC1a selective inhibitor, characterized in that: Comprising the polypeptide according to claim 1.

6. A neuroprotective drug, characterized in that: Comprising the polypeptide according to claim 1.

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