Polypeptides with asic1a-selective inhibitory activity and uses thereof

Abstract

The present application relates to the technical field of active polypeptide, and discloses a polypeptide with ASIC1a selective inhibition activity and application thereof.The amino acid sequence of the polypeptide is EDCIPKWKGCVNRHGDCCEGLECYVRRKSFEVCIPKTPKT.The present application adopts the polypeptide with ASIC1a selective inhibition activity and application thereof, the polypeptide can specifically inhibit the channel current of ASIC1a, can effectively inhibit the channel current of ASIC1b, and has a molecular weight of 4629.40 Da, has a better blood-brain barrier penetration rate compared with a monoclonal antibody with a larger molecular weight, has no significant cytotoxicity through test, has a low possibility of causing human immune response, has relatively less potential unsafe factors, and can be used for preparing a neuroprotective drug for treating ischemic brain injury caused by ischemic stroke.

Description

TECHNICAL FIELD

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

[0002] Stroke is a devastating and pathophysiological complex nervous system disease, with high incidence, high recurrence rate, high disability rate, high mortality rate and other characteristics. Ischemic stroke is the most common type, accounting for about 60% to 70% of all strokes. Therefore, it is of great significance to develop effective neuroprotective drugs for treating ischemic brain injury caused by ischemic stroke.

[0003] When stroke occurs, the cerebral infarction area will cause severe lack of glucose and oxygen due to ischemia, thereby causing excessive release and accumulation of glutamate, triggering excitotoxicity and leading to neuronal death. Recent studies have shown that in ischemic brain injury, glutamate can promote excitotoxicity by increasing the sensitivity and opening degree of acid-sensitive ion channels (ASICs) to acidic environment in addition to the recognized NMDARs (N-methyl-D-aspartate receptor). By comparing the effects of NMDARs antagonists and ASICs knockouts on cerebral infarction volume in stroke mice, studies have shown that blocking ASICs can achieve similar protective effects as blocking NMDARs in reducing ischemic brain injury. This finding suggests that ASICs may be as important as NMDARs in stroke treatment, which indicates that ASICs is a new target for stroke treatment that has not been fully considered.

[0004] Selective inhibition of the channel current of ASIC1a by inhibitors is considered to be a promising direction for the treatment of ischemic brain injury. In vitro studies have shown that the use of non-selective inhibitors (Amiloride) or selective inhibitors (PcTx1) of ASIC1a can effectively reduce intracellular Ca 2+ concentration, thereby effectively reducing neuronal damage; neurons lacking or knocking out ASIC1a gene have significant resistance to acid environment-induced damage. In vivo studies have further verified the role of ASIC1a in brain ischemic injury. In a model of cerebral ischemia, by injecting non-selective or selective inhibitors of ASIC1a into the cerebral ventricle, the infarct area can be reduced by as much as 60%.

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

[0006] Another category of ASIC1a inhibitors is polypeptide toxins extracted from animal venom glands. Compared with small molecule compounds, this kind of polypeptide toxins shows higher selectivity and more significant inhibitory effect, and is more suitable for developing as neuroprotective drugs for treating ischemic brain injury. Currently, there are four active polypeptides that can selectively inhibit the current of ASIC1a channel, among which 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 the current of homologous ASIC1a, and plays an important role in the treatment of ischemic stroke. PcTx1 can selectively inhibit the current of homologous ASIC1a. The IC 50 of PcTx1 to rASIC1a from rats is 1 nM, and the IC 50 of PcTx1 to hASIC1a from humans is 3 nM (inhibiting channel current at pH 7.2). However, it is worth noting that while PcTx1 selectively inhibits the current of ASIC1a, high concentrations of PcTx1 can enhance the channel current of ASIC1b (EC 50 around 50-100 nM). ASIC1b is mainly expressed in peripheral sensory neurons and has been shown to play a role in the nociceptive system of rodents. Therefore, when PcTx1 is developed and applied as a drug molecule for treating ischemic stroke, it may have some predictable adverse reactions on the organism. SUMMARY

[0007] The polypeptide with ASIC1a selective inhibitory activity provided by the application can specifically inhibit the channel current of ASIC1a, can effectively inhibit the channel current of ASIC1b, has a molecular weight of 4629.40 Da, has a better blood-brain barrier penetration rate compared to a monoclonal antibody with a larger molecular weight, has no significant cytotoxicity after test, has a low possibility of causing an immune response of a human body, has relatively few potential unsafe factors, and can be used for preparing a neuroprotective drug for treating ischemic brain damage caused by ischemic stroke.

[0008] To achieve the above object, the application provides a polypeptide with ASIC1a selective inhibitory activity, which has an amino acid sequence of EDCIPKWKGCVNRHGDCCEGLECYVRRKSFEVCIPKTPKT.

[0009] Further, the application further provides application of the polypeptide in preparation of an ASIC1a selective inhibitor.

[0010] Further, the application further provides application of the polypeptide in preparation of a neuroprotective drug for ischemic brain damage.

[0011] Further, the application further provides application of the polypeptide in preparation of a neuroprotective drug for treating ischemic brain damage caused by ischemic stroke.

[0012] Further, the application further provides an ASIC1a selective inhibitor, which comprises the polypeptide.

[0013] Further, the application further provides a neuroprotective drug, which comprises the polypeptide.

[0014] The polypeptide sequence provided by the application can inhibit 37.5% of the channel current of ASIC1a at a concentration of 10 nM. 50 The IC50 is 37.829 nM, the channel current of ASIC1b is not enhanced at a high concentration, the polypeptide can inhibit 31.5% of the channel current of ASIC1b at a concentration of 10 nM, and the IC50 is 83.947 nM. 50

[0015] The polypeptide with ASIC1a selective inhibitory activity provided by the application and the application thereof have the following advantages and positive effects:

[0016] ​The polypeptide can specifically inhibit the channel current of ASIC1a, effectively inhibit the channel current of ASIC1b, has a better blood-brain barrier penetration rate compared to a monoclonal antibody with a larger molecular weight, has no significant cytotoxicity, has a low possibility of causing an immune response of the human body, has relatively few potential unsafe factors, and can be used for preparing a neuroprotective drug for treating ischemic brain damage caused by ischemic stroke.

[0017] The technical solutions of the present application are described in further detail below with reference to the accompanying drawings and examples. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 A mutation site of the polypeptide in the embodiment of the present application is shown in the figure;

[0019] Figure 2 The immunosorbent thermogram of the third selection of the mutant library in the embodiment of the present application and HEK293T-hASIC1a is shown in the figure;

[0020] Figure 3 The inhibition effect of the polypeptide on ASIC1a in the embodiment of the present application is shown in the figure;

[0021] Figure 4 The inhibition effect of the polypeptide on ASIC1b in the embodiment of the present application is shown in the figure;

[0022] Figure 5 The heterologous expression SDS-PAGE of the polypeptide in the embodiment of the present application is shown in the figure;

[0023] Figure 6 The cell activity detection result of the polypeptide in the embodiment of the present application is shown in the figure. DETAILED DESCRIPTION

[0024] The technical solutions of the present application are described in further detail below with reference to the accompanying drawings and examples.

[0025] Unless otherwise defined, the technical terms or scientific terms used in the present application shall have the usual meanings understood by those with ordinary skills in the art to which the present application belongs.

[0026] Based on the embodiments in the present application, all other embodiments obtained by those with ordinary skills in the art without making creative efforts shall fall within the scope of protection of the present application. The experimental methods not specified in the following examples are generally determined according to national standards. The experimental instruments, equipment and reagents not specified in the following examples are all commercially available raw materials.

[0027] Unless otherwise defined, all technical and scientific terms used in the application have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Methods and materials similar or equivalent to those described herein can be used in the practice of the application. It is to be understood that the examples and features of the application can be combined in all permutations.

[0028] Example 1

[0029] The application mutates PcTx1, and first determines that four amino acid residues (Trp24, Lys25, Arg28, Thr37) play a key role in the subtype selection of ion channels by PcTx1. Therefore, the four active sites are used as randomization sites (the amino acid residues marked in green), and the key sites are saturated mutated to construct a random tetrapeptide library. The constructed random tetrapeptide library is displayed on the surface of phage by phage display technology. Figure 1

[0030] In the application, the Sleeping Beauty technology is used to construct a stable cell strain HEK293T-ASIC1a stably expressing voltage-gated ASIC1a ion channels, which is inoculated in a 96-well enzyme plate (1×10 4 cells / well) and cultured in a 37℃, 5% CO2 incubator for 2-3 days, which can be used as a target antigen for detection.

[0031] 5×10 12 CFU of the random peptide library is added to the inoculated and cultured cell plate, and incubated at room temperature for 2h. After washing the plate with PBS for 3 times, Glycine-HCl (pH 7.4) is added to elute the binding liquid, and the eluate is mixed with E. coli XL1-Blue, which is incubated at 37℃ for 30min, and then shaken and cultured overnight at 30℃ to obtain the amplified antibody 1st library.

[0032] The above steps are repeated to obtain the 2nd and 3rd libraries in turn.

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

[0034] Table 1 Binding of mutation library-2nd and mutation library-3rd with stable cell strain HEK293T-ASIC1a

[0035]

[0036] ​The mutant library-3rd has stronger immunosorbent capacity with the stable cell strain HEK293T-ASIC1a, and the screening of single clone is further performed from the mutant library-3rd. The active polypeptide molecule with good ASIC1a selective inhibition effect is screened.

[0037] According to the above detection results, the clones with significant difference in absorbance relative to the negative control are selected for retesting and sequencing, and the polypeptide sequence is obtained, as shown in Table 2.

[0038] Table 2 Polypeptide sequence

[0039]

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

[0041] Example 2

[0042] Inhibitory analysis:

[0043] The polypeptide sequence can inhibit 37.5% of the channel current of ASIC1a at a concentration of 10 nM. The IC 50 is 37.829 nM Figure 3 , and does not enhance the channel current of ASIC1b at a high concentration. The polypeptide sequence can inhibit 31.5% of the channel current of ASIC1b at a concentration of 10 nM, and the IC 50 is 83.947 nM Figure 4 .

[0044] The active polypeptide described in the application can be obtained by chemical synthesis or various obtaining means such as prokaryotic and eukaryotic.

[0045] The amino acid composition and molecular weight of the polypeptide are predicted by Expasy ProtParam tool. The polypeptide is obtained by chemical synthesis or prokaryotic expression, and the molecular weight thereof is identified by SDS-PAGE Figure 5 , which shows that the molecular weight of the polypeptide is 4629.40 Da, and the polypeptide has better blood-brain barrier penetration rate compared with the monoclonal antibody with larger molecular weight. The polypeptide has no significant cytotoxicity Figure 6 , has low possibility of causing human immune response, and has relatively less potential unsafe factors. Therefore, the active polypeptide can be used for developing a neuroprotective drug for treating ischemic brain injury caused by ischemic stroke, and is widely used in the fields of biotechnology, biological medicine, etc.

[0046] Therefore, the application adopts the polypeptide with ASIC1a selective inhibition activity and its application, the polypeptide can specifically inhibit the channel current of ASIC1a, can effectively inhibit the channel current of ASIC1b, and has a molecular weight of 4629.40 Da, has a better blood-brain barrier penetration rate compared with a larger molecular weight monoclonal antibody, has no significant cytotoxicity through test, has a low possibility of causing human immune response, has relatively less potential unsafe factors, and can be used for preparing a neuroprotective drug for treating ischemic brain damage caused by ischemic stroke.

[0047] Finally, it should be noted that: the above examples are only used to illustrate the technical solutions of the present application, but not to limit them, although the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that: it can still modify or equivalently replace the technical solutions of the present application, and these modifications or equivalent replacements cannot make the modified technical solutions deviate from the spirit and scope of the technical solutions of the present application.

Claims

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

2. The use of the polypeptide as described in claim 1 in the preparation of a medicament for treating ischemic stroke.

3. A selective inhibitor of ASIC1a, characterized in that: It contains the polypeptide as described in claim 1.

4. A drug for treating ischemic stroke, characterized in that: It contains the polypeptide as described in claim 1.

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