Polypeptides that inhibit and / or clear beta-amyloid aggregates and uses thereof

Abstract

The present application relates to the technical field of medicine, and more particularly to a polypeptide for inhibiting and / or eliminating beta-amyloid aggregation and application thereof. The present application first develops a functional active polypeptide ANI-1 which can act on diseases caused by beta-amyloid aggregation and deposition such as Alzheimer's disease from multiple targets, so as to effectively prevent and treat the diseases. The polypeptide ANI-1 of the present application has the functions of eliminating A beta aggregation, delaying behavioral decline caused by beta-amyloid toxicity (anti-paralysis, promoting movement), inhibiting neurogenic inflammation related to beta-amyloid deposition and enhancing antioxidant defense, has better overall efficacy and neuroprotective potential, and can provide a new idea for developing a multi-target drug for diseases caused by beta-amyloid aggregation and deposition such as Alzheimer's disease.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and more particularly to a polypeptide that inhibits and / or clears β-amyloid protein aggregation and its applications. Background Technology

[0002] Alzheimer's disease (AD) is an age-related neurodegenerative disease characterized by abnormal deposition of β-amyloid protein (Aβ) in the brain, forming senile plaques; hyperphosphorylation of tau protein leading to neurofibrillary tangles; and accompanying neuroinflammatory responses and oxidative stress damage. Aβ aggregation and toxicity are considered core components of AD pathogenesis. The accumulation of Aβ oligomers and fibers inside and outside neurons can trigger synaptic dysfunction, neuronal death, inflammatory activation, and oxidative damage, ultimately leading to progressive cognitive decline. Currently, there are no drugs that can effectively halt or reverse the course of AD; existing therapies primarily focus on symptom relief and cannot fundamentally intervene in the disease progression. Therefore, developing novel therapeutic strategies that can specifically clear or inhibit Aβ aggregation and alleviate related pathological processes has significant clinical and social value. In recent years, bioactive peptides have shown potential applications in the prevention and treatment of neurodegenerative diseases due to their high safety, good biocompatibility, and diverse pharmacological activities.

[0003] However, current peptides targeting Aβ have relatively limited targets and therefore limited therapeutic effects. Therefore, developing peptides that can target more targets is of great significance for developing potential drugs to treat, prevent, or cure Alzheimer's disease. Summary of the Invention

[0004] Therefore, based on the above background, this invention develops a polypeptide that inhibits and / or clears β-amyloid protein aggregation and its application. The polypeptide developed in this invention can achieve multi-target prevention or treatment of diseases caused by β-amyloid protein aggregation and deposition, such as Alzheimer's disease, by clearing Aβ aggregation, delaying behavioral decline, inhibiting inflammation and enhancing antioxidant defense, thus providing new ideas for the development of drugs for related diseases.

[0005] The technical solution provided by this invention is as follows: One object of the present invention is to provide: a polypeptide ANI-1 that inhibits and / or clears β-amyloid protein aggregation, the amino acid sequence of said polypeptide ANI-1 being as follows: SEQ ID NO.1 As shown: GNLATLISSLLAAAAHDFQSQL.

[0006] A second objective of this invention is to provide the use of polypeptide ANI-1 in the preparation of medicaments for the prevention and / or treatment of diseases caused by β-amyloid aggregation and deposition, wherein the amino acid sequence of polypeptide ANI-1 is as follows: SEQ ID NO.1 As shown: GNLATLISSLLAAAAHDFQSQL.

[0007] Furthermore, diseases caused by the aggregation and deposition of β-amyloid protein include Alzheimer's disease and cerebral amyloid angiopathy.

[0008] A second objective of this invention is to provide that the drug has at least one of the following functions (a1) to (a3), wherein the functions include: a1) Clears β-amyloid protein aggregates; a2) Improves behavioral disorders caused by β-amyloid toxicity; a3) Inhibit and / or improve neuroinflammation and oxidative stress associated with β-amyloid deposition.

[0009] The first objective of this invention is to provide that the behavioral disorders include hemiplegia and dysphagia.

[0010] A third objective of this invention is to provide a drug for the prevention and / or treatment of Alzheimer's disease, wherein the drug contains at least an effective amount of the polypeptide ANI-1, and the amino acid sequence of the polypeptide ANI-1 is as follows: SEQ ID NO.1 As shown.

[0011] Furthermore, the polypeptide ANI-1 is the only active ingredient in the drug.

[0012] Furthermore, the polypeptide ANI-1 is mixed with other active ingredients.

[0013] The fourth objective of this invention is to provide the application of peptide ANI-1 in experiments studying the pathological or therapeutic mechanisms of diseases caused by β-amyloid aggregation and deposition, wherein the amino acid sequence of peptide ANI-1 is as follows: SEQ ID NO.1 As shown.

[0014] The beneficial effects achieved by this invention are as follows: This invention first developed a functional active peptide, ANI-1, which can exert multi-target effects on diseases caused by β-amyloid protein aggregation and deposition, such as Alzheimer's disease, to effectively prevent and treat these diseases. The peptide ANI-1 of this invention has the functions of clearing Aβ aggregation, delaying behavioral decline caused by β-amyloid protein toxicity (anti-paralysis, promoting movement), inhibiting neuroinflammation related to β-amyloid protein deposition, and enhancing antioxidant defense. It has better overall efficacy and neuroprotective potential, and can provide new ideas for developing multi-target drugs for diseases caused by β-amyloid protein aggregation and deposition, such as Alzheimer's disease.

[0015] This invention offers a targeted and highly effective approach. The peptide ANI-1 can effectively inhibit or eliminate β-amyloid protein aggregation, intervening in the disease process at its source. Validated using an AD nematode model induced with Aβ expression at 25°C, peptide ANI-1 significantly reduces the number of Aβ deposition points in the neural ring region of the nematode's head, demonstrating its clearing or inhibitory effect on Aβ aggregation. Furthermore, peptide ANI-1 intervention significantly slows the paralysis process in nematodes, increases the non-paralysis rate, and improves the nematode's choking ability. At the molecular level, peptide ANI-1 reduces inflammatory marker levels, upregulates the expression of transcription factor daf-16, and thereby enhances the activity of its downstream product, superoxide dismutase (SOD), strengthening the body's antioxidant defense capabilities and exhibiting excellent anti-inflammatory and anti-stress abilities. Attached Figure Description

[0016] Figure 1 The effects of polypeptide ANI-1 on the growth, development, movement, and feeding of nematodes, as described in this embodiment of the invention.

[0017] Figure 2 The figures show the behavioral performance and verification of the AD nematode model and the results of the antioxidant stress experiment in this embodiment of the invention. Figure 2 A represents the experimental results of the non-paralysis rate; Figure 2 B represents the result of the swallowing test; Figure 2 C represents the expression results of inflammatory markers; Figure 2 D represents the result of superoxide dismutase (SOD) activity assay.

[0018] Figure 3 The Aβ scavenging effect of peptide ANI-1 in the nematode model of this invention is illustrated in this embodiment. Figure 3 A represents the Aβ deposition fluorescence effect in different groups of nematodes. Figure 3 B represents the statistical analysis of the number of points (*p<0.05, **p<0.01, n=10).

[0019] Figure 4 Molecular binding analysis of the polypeptide ANI-1 and Aβ oligomer of the present invention.

[0020] Figure 5 The images show the CL4176 nematode in both non-paralyzed and paralyzed states according to an embodiment of the present invention, with the upper right and lower right images showing the paralyzed state. Detailed Implementation

[0021] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0022] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods; the experiments used in the following embodiments... Unless otherwise specified, all materials were purchased from commercial channels.

[0023] The polypeptide AN1 used in the following examples was synthesized using a solid-phase polypeptide synthesis method.

[0024] The synthesis steps are as follows: The resin was swollen in DMF, and Fmoc-protected amino acids, DIEA, and DMF were added sequentially for reaction. The Fmoc groups were removed using piperidine solution, and the reaction success was detected by ninhydrin. During the synthesis process, the washing, condensation, and detection steps were repeated multiple times until the polypeptide chain was completely synthesized. Finally, 5-FITC was added for labeling. After synthesis, small amounts of the finished polypeptide were taken for molecular weight identification by mass spectrometry (MS) and purity identification by high-performance liquid chromatography (HPLC). The target ANI-1 with a synthetic purity >95% was obtained.

[0025] In existing AD research, nematode models (such as transgenic nematodes expressing human Aβ) have been widely used to preliminarily evaluate the effects of drugs on Aβ toxicity, paralytic phenotype, motor behavior, and related biochemical indicators. In this model, Aβ accumulation can lead to progressive paralysis in nematodes, weakened pharyngeal pump movements (spitting), increased oxidative stress levels, and activated inflammatory responses. These phenotypes share certain similarities with the neuropathological features of AD, providing an effective platform for rapidly screening lead compounds with neuroprotective effects.

[0026] To evaluate the improvement and therapeutic effect of peptide ANI-1 in an Alzheimer's disease model, a series of in vivo experiments on AN1 of the peptide were conducted using a nematode model.

[0027] 1. Materials and Methods 1.1 Nematode strains and culture conditions Transgenic CL4176 strain of Caenorhabditis elegans (dv Is27[myo-3p:A-Beta(1-42):let-851 3'UTR+rol-6(su1006)]X), CL2006 (dv Is2[p CL12(unc-54 / human Abeta(1–42) minige-ne)+rol-6(su1006)]) and wild-type N2 were all purchased from Shanghai Southern Model Biotechnology Co., Ltd.

[0028] Nematodes were routinely cultured on solid nematode growth medium (NGM) with Escherichia coli OP50 as their food source. The N2 strain was cultured at 25°C, while the CL4176 and CL2006 strains were cultured at 16°C.

[0029] 1.2 Preparation of NGM culture plates for nematodes Weigh 2.26 g of NGM powder into an Erlenmeyer flask, dissolve it in 100 mL of purified water, and autoclave at 120°C for 15 min. After cooling to approximately 50°C, add 800 µL each of autoclaved 1M MgSO4, 5 mg / mL cholesterol (dissolved in anhydrous ethanol, not sterilized), and 1M CaCl2. Shake well and pour into a sterile 6 cm culture plate. After solidification, allow it to stand at room temperature for 2 days without contamination before use.

[0030] 1.3 Nematode Culture and Synchronization Adult nematodes and eggs in the plate were washed into EP tubes using M9 liquid medium, and nematode synchronization was performed using the lysis method. Synchronization was achieved when the nematodes in the tubes hatched into larvae (L1 stage). All nematodes were fed with E. coli OP50. N2 strain nematodes were cultured at 25°C (N2 nematodes are normal wild-type nematodes), while CL4176 and CL2006 strains were cultured at 16°C (CL4176 and CL2006 are two temperature-sensitive transgenic nematodes; under normal conditions, when cultured at 16°C, their bodies exhibit a C-shaped curvature and they can move normally in the culture medium. When the temperature rises to 25°C, the serine / threonine protein kinase system is inactivated, leading to overexpression of Aβ toxic proteins in the muscle, thus inducing a paralytic phenotype).

[0031] 1.4 Effects of peptide ANI-1 on the growth and development of wild-type N2 nematodes 1.4.1 Reproductive capacity determination N2 nematodes of the same instar were selected (one nematode per plate, five plates per group). Under an inverted microscope, 2.5, 5, and 10 μM of the peptide ANI-1 were evenly dropped onto the plates. A blank control group (no intervention) was set up. Observations were made at regular intervals, and the time of the first oviposition and the daily oviposition amount of each nematode were recorded until it was impossible to distinguish whether the eggs came from the observed nematode or its progeny. The experiment was repeated independently three times.

[0032] 1.4.2 Measurement of athletic ability The nematodes used in the following experiment were N2 nematodes. Body bending frequency: Nematodes of the same age were selected and treated with 2.5, 5, and 10 μM peptide ANI-1 (at room temperature, ANI-1 was evenly added to NGM plates coated with OP50). After 24 hours of intervention, nematodes of the same age were placed on clean, sterile NGM plates (one nematode per plate, 5 plates per group). After the nematodes crawled for 1 minute to remove the OP50 adhering to their bodies, one complete sinusoidal movement of the nematode's body was counted as one body bending. The number of body bending events within 1 minute was recorded. The experiment was independently repeated 3 times.

[0033] Pharyngeal pump rhythmic movement assay: Nematodes of the same age, untreated, or treated with 2.5, 5, and 10 μM peptide ANI-1, were placed on clean, sterile NGM plates (one nematode per plate, five plates per group). The number of swallowing movements within 30 seconds was measured. The method involved observing the number of twitches of the nematode's pharyngeal muscles within 30 seconds under a microscope. The experiment was independently repeated three times.

[0034] See results Figure 1 ,Depend on Figure 1 It is evident that the polypeptide ANI-1 has no effect on the growth, development, movement, or feeding of nematodes (n=15).

[0035] Figure 1 The average oviposition rate was calculated as follows: N2 nematodes of the same instar were selected (one nematode per plate, five plates per group). Under an inverted microscope, 2.5, 5, and 10 μM of the polypeptide ANI-1 were evenly dropped onto the plates. The nematodes were observed under the microscope until they reached sexual maturity (eggs were visible inside). After 4-5 hours, the nematodes began to lay eggs. The number of eggs was manually counted under the microscope using a counter. The average oviposition rate was calculated for five nematodes from each group of five plates. The experiment was independently repeated three times.

[0036] When calculating differences, Figure 1 In group A, the p-values ​​of the 2.5 μM and 5 μM groups compared with the intervention group were 0.1106 and 0.1287, respectively. Figure 2In C, the p-value between the 2.5 μM group and the unintervention group was 0.0637. At this point, p > 0.05, and there was no significant difference between the intervention group and the experimental group. Therefore, it can be concluded that ANI-1 intervention has no significant effect on the normal growth, development, movement, and feeding of nematodes.

[0037] 1.5 Effects of peptide ANI-1 on the AD nematode model 1.5.1 Paralysis Experiment Three CL4176 nematodes of the same age were selected (three nematodes per plate, three plates per group), and cultured at 25℃ for 10 h. Then, 2.5, 5, and 10 μM of the peptide ANI-1 were added to the plates, with a blank control group included. The paralysis status of the nematodes was observed every 2 h, and the paralysis rate was calculated at 24, 36, 48, 60, and 72 h until all nematodes were paralyzed. The experiment was independently repeated three times. Results are shown below. Figure 2 A.

[0038] Figure 5 The images show the CL4176 nematode's condition before and after paralysis, with the upper right and lower right images showing the paralyzed state. At 16°C and before complete paralysis, the CL4176 nematode exhibits a curved C-shape and can move normally in the culture medium. When the temperature is raised to 25°C, Aβ gradually accumulates in the muscle cells, causing the nematode's body to stiffen and become immobile. It can only prey on op50 near its head. Once it has consumed all the op50, a halo forms around its head, at which point the nematode is paralyzed.

[0039] 1.5.2 Pharyngeal pump rhythmic movement experiment Three CL4176 nematodes of the same instar were selected (three nematodes per plate, three plates per group), incubated at 25°C for 10 h, and then 2.5, 5, and 10 μM ANI-1 were added to the plates. A control group was also included. Swallowing was observed every 2 h, and the number of swallows within 30 seconds was counted at 24, 36, 48, 60, and 72 h. The counting method involved observing the number of twitches of the nematode's pharyngeal muscles within 30 seconds under a microscope. The experiment was independently repeated three times. Results are shown below. Figure 2 B.

[0040] 1.5.3 Thiamine S staining experiment on Aβ sediments CL2006 nematodes synchronized to the L4 stage were cultured at 25℃ for 8-12 h. 5 and 10 μM of the peptide ANI-1 were added to plates for 2-3 days of intervention (at room temperature) as experimental groups, while an N2 nematode control group (at room temperature) was set up. The immunofluorescence staining image of the N2 nematodes served as the control group, aiming to present the fluorescence of normal nematodes. Nematodes were collected in EP tubes, and 1 mL of 4% paraformaldehyde solution was added for fixation at 4℃ for 24 h. The fixative was discarded, and 1 mL of permeabilization buffer (1% Triton X-100) was added for incubation at 37℃ for 24 h. After permeabilization, the nematodes were washed three times with TBST solution (PBS solution containing 0.1% Tween 20), stained with the fluorescent dye thioflavin S (0.125%) at room temperature for 30 min, centrifuged, and the staining solution was discarded. The stain was then destained three times with 50% ethanol. Photographs of CL2006 nematodes were taken under a laser confocal microscope, and the amount of Aβ protein deposited in the head neural ring (the part before the pharyngeal pump) was counted. Each group contained at least 15-20 nematodes. The experiment was independently repeated three times.

[0041] 1.5.4 Real-time quantitative PCR experiment AD nematode models of CL4176 nematodes (untreated AD nematode model, AD nematode models treated with 2.5 μM and 5 μM ANI-1) were collected in EP tubes. Total RNA was extracted from the nematodes using TRIzol reagent, and cDNA was synthesized by reverse transcription. The mRNA expression of genes related to inflammation and oxidative stress, such as tir-1, skn-1, sod-3, and daf-16, in nematodes was detected by real-time quantitative PCR. -△△CT The expression differences were calculated using the algorithm, plotted using GraphPad 9.0, and N2 nematodes were used as a control group to indicate the lifespan and metabolic status of nematodes. The results are shown in [Figure 1]. Figure 2 C and Figure 2 D.

[0042] The above experiments show that ANI-1 has no effect on the growth, development, movement, or feeding of nematodes; combining experiments on paralysis, feeding, and molecular results, the intervention with 5 μM ANI-1 showed the most significant effect. Figure 2 D. The effect of 5 μM ANI-1 on the activity of DAF-16, a longevity-related transcription factor in nematodes, and its downstream effector molecule superoxide dismutase (SOD) was detected.

[0043] CL4176 nematodes were transferred and cultured at 25℃ for 8–12 h. Then, 2.5, 5, and 10 μM ANI-1 were added to the plates for intervention. A second intervention was performed 24 h later. After 12 h, the nematodes were collected in EP tubes. Total RNA was extracted from the nematodes using TRIzol reagent, and cDNA was synthesized by reverse transcription. Real-time quantitative PCR was used to detect the mRNA expression of genes related to inflammation and oxidative stress pathways, such as tir-1, skn-1, sod-3, and daf-16. Two... -△△CT The method calculates the differences in expression, and plots the graph using GraphPad 9.0.

[0044] Depend on Figure 2 It is evident that the peptide ANI-1 significantly improved the behavioral phenotype of the AD nematode model. After temperature-induced Aβ expression, compared with the blank control group (AD nematode model), the paralysis process in nematodes treated with 5 μM peptide ANI-1 was most significantly delayed. Figure 2 A). Statistical analysis showed that throughout the observation period, the non-paralysis rate in all groups treated with peptide ANI-1 remained at a high level (*p < 0.05, n=15), indicating that peptide ANI-1 could effectively counteract the loss of motor function caused by Aβ toxicity. Simultaneously, peptide ANI-1 treatment also significantly increased the frequency of pharyngeal pumping movements in nematodes (…). Figure 2 (B) This indicator is a key parameter for assessing its food intake and neuromuscular function (*p < 0.05, **p < 0.01, n=30). These two behavioral results together confirm that the peptide ANI-1 has a certain neuroprotective effect and can alleviate the functional decline caused by Aβ aggregation.

[0045] And by Figure 2 C and Figure 2 As can be seen from D, the peptide ANI-1 also exerts anti-inflammatory and antioxidant effects by regulating key signaling pathways. Biochemical detection results show ( Figure 2 C) The levels of inflammatory markers in nematodes treated with ANI-1 were significantly reduced. Furthermore, it was found that the peptide ANI-1 upregulated the expression of the longevity-related transcription factor DAF-16, and consequently significantly enhanced the activity of its downstream effector molecule, superoxide dismutase (SOD) (*p < 0.05, **p < 0.01, ***p < 0.001). Increased SOD activity signifies enhanced free radical scavenging capacity. Analysis suggests that the neuroprotective mechanism of peptide ANI-1 may partly depend on activating the DAF-16-mediated antioxidant stress pathway, thereby alleviating the inflammation and oxidative damage associated with Aβ deposition. Figure 3 It is evident that the peptide ANI-1 directly reduces the pathological deposition of Aβ in the neural ring region. This was demonstrated by observing the neural ring in the head of the nematode (a key affected area in the AD model) using thioflavone S staining. Figure 3 As can be seen from ANI-1 treatment, the green fluorescence signal (indicating Aβ deposition) was significantly weaker than that in the model control group. Quantitative analysis of the number of Aβ deposition sites further confirmed that ANI-1 significantly reduced the formation of Aβ aggregates (*p < 0.05, **p < 0.01, n=10). This result directly demonstrates from a pathological morphological perspective that ANI-1 has the core function of clearing or inhibiting Aβ protein aggregation.

[0046] Figure 4 This is the amino acid binding site where ANI-1 interacts with Aβ protein, suggesting that ANI-1 can bind to Aβ and affect Aβ aggregation.

[0047] As can be seen from the above results, the polypeptide ANI-1 of the present invention can significantly improve Aβ-induced behavioral disorders in nematodes, reduce inflammation and oxidative stress levels, and directly reduce the pathological deposition of Aβ protein.

[0048] The present invention and its embodiments have been described above. This description is not restrictive, and the embodiments shown are only one of the embodiments of the present invention. The actual structure is not limited to this. In conclusion, if those skilled in the art are inspired by this description and design similar structures and embodiments without departing from the spirit of the present invention, they should all fall within the protection scope of the present invention.

Claims

1. A polypeptide ANI-1 that inhibits and / or clears β-amyloid protein aggregation, characterized in that, The amino acid sequence of the polypeptide ANI-1 is as follows: SEQ ID NO.1 As shown: GNLATLISSLLAAAAHDFQSQL.

2. The use of polypeptide ANI-1 in the preparation of medicaments for the prevention and / or treatment of diseases caused by β-amyloid aggregation and deposition, characterized in that, The amino acid sequence of the polypeptide ANI-1 is as follows: SEQ ID NO.1 As shown: GNLATLISSLLAAAAHDFQSQL.

3. The application according to claim 2, characterized in that, Diseases caused by the aggregation and deposition of β-amyloid protein include Alzheimer's disease and cerebral amyloid angiopathy.

4. The application according to claim 2, wherein the drug has at least one of the following functions (a1) to (a3), the functions including: a1) Clears β-amyloid protein aggregates; a2) Improves behavioral disorders caused by β-amyloid toxicity; a3) Inhibit and / or improve neuroinflammation and oxidative stress associated with β-amyloid deposition.

5. The application according to claim 4, characterized in that, The behavioral disorders include hemiplegia and dysphagia.

6. A drug for the prevention and / or treatment of Alzheimer's disease, characterized in that, The drug contains at least an effective amount of polypeptide ANI-1, the amino acid sequence of which is as follows: SEQ ID NO.1 As shown.

7. A medicament for the prevention and / or treatment of Alzheimer's disease according to claim 6, characterized in that, The drug contains polypeptide ANI-1 as its sole active ingredient.

8. A medicament for the prevention and / or treatment of Alzheimer's disease according to claim 6, characterized in that, The polypeptide ANI-1 is mixed with other active ingredients.

9. The application of polypeptide ANI-1 in experiments studying the pathological and / or therapeutic mechanisms of diseases caused by β-amyloid aggregation and deposition, characterized in that, The amino acid sequence of the polypeptide ANI-1 is as follows: SEQ ID NO.1 As shown.

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