Use of an inhibitor or a target gene knockdown agent in the manufacture of a medicament for the treatment of the disease SCA3

Abstract

The application belongs to the field of biological medicine, and particularly relates to application of an inhibitor or a target gene knockdown reagent in preparation of a medicine for treating a disease SCA3, wherein the SCA3 is a type 3 spinal and cerebellar ataxia, the inhibitor is an inhibitor of a gene PIK3CA or a kinase PI3K alpha, and the inhibitor is a compound HS173; a target gene in the target gene knockdown reagent is the PIK3CA. The application shows through experiments that, compared with a control group, accumulation of pathogenic ATXN3 protein of a mouse in an HS173 treatment group is significantly reduced, a degree of neuron loss is significantly reduced, cerebellar atrophy is improved, and a cerebellar inflammation level is improved; meanwhile, motor coordination, balance ability and muscle strength of the mouse are all significantly improved. The HS173 can specifically inhibit PIK3CA activity, and can be used as a potential medicine for treating the SCA3.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to the application of an inhibitor or target gene knockdown reagent in the preparation of drugs for treating the disease SCA3. Background Technology

[0002] Spinocerebellar ataxia type 3 (SCA3) is an autosomal dominant inherited neurodegenerative disease and the most common subtype of spinocerebellar ataxia (SCA). It is caused by an abnormal amplification of the CAG repeat sequence in the ATXN3 gene, leading to damage to nerve cells in the cerebellum, brainstem, and other regions, resulting in progressive motor incoordination, nystagmus, dysphagia, and other symptoms. Currently, there is no cure. Among hereditary neurodegenerative diseases, SCA3 has the second highest global prevalence after Huntington's disease, accounting for approximately 60% to 70% of all SCA cases in the Chinese population.

[0003] Mutant ATXN3 is prone to misfolding, accumulation, and aggregation, leading to the formation of intranuclear inclusion bodies in affected neural tissue. The resulting polyglutamine (polyQ) toxicity disrupts multiple cellular processes, including protein quality control, transcriptional regulation, and nuclear-cytoplasmic transport, resulting in progressive neuronal dysfunction and degeneration. Although various molecular abnormalities have been reported in SCA3, polyQ-amplified ATXN3 itself is widely considered a major driver of pathogenesis. Consistent with this view, reducing polyQ protein burden in disease models can improve aggregation and neuronal phenotypes, supporting mutant protein burden as a manageable therapeutic target. However, the upstream regulators of mutant ATXN3 homeostasis have not been systematically explored. Identifying these mechanisms is crucial for identifying druggable upstream pathways that can be used to reduce toxic ATXN3 burden and alter disease progression.

[0004] Therefore, there is a need in the art for a new drug for treating the neurodegenerative disease SCA3, or for expanding the use of existing drugs for treating the neurodegenerative disease SCA3. Summary of the Invention

[0005] Given the central role of kinases in signal transduction and protease inhibition, as well as their established pharmacological effects, they are a particularly attractive class of regulators in SCA3. Therefore, we screened for a kinase, PI3Kα, the kinase corresponding to PIK3CA, which can regulate ATXN3 protein expression and aggregation.

[0006] The present invention first provides the application of an inhibitor in the preparation of a drug for treating the disease SCA3, wherein SCA3 is spinocerebellar ataxia type 3, and the inhibitor is an inhibitor of gene PIK3CA or kinase PI3Kα, and the inhibitor is compound HS173.

[0007] In this invention, the compound HS173 has the CAS number 1276110-06-5 and its structural formula is as follows.

[0008] In one specific embodiment, the inhibitor is used in the preparation of a medicament for treating SCA3 in humans or animals, wherein the medicament is in the form of an oral or injectable formulation.

[0009] This invention also provides the application of a target gene knockdown reagent in the preparation of a drug for treating the disease SCA3, wherein SCA3 is spinocerebellar ataxia type 3, and the target gene is PIK3CA; the target gene knockdown reagent is a nucleic acid silencing reagent and / or a gene editing reagent; the nucleic acid silencing reagent is selected from at least one of small interfering RNA, short hairpin RNA, microRNA, and antisense oligonucleotides; the target gene knockdown reagent is used to reduce the expression levels of target gene mRNA and protein.

[0010] In one specific embodiment, the target gene knockdown reagent is AAV-shPIK3CA.

[0011] In one specific implementation, the drug is a neuro-targeted therapy drug used to intervene in the early, middle, and late stages of SCA3 disease progression, delay disease progression, and improve the patient's neuromotor function.

[0012] In one specific implementation, the delay in disease progression includes reducing the accumulation of the SCA3 pathogenic protein ATXN3, mitigating the degree of neuronal loss, and improving cerebellar atrophy and cerebellar inflammation levels; the neuromotor function includes the patient's motor coordination, balance, and muscle strength.

[0013] In one specific embodiment, the drug further comprises a pharmaceutically acceptable carrier and / or functional excipients, wherein the functional excipients are selected from at least one of buffers and stabilizers; the carrier and / or functional excipients do not affect the activity of the inhibitor or the function of the target gene knockdown reagent.

[0014] The present invention has the following beneficial effects:

[0015] 1. This application discovered through kinase screening that PIK3CA is involved in the expression and aggregation regulation of mutant ATXN3 protein, revealing the important role of PIK3CA in the occurrence of spinocerebellar ataxia type 3 (SCA3), and providing a new target and strategy for the treatment of spinocerebellar ataxia type 3 (SCA3).

[0016] 2. HS173 alleviates neuronal damage by effectively inhibiting PIK3CA activity and reducing the level of mutant ATXN3 protein, thus explaining the pharmacological mechanism and target of HS173 as a therapeutic agent for SCA3; at the same time, it provides a new supplement to the clinical application of HS173.

[0017] 3. This invention experimentally demonstrates that the level of mutant ATXN3 protein in the brains of mice treated with HS173 is reduced, the degree of neuronal loss is significantly alleviated, cerebellar atrophy is improved, and the level of cerebellar inflammation is reduced; simultaneously, the mice's motor coordination, balance, and muscle strength are significantly improved. This further confirms that HS173 and the PIK3CA knockdown reagent have good application prospects in the clinical treatment of SCA3. Attached Figure Description

[0018] Figure 1 A schematic diagram illustrating how the PIK3CA inhibitor HS173 reduces ATXN3-66Q protein levels. Figure 1 Image A shows the Western blot analysis results. Image B shows the grayscale analysis results of the Western blot, with Actin as an internal reference for quantitative analysis of ATXN3-66Q-GFP protein expression level.

[0019] Figure 2 This diagram illustrates how the PIK3CA inhibitor HS173 reduces the formation of pathogenic ATXN3-66Q protein aggregates. In the diagram, A is an immunofluorescence micrograph, and B represents the statistical results of A.

[0020] Figure 3 This diagram illustrates how PIK3CA overexpression increases ATXN3-66Q protein levels. A represents the Western blot analysis result. B shows the results of the Western blot grayscale analysis, with GAPDH used as an internal control to quantify the ATXN3-66Q-GFP protein expression level.

[0021] Figure 4 This diagram illustrates how PIK3CA overexpression increases the formation of pathogenic ATXN3-66Q protein aggregates. In the diagram, A is a fluorescence micrograph, and B represents the statistical results of A.

[0022] Figure 5 This is a schematic diagram of a control group or a PIK3CA inhibitor HS173 group of mice that received intraperitoneal injection of DMSO.

[0023] Figure 6 This is a schematic diagram illustrating how HS173 reduces the expression of pathogenic ATXN3 protein in the deep cerebellar nuclei of SCA3 mice. A shows the staining of pathogenic ATXN3 and NeuN (neuronal nuclear marker) in the deep cerebellar nuclei, derived from the WT-DMSO, SCA3-DMSO, and SCA3-HS173 treatment groups, respectively. B is a statistical graph of A.

[0024] Figure 7 This is a schematic diagram illustrating how HS173 reduces the expression of pathogenic ATXN3 protein in the pontine region of SCA3 mice. A shows the staining of pathogenic ATXN3 and NeuN in the pontine region, derived from the WT-DMSO, SCA3-DMSO, and SCA3-HS173 treatment groups, respectively. B is a statistical graph of A.

[0025] Figure 8 This is a schematic diagram illustrating how HS173 rescues the loss of Purkinje cells in SCA3 mice. A shows Calbindin 28K (Purkinje cell marker) staining, from WT-DMSO, SCA3-DMSO, and SCA3-HS173 treatment groups, respectively; B is a statistical graph of A.

[0026] Figure 9 This is a schematic diagram of longitudinal atrophy of the cerebellar vermis in HS173-rescue SCA3 mice. A is a fluorescence image of the cerebellar vermis in mice, and B is a quantitative analysis of the cerebellar vermis length L in each group.

[0027] Figure 10 This diagram illustrates how HS173 rescued cerebellar molecular layer atrophy in SCA3 mice. A represents fluorescence images of the cerebellar molecular layer, from the WT-DMSO, SCA3-DMSO, and SCA3-HS173 treatment groups, respectively. B shows the quantitative analysis of cerebellar molecular layer thickness in each group (A).

[0028] Figure 11 This diagram illustrates how HS173 treatment reduces microglia expression in SCA3 mice. A represents fluorescence images of cerebellar microglia in mice, with Iba1 being a microglia marker, derived from mice treated with WT-DMSO, SCA3-DMSO, and SCA3-HS173, respectively. B represents a statistical graph of microglia fluorescence intensity from the three groups of mice in A.

[0029] Figure 12 This diagram illustrates how HS173 treatment reduces astrocyte expression in SCA3 mice. A represents fluorescence images of mouse cerebellar astrocytes, with GFAP representing astrocyte markers from mice treated with WT-DMSO, SCA3-DMSO, and SCA3-HS173, respectively. B represents a statistical graph of microglia fluorescence intensity from the three groups of mice in A.

[0030] Figure 13This is a schematic diagram illustrating how RNA-seq analysis shows that PIK3CA inhibition reverses abnormal transcriptomic features in SCA3 mice.

[0031] Figure 14 This diagram illustrates how PIK3CA inhibition reverses inflammation and innate immune-related transcriptional abnormalities in SCA3 mice. Figure A shows the KEGG enrichment analysis, and figure B shows the GO analysis.

[0032] Figure 15 A schematic diagram illustrating AAV-shPIK3CA-mediated knockdown of PIK3CA in SCA3 mice.

[0033] Figure 16 This diagram illustrates how AAV-shPIK3CA-mediated knockdown of PIK3CA in SCA3 mice can restore Purkinje cells lost in the mice. A represents immunofluorescence staining, and B shows the statistical results of Purkinje cell counts.

[0034] Figure 17 The diagram shows the rotarod experiment in mice, where A is a schematic diagram of the rotarod experiment; B is an analysis of the rotarod experiment results, specifically a comparison of the duration of the fall latency in mice.

[0035] Figure 18 This is a data graph of the forelimb gripping force experiment in mice, specifically a comparison of the peak maximum gripping force of the mice.

[0036] Figure 19 This is a schematic diagram of the mouse balance beam experiment.

[0037] Figure 20 The data graph shows the time it takes for mice to reach the dark box, and B shows the number of times the mice slipped on their hind limbs.

[0038] Figure 21 The data graphs show the time it takes for mice to reach the dark box, where A represents a comparison of the time required for mice to reach the dark box, and B represents a comparison of the number of times mice slipped on their hind limbs.

[0039] Figure 22 The results are from a mouse mining experiment, specifically a comparison of the time mice spent at rest and moving. Detailed Implementation

[0040] Experimental methods in the following embodiments of the present invention, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer. All commonly used chemical reagents used in the embodiments are commercially available products. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to limit the invention.

[0041] Those skilled in the art will recognize that compound HS173 is a highly effective and selective PI3Kα inhibitor, primarily used in research experiments on cancer and fibrosis. The HS173 used in this invention is commercially available.

[0042] Example 1

[0043] This example demonstrates how PIK3CA regulates the expression and protein aggregation of pathogenic ATXN3.

[0044] To elucidate the functional role of PIK3CA in regulating the pathology of mutant ATXN3, we investigated its effects on the expression level and aggregate number of ATXN3-66Q protein.

[0045] To assess the effect of PIK3CA inhibition on ATXN3 protein expression, HEK293T cells were transfected with the mutant ATXN3-66Q-GFP plasmid and treated with either DMSO or the PIK3CA inhibitor HS173. (See below) Figure 1 .

[0046] Figure 1 A schematic diagram illustrating how the PIK3CA inhibitor HS173 reduces ATXN3-66Q protein levels. Figure 1 Figure A shows the Western blot analysis results. Figure B shows the results of the Western blot grayscale analysis, which quantifies the expression level of ATXN3-66Q-GFP protein using Actin as an internal control. * indicates p < 0.05. Specifically, Figure A shows the expression of ATXN3-66Q protein detected by Western blot after cells were transfected with empty vector control and ATXN3-66Q-GFP plasmid, respectively, and treated with DMSO or HS173. In the figure, DMSO is the control group, HS173 is the inhibitor treatment group, and Actin is used as an internal control. Figure B shows the numerical comparison of the Western blot grayscale analysis results between the DMSO control group and the HS173 inhibitor treatment group. Each point in B represents an independent experiment.

[0047] Figure 1 Western blot results showed that HS173 treatment significantly reduced ATXN3-66Q-GFP protein levels. Gray-scale quantitative analysis results were consistent with the Western blot results, suggesting that PIK3CA inhibition can reduce the expression of mutant ATXN3 protein. In other words, the specific inhibition of PIK3CA activity by the PIK3CA inhibitor HS173 significantly reduced the protein level of ATXN3-66Q.

[0048] To assess the effect of PIK3CA inhibition on the number of mutant ATXN3 protein aggregates, HEK293T cells were transfected with the ATXN3-66Q-GFP plasmid and treated with either DMSO or HS173. ATXN3-66Q-GFP aggregate formation was observed using fluorescence microscopy. The ATXN3 aggregate level was quantitatively analyzed by counting the total number of aggregates in each cell within each field of view. (See details below.) Figure 2 .

[0049] Figure 2 This diagram illustrates how the PIK3CA inhibitor HS173 reduces the formation of ATXN3-66Q protein aggregates. In the diagram, A represents an immunofluorescence micrograph, B represents the statistical results of A, and * indicates p < 0.05. Specifically, A shows the number of ATXN3-66Q-GFP protein aggregates observed under a fluorescence microscope after cells were transfected with the empty vector control (Vector) and ATXN3-66Q-GFP plasmid, respectively, and treated with DMSO or HS173. DAPI represents cell nuclei, and Merge represents merged images.

[0050] and Figure 1 These observations are consistent. Figure 2 Immunofluorescence imaging showed that treatment with the HS173 inhibitor significantly reduced the formation of pathogenic ATXN3-66Q aggregates.

[0051] To evaluate the effect of PIK3CA overexpression on ATXN3 protein expression, HEK293T cells were co-transfected with the ATXN3-66Q-GFP plasmid and either an empty control vector or the HA-PIK3CA overexpression plasmid. Cellular proteins were collected 48 h after transfection, and ATXN3 protein expression levels were detected by Western blotting. Gray-scale quantitative analysis was used to statistically analyze the expression level of mutant ATXN3-66Q protein; see details below. Figure 3 .

[0052] Figure 3 This diagram illustrates how PIK3CA overexpression increases ATXN3-66Q protein levels. A represents the Western blot analysis result. B shows the grayscale analysis results of the Western blot, with GAPDH used as an internal control to quantify ATXN3-66Q-GFP protein expression levels. In the diagram, HA-PIK3CA signifies PIK3CA overexpression. Specifically, Figure 3 In Figure A, cells co-expressed ATXN3-66Q-GFP and empty vector control, ATXN3-66Q-GFP and HA-PIK3CA, respectively. The expression of ATXN3-66Q was detected by Western blotting, and GAPDH was used as an internal control. Figure 3In Figure B, the expression levels of ATXN3-66Q protein are compared between the empty vector control and the PIK3CA group. Each point in B represents an independent experiment. * indicates p < 0.05.

[0053] from Figure 3 It can be seen that, with Figure 1 and Figure 2 Conversely, overexpression of PIK3CA led to a significant upregulation of ATXN3-66Q expression.

[0054] To evaluate the effect of PIK3CA overexpression on ATXN3 protein expression, HEK293T cells were co-transfected with the ATXN3-66Q-GFP plasmid and either an empty control vector or the HA-PIK3CA overexpression plasmid. Forty-eight hours after transfection, the formation of ATXN3-66Q-GFP aggregates was observed using fluorescence microscopy. The ATXN3 aggregate level was quantitatively analyzed by counting the total number of aggregates in each cell within each field of view. (See details below.) Figure 4 .

[0055] Figure 4 This diagram illustrates the increased formation of ATXN3-66Q protein aggregates due to PIK3CA overexpression. A represents an immunofluorescence micrograph, B represents the statistical results of A, and * indicates p < 0.05. Figure 4 In the image, A represents cells co-expressing ATXN3-66Q-GFP and empty vector control, ATXN3-66Q-GFP and HA-PIK3CA, respectively. The amount of ATXN3-66Q-GFP protein aggregation was observed using a fluorescence microscope. DAPI represents the cell nucleus, and Merge represents the merged image.

[0056] Figure 4 Immunofluorescence analysis further confirmed that PIK3CA overexpression significantly enhanced the aggregation of pathogenic ATXN3-66Q protein.

[0057] Example 2

[0058] This example illustrates that the PIK3CA inhibitor HS173 can reduce the accumulation of ATXN3 protein in neurons of SCA3 model mice.

[0059] To evaluate the effect of PIK3CA inhibition on pathological changes in SCA3 mice, male SCA3 mice and WT mice (both 5 months old, weighing 26-30g) were treated with the PIK3CA inhibitor HS173. The experimental animals were randomly divided into three groups: WT mice (wild-type mice) received DMSO treatment, SCA3 mice received DMSO treatment, and a portion of the SCA3 mice received HS173 (10mg / kg) via intraperitoneal injection once daily for one month. After one month of continuous intraperitoneal injection, the therapeutic effect of HS173 was investigated by evaluating behavioral and pathological changes in the mice; detailed experimental procedures are described below. Figure 5 .

[0060] Figure 5 This diagram illustrates either the control group (receiving DMSO) or the PIK3CA inhibitor HS173 group in mice that received intraperitoneal injection. Five-month-old mice were injected intraperitoneally with 10 mg / kg DMSO or a PIK3CA inhibitor daily for one month. Motor function, coordination, and pathological changes in the mice were assessed.

[0061] To assess the effect of PIK3CA inhibition on ATXN3 protein accumulation in SCA3 mouse neurons, WT and SCA3 mice were treated with DMSO or HS173, and then immunofluorescence staining was performed using ATXN3 and NeuN (neuronal nuclear markers). ATXN3 expression in deep cerebellar nuclei was observed using fluorescence microscopy, and the fluorescence intensity of ATXN3 in NeuN-positive neurons was measured using ImageJ software for quantitative analysis. See details... Figure 6 .

[0062] Figure 6 This diagram illustrates how HS173 reduces the expression of pathogenic ATXN3 protein in the deep cerebellar nuclei of SCA3 mice. Figure A shows the staining of pathogenic ATXN3 and NeuN in the deep cerebellar nuclei, from the WT-DMSO, SCA3-DMSO, and SCA3-HS173 treatment groups, respectively. Figure B is a statistical graph of A, where each white circle represents one mouse. *p<0.05, **p<0.01. Specifically, Figure A shows the immunofluorescence detection of pathogenic ATXN3 in the deep cerebellar nuclei (green) and NeuN-labeled neuronal nuclei (red) after DMSO and HS-173 treatment of WT mice and spinocerebellar ataxia type 3 (SCA3) mice, respectively. The fluorescence intensity of ATXN3 in the NeuN nuclei was detected.

[0063] from Figure 6The ATXN3 and NeuN (neuronal nuclear marker) staining results showed that, compared with the WT group mouse control, the expression level of ATXN3 in the deep cerebellar nuclei of the spinocerebellar ataxia type 3 (SCA3) group was significantly increased. After HS173 treatment, the expression level of ATXN3 in the SCA3 group was significantly decreased, approaching the WT level. These results indicate that ATXN3 expression is increased in the deep cerebellar nuclei of SCA3 mice, while treatment with the PIK3CA inhibitor HS173 can reduce ATXN3 expression in the deep cerebellar nuclei of SCA3 mice.

[0064] To assess the effect of PIK3CA inhibition on ATXN3 protein accumulation in SCA3 mouse neurons, WT and SCA3 mice were treated with DMSO or HS173, and then immunofluorescence staining was performed using ATXN3 and NeuN. ATXN3 expression in the pontine region was observed using fluorescence microscopy, and the fluorescence intensity of ATXN3 in NeuN-positive neurons was measured using ImageJ software for quantitative analysis. See details... Figure 7 .

[0065] Figure 7 This diagram illustrates how HS173 reduces pathogenic ATXN3 protein expression in the pontine region of SCA3 mice. Figure A shows the staining of pathogenic ATXN3 and NeuN in the pontine region, from the WT-DMSO, SCA3-DMSO, and SCA3-HS173 treatment groups, respectively. Figure B is a statistical graph of A, where each white circle represents one mouse (p < 0.05). Specifically, Figure A shows the immunofluorescence detection of pathogenic ATXN3 in the pons (green) and NeuN-labeled neuronal nuclei (red) after DMSO and HS173 treatment of WT mice and spinocerebellar ataxia type 3 (SCA3) mice, respectively, and the detection of ATXN3 fluorescence intensity in the NeuN nuclei.

[0066] from Figure 7 The ATXN3 and NeuN staining results showed that, compared with the WT group mouse control, the ATXN3 expression level in the pons of the spinocerebellar ataxia type 3 (SCA3) group was significantly increased. After HS173 treatment, the ATXN3 expression level in the SCA3 group significantly decreased, approaching the WT level. These results indicate that ATXN3 expression is increased in the pons of SCA3 mice, while treatment with the PIK3CA inhibitor HS173 can reduce ATXN3 expression in the pons of spinocerebellar ataxia type 3 (SCA3) mice.

[0067] Example 3

[0068] This example illustrates that the PIK3CA inhibitor HS173 can improve cerebellar structural degeneration in SCA3 model mice.

[0069] To evaluate the effect of PIK3CA inhibition on cerebellar pathological changes in SCA3 mice, 5-month-old WT mice and SCA3 mice were treated with DMSO or HS173 for one month, respectively. Cerebellar tissue was harvested and frozen sections were prepared. Immunofluorescence staining with Calbindin 28K antibody was used to label Purkinje cells, and the number of Calbindin-positive Purkinje cells was counted. See details... Figure 8 .

[0070] Figure 8 This diagram illustrates how HS173 rescued Purkinje cells lost in SCA3 mice. Figure A shows Calbindin 28K (Purkinje cell marker) staining, from the WT-DMSO, SCA3-DMSO, and SCA3-HS173 treatment groups, respectively. Figure B is a statistical graph of A, where each white circle represents one mouse, *p<0.05, **p<0.01. Specifically, Figure A shows the immunofluorescence detection of Purkinje neurons (green) labeled with calbindin 28K antibody after DMSO and HS173 treatment of WT mice and spinocerebellar ataxia type 3 (SCA3) mice, respectively, to determine the number of Purkinje cells.

[0071] from Figure 8 Calbindin 28K staining revealed abundant Purkinje cells in the WT-DMSO group, arranged in a well-ordered monolayer, while the SCA3-DMSO group showed a significant reduction in Purkinje cells accompanied by disordered cell arrangement. Treatment with the PIK3CA inhibitor HS173 restored the Purkinje cell density in SCA3 mice (indicated by the white arrow in A). Quantitative analysis in B confirmed that the number of Purkinje cells in SCA3-DMSO mice was significantly lower than that in the WT control group, while HS173 treatment significantly increased the number of Purkinje cells in spinocerebellar ataxia type 3 (SCA3) mice.

[0072] To evaluate the effect of PIK3CA inhibition on cerebellar pathological changes in SCA3 mice, 5-month-old WT mice and SCA3 mice were treated with DMSO or HS173 for one month, respectively. The overall morphology of the cerebellum was observed, and the length of the cerebellar vermis was measured to assess the degree of cerebellar atrophy. See details below. Figure 9 .

[0073] Figure 9This diagram illustrates the longitudinal atrophy of the cerebellar vermis in SCA3 mice rescued by HS173. A shows fluorescence images of the cerebellar vermis in mice, and B shows the quantitative analysis of the cerebellar vermis length L (in μm) in each group. The fluorescence images in A are from the WT-DMSO, SCA3-DMSO, and SCA3-HS173 treatment groups, respectively. The white line L in the diagram indicates the vermis length L measured along the axis from the 5th to the 8th cerebellar lobule. B is a statistical graph of A, where each white circle represents one mouse, *p < 0.05.

[0074] from Figure 9 The results showed that, compared with the WT group mice, the length (L) of the cerebellar vermis in the spinocerebellar ataxia type 3 (SCA3) group was significantly shortened. After treatment with HS173, the vermis length in the SCA3 group recovered and approached the WT level. These results indicate that SCA3 causes longitudinal atrophy of the cerebellar vermis, and treatment with the PIK3CA inhibitor HS173 can alleviate vermis atrophy in SCA3 mice.

[0075] To assess the effect of PIK3CA inhibition on cerebellar pathological changes in SCA3 mice, 5-month-old WT mice and SCA3 mice were treated with DMSO or HS173 for one month, respectively. DAPI (nuclear, blue) and Calbindin 28K (Purkinje cells, green) staining were used to visualize the cerebellar cortex structure. Measurements were taken between the outer edge of the molecular layer (ML) and the granular layer, and the ML thickness was calculated to evaluate the effect of HS173 on the histopathological changes in the cerebellum of SCA3 mice. See details below. Figure 10 .

[0076] Figure 10 This diagram illustrates the role of HS173 in rescuing cerebellar molecular layer atrophy in SCA3 mice. A represents fluorescence images of the cerebellar molecular layer (ML), from the WT-DMSO, SCA3-DMSO, and SCA3-HS173 treatment groups, respectively. B represents the quantitative analysis of the cerebellar molecular layer thickness (ML, μm) for each group in A; that is, B is a statistical graph of A, where each white circle represents one mouse. Specifically, A represents the thickness of the cerebellar slices after DAPI and Calbindin 28K staining, measured vertically from the outer edge of the molecular layer to the starting point of the granular layer.

[0077] from Figure 10 It was found that the molecular layer thickness of mice in the spinocerebellar ataxia type 3 (SCA3) group was significantly shorter than that in the WT group. Treatment with the PIK3CA inhibitor HS173 could partially restore the cerebellar molecular layer thickness in SCA3 mice (*p<0.05, **p<0.01). In other words, this invention further measured the cerebellar molecular layer (ML) thickness in mice. Figure 10The results showed that the molecular layer of spinocerebellar ataxia type 3 (SCA3) mice was significantly thinner, and HS173 treatment could restore the molecular layer thickness of SCA3 mice.

[0078] In summary, these results indicate that treatment with the PIK3CA inhibitor HS173 reduced the expression level of ATXN3 in the cerebellum and pons, alleviated the loss of Purkinje cells in spinocerebellar ataxia type 3 (SCA3) mice, and improved the atrophy of the vermis and molecular layer structures of the cerebellum in SCA3 mice.

[0079] Example 4

[0080] This example illustrates that the PIK3CA inhibitor HS173 can reduce the neuroinflammatory response in SCA3 model mice.

[0081] Neurodegenerative diseases in SCA3 mice are typically accompanied by the proliferation and activation of glial cells. Microglia, as innate immune cells of the central nervous system, maintain brain homeostasis by phagocytizing and clearing damaged neurons and abnormal protein aggregates; however, their continued activation leads to increased release of pro-inflammatory factors, thereby exacerbating neuroinflammation and nerve damage. Simultaneously, astrocytes also undergo reactive activation in disease states, participating in the regulation of inflammatory responses, alterations in neuronal support functions, and the progression of neurodegenerative diseases. Therefore, to assess the neuroinflammatory status of the cerebellum in SCA3 mice, we used immunofluorescence staining analysis with the microglia marker Iba1 and the astrocyte marker GFAP, respectively.

[0082] To assess the effect of PIK3CA inhibition on neuroinflammatory responses in SCA3 mice, 5-month-old WT mice and SCA3 mice were treated with DMSO or HS173 for one month, respectively. Cerebellar tissue was harvested and frozen sections were prepared. Immunofluorescence staining was performed using the microglial marker Iba1, and DAPI was used to label cell nuclei. Images were acquired using a fluorescence microscope, and the fluorescence intensity of Iba1 was quantitatively analyzed using ImageJ software to evaluate the effect of HS173 on glial cell activation and neuroinflammatory levels in the cerebellar region. See details below. Figure 11 .

[0083] Figure 11This diagram illustrates the reduction of microglia expression in SCA3 mice by HS173 treatment. In the diagram, A represents fluorescence images of cerebellar microglia (IBA1) from mice treated with WT-DMSO, SCA3-DMSO, and SCA3-HS173, respectively. Green indicates IBA1-labeled microglia, blue indicates DAPI-labeled nuclei, and Merge indicates a combined green and blue image. B represents a statistical graph of microglia fluorescence intensity from the three groups in A, with each white circle representing one mouse. *p<0.05, **p<0.01.

[0084] Figure 11 The results showed that the fluorescence intensity of microglia (IBA1) in SCA3 mice was significantly increased, and treatment with the PIK3CA inhibitor HS173 could reduce the fluorescence intensity of IBA1. This indicates that PIK3CA inhibition alleviates the expression of inflammatory cells, namely microglia.

[0085] To assess the effect of PIK3CA inhibition on neuroinflammatory responses in SCA3 mice, 5-month-old WT mice and SCA3 mice were treated with DMSO or HS173 for one month, respectively. Cerebellar tissue was harvested and frozen sections were prepared. Immunofluorescence staining was performed using the astrocyte marker GFAP, and DAPI was used to label cell nuclei. Images were acquired using fluorescence microscopy, and the fluorescence intensity of GFAP was quantitatively analyzed using ImageJ software to evaluate the effect of HS173 on glial cell activation and neuroinflammatory levels in the cerebellar region. See details below. Figure 12 .

[0086] Figure 12 This diagram illustrates how HS173 treatment reduces astrocyte expression in SCA3 mice. In this diagram, A represents fluorescence images of cerebellar astrocytes (GFAP) from mice treated with WT-DMSO, SCA3-DMSO, and SCA3-HS173, respectively. Green GFAP labels the astrocytes, blue DAPI represents the nuclei, and Merge represents the merged green and blue images. B represents a statistical graph of astrocyte fluorescence intensity from the three groups in A. Each white circle represents one mouse, *p<0.05.

[0087] Figure 12 The results showed that the fluorescence intensity of astrocytes (GFAP) in SCA3 mice was significantly increased, and treatment with the PIK3CA inhibitor HS173 could reduce the fluorescence intensity of GFAP. This indicates that PIK3CA inhibition reduces the expression of inflammatory cells, namely astrocytes.

[0088] To investigate the effects of PIK3CA inhibition on transcriptomic alterations in SCA3 mice, we collected cerebellar tissues from mice in the WT-DMSO, SCA3-DMSO, and SCA3-HS173 groups for RNA sequencing (RNA-seq) analysis. By comparing transcriptomic data from the WT-DMSO and SCA3-DMSO groups, and the SCA3-DMSO and SCA3-HS173 groups, we screened differentially expressed genes that were abnormally expressed in SCA3 mice and whose expression was reversed after HS173 treatment. Venn analysis was then used to screen for these reversed genes.

[0089] First, to further investigate the effect of the PIK3CA inhibitor HS173 on the neuroinflammatory response in SCA3 mice, we collected cerebellar tissue from mice in the WT-DMSO, SCA3-DMSO, and SCA3-HS173 groups for RNA sequencing (RNA-Seq) analysis. See details below. Figure 13 and Figure 14 .

[0090] Figure 13 RNA-seq analysis was used to reveal the reversal of abnormal transcriptomic features in SCA3 mice by PIK3CA inhibition. Specifically, Venn diagrams of differentially expressed genes were used to screen for reverse genes that were upregulated in the SCA3-DMSO group compared to the WT-DMSO group and downregulated after HS173 treatment, as well as reverse genes that were downregulated in the SCA3-DMSO group compared to the WT-DMSO group and upregulated after HS173 treatment. A total of 142 genes that were abnormally upregulated in SCA3 mice and restored after HS173 treatment were identified.

[0091] We Figure 13 Differentially expressed genes that were reversed after HS173 treatment were subjected to KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway enrichment analysis and Gene Ontology (GO) functional enrichment analysis to assess the impact of PIK3CA repression of HS173 on SCA3-related biological processes and signaling pathways. See details below. Figure 14 .

[0092] Figure 14This diagram illustrates how PIK3CA inhibition reverses inflammation and innate immune-related transcriptional abnormalities in SCA3 mice. A represents KEGG enrichment analysis, and B represents GO analysis. KEGG enrichment analysis showed that these reversal genes were primarily enriched in inflammatory and innate immune-related pathways, including cytoplasmic DNA recognition, phagosome pathway, TNF signaling pathway, NF-κB signaling pathway, NOD-like receptor signaling pathway, Toll-like receptor signaling pathway, chemokine signaling pathway, and cytokine-receptor interaction pathway. GO analysis (B) evaluated the functional characteristics of differentially expressed genes at three levels: biological process, cellular component, and molecular function. Results showed that at the biological process level, reversal genes were mainly enriched in processes such as positive regulation of inflammatory responses, cytokine-mediated signaling pathways, chemokine-mediated signaling pathways, and phagocytosis regulation; at the cellular component level, they were mainly enriched in structures such as lysosomal membranes, endocytic vesicle membranes, membrane rafts, and the NADPH oxidase complex; and at the molecular function level, they were mainly involved in cytokine activity, cytokine receptor activity, chemokine receptor binding, and chemokine activity.

[0093] from Figure 13-14 RNA-Seq results showed that treatment with the PIK3CA inhibitor HS173 could inhibit cerebellar inflammatory response and activation of cytokines and chemokine responses in SCA3 mice.

[0094] Example 5

[0095] This example illustrates that knocking down PIK3CA can reproduce the neuroprotective effect in SCA3 model mice.

[0096] To verify the effect of PIK3CA knockdown on Purkinje cell lesions in SCA3 mice, 5-month-old WT mice and SCA3 knock-in mice under anesthesia underwent stereotactic injection. AAV-shPIK3CA (or shPIK3CA-AAV) or the control virus AAV-shNC was injected into the V–VI lobes of the cerebellum. One month after viral expression, the mice were sacrificed and cerebellar tissue was collected for further analysis. See details below. Figures 15-16 .

[0097] Figure 15This diagram illustrates the shPIK3CA-AAV-mediated knockdown of PIK3CA expression in SCA3 mice. Specifically, 5-month-old WT mice and SCA3 knock-in mice were stereotactically injected with AAV virus into the V-VI lobes of the cerebellum. WT mice were injected with AAV-shNC-mCherry, while SCA3 mice were injected with either AAV-shNC-mCherry or AAV-shPIK3CA-mCherry. Cerebellar tissue was collected one month after viral expression for pathological analysis. Both AAV-shPIK3CA and the control virus AAV-shNC were commercially available. AAV: Adeno-associated virus, a commonly used gene delivery vector with high safety and low immunogenicity, capable of efficiently introducing genetic material into host cells. shPIK3CA: Short hairpin RNA targeting the PIK3CA gene; this is an artificially designed RNA sequence that, upon entering the cell, specifically silences or knocks down the expression of the PIK3CA gene through RNA interference, thereby inhibiting the function of the protein encoded by this gene. AAV-shNC is a commonly used negative control viral vector in gene function studies, namely an adeno-associated virus carrying a short hairpin RNA for negative control. The "mCherry" in this vector represents the cherry-colored fluorescent protein.

[0098] We collected the process Figure 15 Mouse brain tissue and cerebellum tissue were treated. After fixation and sectioning, Purkinje cells were labeled using immunofluorescence staining with Calbindin28K antibody. mCherry signal was used to indicate the virus-infected area, and DAPI was used to label the cell nucleus. Images were acquired using fluorescence microscopy, and the number of Calbindin-positive Purkinje cells in the V-VI lobes of the cerebellum was statistically analyzed to assess the effect of PIK3CA knockdown on Purkinje cell loss in SCA3 mice. See details below. Figure 16 .

[0099] Figure 16Figure 1 shows the results of shPIK3CA-AAV-mediated PIK3CA knockdown restoring Purkinje cells lost in SCA3 mice. A represents the immunofluorescence assay results. Purkinje neurons were labeled with Calbindin 28K antibody (green), mCherry signal (red) was used to indicate the viral infection area, DAPI (blue) was used to label the cell nucleus, Merge is the merged image, and Zoom on the right is a magnified view of the local area. The results showed that the WT-AAV-shNC group had abundant and well-organized Purkinje cells, forming a continuous monolayer structure; the SCA3-AAV-shNC group showed a significant decrease in Purkinje cell number, accompanied by disordered cell arrangement and damaged dendritic structure; after PIK3CA knockdown, the number of Purkinje cells significantly increased, the cell arrangement became more regular, and the dendritic morphology improved, suggesting that PIK3CA knockdown can alleviate the degenerative changes in Purkinje neurons of SCA3 mice. B shows the quantitative analysis of Purkinje cell number; each white circle represents one mouse, *p<0.05, **p<0.01. The results showed that, compared with the WT-AAV-shNC group, the number of Purkinje cells in the V-VI lobes of the cerebellum was significantly reduced in the SCA3-AAV-shNC group; while after PIK3CA knockdown, the number of Purkinje cells in SCA3 mice increased significantly and returned to the WT level.

[0100] from Figure 15-16 The results showed that targeted inhibition of PIK3CA could effectively improve the loss of Purkinje cells in SCA3 mice, providing histological evidence for its neuroprotective effect.

[0101] Example 6

[0102] This example illustrates that the PIK3CA inhibitor HS-173 can improve motor dysfunction in SCA3 model mice.

[0103] To assess the effect of PIK3CA inhibition on motor function in SCA3 mice, rotarod, grip strength, balance beam, and minefield tests were performed after HS173 treatment. See [link to relevant documentation]. Figures 17-22 .

[0104] Figure 17 The experiment involved placing mice on a rotundus apparatus. The rotation speed was gradually increased from 5 rpm to 40 rpm over 90 seconds, and then maintained at 40 rpm. The latency period before the mouse fell off the rotundus was recorded, with the longest test time being 300 seconds.

[0105] Figure 17This diagram illustrates how the PIK3CA inhibitor HS173 improves motor function in SCA3 mice using a rotarod experiment. A is a schematic diagram of the rotarod experiment. The longer a mouse stays on the rotarod, the better its motor coordination and exercise tolerance. B compares the rotarod dwell times of mice; each circle represents one mouse, *p<0.05, **p<0.01. The rotarod experiment results show that SCA3 mice spent significantly less time on the rotarod than wild-type mice, suggesting impaired motor coordination. After HS173 treatment, the dwell time in SCA3-HS173 mice was significantly prolonged (p<0.05), indicating that PIK3CA inhibitor HS173 treatment can improve motor coordination and exercise tolerance in SCA3 mice.

[0106] In the forelimb grip strength test of mice, the maximum grip strength of the mice's forelimbs was measured using a grip strength meter. Multiple measurements were taken consecutively, and the average value was statistically analyzed. See details... Figure 18 .

[0107] Figure 18 This is a graph showing the data from the forelimb gripping force experiment in mice, specifically a comparison of the peak maximum gripping force. The graph includes the WT-DMSO, SCA3-DMSO, and SCA3-HS173 treatment groups. Each circled dot represents one mouse. *p<0.05, ***p<0.001. Figure 18 The results of the forelimb grip strength test in mice also showed that the forelimb grip strength of SCA3 mice was significantly reduced, while HS173 treatment significantly improved the maximum grip strength of mice.

[0108] Figures 19-21 All experiments involved balance beam tests on mice. Balance beams with widths of 1 cm and 0.5 cm, and lengths of 100 cm, were used for testing. The time required for the mice to reach the end point of the dark box from the starting point and the number of times their hind limbs slipped during the test were recorded to evaluate motor coordination and balance ability.

[0109] Figure 19 This is a schematic diagram of the mouse balance beam experiment. The shorter the time it takes for a mouse to cross the 100cm balance beam to reach the dark box, the better the mouse's motor function. The fewer times the hind limbs slip, the better the mouse's balance ability.

[0110] Figure 20 The graph shows the results of the mouse test on a 1cm wide balance beam. Figure A compares the time it takes for a mouse to reach the dark box from a 100cm long balance beam; Figure B compares the number of times a mouse slips on its hind legs while walking on the balance beam. Each circled dot in the graph represents one mouse, and each dot represents the average of at least three independent trials for that mouse. *p<0.05.

[0111] Figure 21The graph shows the results of the mouse balance beam experiment on a 0.5 cm wide beam. Figure A compares the time it takes for a mouse to reach the dark box from a 100 cm long beam; Figure B compares the number of times a mouse slips on its hind legs while walking on the beam. Each circled dot in the graph represents one mouse, and each dot represents the average of at least three independent experiments for that mouse. *p<0.05, ***p<0.001, ****p<0.0001.

[0112] from Figures 19 to 21 The results showed that in the balance beam experiment, SCA3 mice took longer to traverse the 0.5cm and 1cm wide balance beams, and the number of times their hind limbs slipped off was also significantly increased. However, after treatment with HS173, these effects were significantly reduced, suggesting that treatment with the PIK3CA inhibitor HS173 can improve the motor function and balance ability of SCA3 mice.

[0113] Figure 22 For the mine experiment, mice were placed in the center of a mine to explore freely, and their movement trajectories were recorded using a video tracking system. The total movement time and rest time of the mice were counted to assess their ability to move independently.

[0114] Figure 22 This figure shows the results of resting and mobilization times in mice during a mining experiment. Statistical analysis of resting and mobilization times revealed that mice in the SCA3 group exhibited decreased mobilization ability (decreased mobilization time, increased resting time), while HS173 treatment partially restored mobilization ability (prolonged mobilization time, decreased resting time). From... Figure 22 It is evident that treatment with the PIK3CA inhibitor HS173 can improve motor function in SCA3 mice.

[0115] In summary, this invention relates to the application of an inhibitor of the activity of the catalytic subunit (p110α) of phosphatidylinositol-4,5-bisphosphate 3-kinase α (PI3Kα). PI3Kα is a protein complex encoded by the PIK3CA gene. Based on a whole-kinase-based CRISPR-Cas9 knockout library screening technique, this invention identified a novel gene, PIK3CA, that can regulate the aggregation of mutant ATXN3-polyQ protein, and systematically validated its function. To investigate the effect of PIK3CA on the aggregation of mutant ATXN3-polyQ protein, this invention used the PIK3CA inhibitor HS173 to inhibit its activity in human embryonic kidney 293T cells (HEK293T). The results showed that the expression level and the number of aggregates of mutant ATXN3 protein were significantly reduced; conversely, when PIK3CA protein was overexpressed, the expression level and the number of aggregates of ATXN3 protein increased. Furthermore, SCA3 mice were treated with HS173 via intraperitoneal injection. Results showed that, compared to the DMSO control group, the HS173-treated group exhibited decreased expression levels of mutant ATXN3 protein in the cerebellum and pons, significantly reduced cerebellar neuronal loss, improved cerebellar atrophy, and reduced cerebellar inflammation. Simultaneously, the SCA3 mice showed significant improvements in motor coordination, balance, and muscle strength. In summary, we found that PIK3CA activity promotes the expression and accumulation of mutant ATXN3 protein, and HS173 can specifically inhibit PIK3CA activity, reduce the expression and accumulation of mutant ATXN3 protein, and improve inflammation, neuronal damage, cerebellar atrophy, and motor dysfunction in SCA3 mice; therefore, it may serve as a potential drug for the treatment of SCA3.

[0116] This invention relates to the application of an inhibitor in the preparation of a drug for treating SCA3, namely spinocerebellar ataxia type 3. The inhibitor is an inhibitor of the gene PIK3CA or the kinase PI3Kα, and the inhibitor is the compound HS173. This invention also relates to the application of a target gene knockdown reagent in the preparation of a drug for treating SCA3, wherein the target gene is PIK3CA. Experiments show that, compared to the control group, mice treated with HS173 showed significantly reduced accumulation of pathogenic ATXN3 protein, significantly reduced neuronal loss, improved cerebellar atrophy, and improved cerebellar inflammation; simultaneously, the mice's motor coordination, balance, and muscle strength were significantly improved. HS173 can specifically inhibit PIK3CA activity and can be considered a potential drug for treating SCA3.

[0117] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. Use of an inhibitor for the manufacture of a medicament for the treatment of the disease SCA3, characterized in that, The SCA3 refers to spinocerebellar ataxia type 3, and the inhibitor is an inhibitor of the gene PIK3CA or the kinase PI3Kα, and the inhibitor is the compound HS173.

2. The application according to claim 1, characterized in that, The inhibitor is used in the preparation of a medicament for treating SCA3 in humans or animals, wherein the medicament is in the form of an oral or injectable formulation.

3. The application according to claim 1 or 2, characterized in that, The drug further comprises a pharmaceutically acceptable carrier and / or functional excipients, wherein the functional excipients are selected from at least one of buffers and stabilizers; the carrier and / or functional excipients do not affect the activity of the inhibitor.

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