Use of aurantiamacartin for preparing a high-affinity GLUT1 protein targeting inhibitor

Abstract

The application discloses application of aurantiamarin in preparation of a GLUT1 protein targeting inhibitor with high affinity, and belongs to the technical field of medicinal chemistry. It is found that aurantiamarin shows micromolar (muM) level high-efficiency inhibitory activity on GLUT1, and the aurantiamarin may play a role through a new mechanism different from existing competitive inhibitors, such as allosteric inhibition or state-dependent blockage, effectively blocks glucose uptake, and is not prone to drug resistance.

Description

Technical Field

[0001] This invention belongs to the field of medicinal chemistry technology, specifically the use of cassia aurantium in the preparation of a high-affinity GLUT1 protein-targeting inhibitor. Background Technology

[0002] With global population growth and aging, and the increase in risk factors such as environmental, metabolic, and lifestyle factors, the number of people worldwide suffering from neurological diseases such as stroke, Alzheimer's disease, and meningitis has risen sharply. Most central nervous system diseases can directly or indirectly lead to cognitive impairment, which is often the core or early symptom of these diseases.

[0003] Manganese is an essential trace element for the human body, participating in various physiological processes. Excessive manganese accumulation in the brain due to occupational and environmental exposure leads to a unique extrapyramidal syndrome, primarily manifested as cognitive impairment. Astrocytes are the main glial cells in the central nervous system, playing a crucial role in maintaining homeostasis, supporting neuronal function, and regulating synaptic activity. Morphological and functional changes in astrocytes play a key role in the occurrence and development of neurocognitive dysfunction. The manganese exposure model, a classic model for studying environmentally driven neurodegenerative diseases, is believed to be associated with cognitive impairment due to the abnormal activation of astrocytes caused by excessive accumulation; however, the specific mechanisms and targets of its damaging effects remain unclear.

[0004] Glucose is a core substance for maintaining cellular energy metabolism and proliferation, and the availability of glucose in tissues is crucial for maintaining homeostatic energy. GLUT1 is a highly conserved and widely distributed glucose transporter in mammalian cells, highly expressed in various human cancers, the blood-brain barrier, erythrocytes, and various epithelial tissues. It is responsible for basal glucose uptake and is essential for maintaining brain energy supply. Under physiological conditions, GLUT1 achieves transmembrane glucose transport through conformational changes, and its function is regulated at multiple levels, including transcription, post-translational modifications, and inhibitors. Dysfunctional mutations may lead to diseases such as GLUT1 deficiency syndrome, epilepsy, and diabetic complications, while GLUT1 overexpression is a prognostic indicator for cancer. Previous studies have shown that increased GLUT1 protein expression in a manganese exposure model is closely related to astrocyte activation, suggesting that abnormal GLUT1 expression may be a potential target for manganese-induced astrocyte activation leading to central nervous system cognitive dysfunction.

[0005] Existing small-molecule GLUT1 inhibitors, such as STF-31, can effectively reduce glucose uptake, inhibit lactate, suppress glycolysis, and induce tumor cell death. Additionally, BAY-867 can also serve as a small-molecule inhibitor effectively reducing glycolysis rate and tumor volume, thereby delaying tumor progression. However, no highly selective GLUT1 inhibitors are currently widely used in the neurological field. While existing inhibitors (such as BAY-876 and STF-31) have shown anti-tumor potential in animal and in vitro studies, they still suffer from insufficient activity, poor selectivity, and unsatisfactory pharmacokinetic properties when targeting manganese-induced astrocyte activation. For example, STF-31 showed low inhibitory efficacy against glucose uptake in GLUT1 during the evaluation process. GLUT4-IN-2 and KL-11743, among others, simultaneously interfere with other GLUT subtypes (such as GLUT2 and GLUT3), leading to potential off-target toxicity; others, due to poor metabolic stability or poor tissue distribution, are difficult to achieve effective in vivo efficacy.

[0006] Therefore, exploring novel mechanisms of action against GLUT1 and developing small-molecule inhibitors with novel chemical structures, high inhibitory activity, high subtype selectivity, and excellent pharmacokinetic properties is of great significance for overcoming the current challenges of neurodegenerative diseases caused by manganese exposure-induced astrocyte activation. There is an urgent need to develop therapeutic drugs with novel mechanisms of action. Summary of the Invention

[0007] The purpose of this invention is to provide the use of cassia seed extract in the preparation of a high-affinity GLUT1 protein-targeting inhibitor, in order to solve the problems mentioned in the background art.

[0008] To solve the above-mentioned technical problems, the present invention is achieved through the following technical solution:

[0009] The purpose of this invention is to provide the use of cassia seed extract in the preparation of a high-affinity GLUT1 protein-targeting inhibitor.

[0010] This invention compares the binding ability of GluUT1 to the positive control drug STF-31. Docking was performed on a library of 10,000 drug-like small molecules, and approximately 970 active small molecules were found to successfully dock to the GluUT1 binding site. After evaluating the hydrogen bonding mode and drug-likeness of the catalytic residues Gln282 and Trp388 at the active site, small molecule compounds with higher binding fractions to GluUT1 were screened, and five candidate small molecules were selected for in vitro experimental validation. The results showed that Cassia tora extract (C...)... 17 H 14 O7, Figure 2 A) It can significantly inhibit GLUT1 activity and manganese exposure-induced astrocyte activation and cognitive impairment.

[0011] Another object of the present invention is to provide a high-affinity GLUT1 protein targeting inhibitor comprising the above-mentioned cassia seed extract and its pharmaceutically acceptable adjuvant.

[0012] Furthermore, the dosage forms of GLUT1 protein-targeting inhibitors are tablets, powders, pills, capsules, or injections.

[0013] Another object of the present invention is to provide the use of the above-mentioned cassia seed extract or GLUT1 protein targeting inhibitor in the preparation of a drug for alleviating neurotoxicity.

[0014] Furthermore, the neurotoxicity is caused by astrocyte activation resulting from manganese exposure.

[0015] Furthermore, the effective dose of cassia seed extract in the drug is 5-25 mg / kg.

[0016] Another object of the present invention is to provide a medicament for alleviating neurotoxicity, comprising the above-mentioned cassia seed extract and pharmaceutically acceptable excipients thereof.

[0017] Furthermore, the dosage form of the drug is tablets, powders, pills, capsules, or injections.

[0018] Another object of the present invention is to provide the use of the above-mentioned cassia seed extract or GLUT1 protein targeting inhibitor in the preparation of a medicament for treating cognitive impairment.

[0019] Furthermore, the effective dose of cassia seed extract in the drug is 5-25 mg / kg.

[0020] The present invention has the following beneficial effects:

[0021] This invention reveals that cassia seed extract exhibits highly potent inhibitory activity against GLUT1 at the micromolar (μM) level. It may exert its effect through novel mechanisms different from existing competitive inhibitors, such as allosteric inhibition or state-dependent blockade, effectively blocking glucose uptake and rarely inducing drug resistance. Furthermore, this invention demonstrates its clear in vitro and in vivo efficacy. In cell models, cassia seed extract effectively inhibits glucose uptake by astrocytes. In an in vivo animal model of manganese exposure, it also significantly improves manganese-induced astrocyte activation, thereby alleviating cognitive impairment. Attached Figure Description

[0022] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1 Flow cytometry plot showing the inhibition of glucose uptake by cassia aurantium;

[0024] Figure 2 This is a binding assay for cassia seed extract and its target protein GLUT1; where A is the structural formula of cassia seed extract; B is a molecular docking pattern of cassia seed extract and GLUT1; C is an interaction diagram of cassia seed extract and GLUT1; D is a root mean square deviation (RMSD) pattern of cassia seed extract and GLUT1; and E is a mean aromatic root fluctuation (RMSF) pattern of cassia seed extract and GLUT1.

[0025] Figure 3 The resonance binding diagram of cassia aurantium and GLUT1 plasma is shown.

[0026] Figure 4 The CETSA diagram of cassia seed extract;

[0027] Figure 5 The results of the Y-maze test in manganese-exposed mice after injection of different doses of cassia seed extract;

[0028] Figure 6 The image shows the results of the cross maze test on manganese-exposed mice after injection of different doses of cassia seed extract. Detailed Implementation

[0029] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and 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.

[0030] Example 1

[0031] 1. Inhibitory activity of au cassia seed extract (AO) against GLUT1

[0032] To evaluate the inhibitory activity of small molecule compounds on GLUT1, we used an astrocyte model with high GLUT1 expression and quantified the inhibitory effect by measuring the uptake of 2-NBDG using flow cytometry. The results are shown in [Figure number missing]. Figure 1 .

[0033] like Figure 1As shown, the small molecule compound (AO) in this invention exhibits inhibitory activity against glucose uptake, indicating that the ability of cassia aurantium to inhibit glucose uptake is significantly better than that of the positive control drug.

[0034] 2. The binding mechanism between au cassia seed extract (AO) and GLUT1

[0035] To elucidate the binding mode, interaction mechanism, and binding stability of the compounds of this invention with the target protein GLUT1 (GLUT1) at the atomic level, the small molecule compound AO ( Figure 2 A) Molecular docking was performed between the 3D structures of STF-31 (a) and the positive control STF-31 and the prepared GLUT1 structure. The results are shown in [Figure A]. Figure 2 .

[0036] like Figure 2 As shown in Figure B, the small molecule compound AO can stably bind in the substrate-binding pocket of GLUT1. Docking analysis indicates that the docking conformation of cassia aurantium can stably bind to the active site of GLUT1. Consistent with the original substrate, the hydrogen bond lengths formed by cassia aurantium with the key residues Gln282 and Trp388 are both 2.2 Å. Figure 2 (See C). Furthermore, cassia aurantium forms stable π-π stacking interactions with Phe26 and Phe379, thereby enhancing its binding to GLUT1. Simultaneously, daunorubicin also forms hydrogen bonds with Gln283, Asn288, and Glu380 at the GLUT1 binding site, with bond lengths of 2.0 Å, 2.3 Å, and 2.4 Å, respectively (see C). Figure 2 The presence of C in the middle of the molecule further contributes to its stability at the binding site. To further evaluate the stability and binding mode of the GLUT1-cytochalasin complex, MD simulations were performed on the docking complex. After a simulation time of 200 ps, ​​the RMSD of the GLUT1-cytochalasin and GLUT1-cytochalasin B complex mainly fluctuated between 1.2 and 2.2, indicating that there were no significant conformational changes in either complex, and both were in a stable state. Figure 2 (D). The mean RMSD value of the GLUT1-cytochalasin complex was 1.523, close to that of the GLUT1-cytochalasin B complex (1.786). Furthermore, the RMSF changes of amino acid residues in the GLUT1-cytochalasin and GLUT1-cytochalasin B complexes were highly similar (D). Figure 2 The low fluctuation levels of both complexes at Gln282, Gln283, and Glu380 indicate that these interactions are very persistent and stable. In summary, these results demonstrate that cassia seed extract has a high binding affinity for GLUT1.

[0037] 3. Affinity of Cassia oleracea extract (AO) to GLUT1

[0038] Kinetic analysis parameters indicate that AO has a high binding affinity for cassia aurantium, as demonstrated by SPR (see...). Figure 3 The assay confirmed that the compound AO of this invention has a clear targeted binding ability to the GLUT1 protein (KD=3.92 μM). Although this moderate binding strength of AO is higher than the activity of the STF-31 target (1 μM), it can effectively and selectively inhibit glucose uptake in astrocytes. Although its affinity still has room for improvement compared to the final target, it has shown great potential for optimization as a GLUT1 inhibitor in alleviating the neurotoxic effects caused by manganese exposure-induced astrocyte activation.

[0039] Example 2

[0040] 1. Cell experiments

[0041] Cell lysates were prepared using astrocytes with high expression of endogenous GLUT1. The lysates were co-incubated with either the compound AO or DMSO (controls of this invention). Protein denaturation was induced by subjecting the samples to a series of temperature gradients (e.g., 37°C to 65°C) using real-time quantitative PCR. Subsequently, the amount of remaining undenatured GLUT1 protein was detected by Western blotting, and its melting temperature was calculated. The results are shown in [Figure number missing]. Figure 4 .

[0042] like Figure 4 As shown, compared with the DMSO control group, treatment with compound AO resulted in a significant positive shift in the melting temperature of the GLUT1 protein. This dose-dependent increase in Tm value is direct evidence that small molecules bind to proteins, making their structure more stable and less susceptible to thermal denaturation, confirming that AO can directly bind to the GLUT1 protein in the endogenous environment.

[0043] 2. Animal experiments

[0044] C57BL / 6J mice were randomly divided into a model group and three treatment groups. The treatment groups were administered different doses of compound AO (1, 5, and 25 mg / kg / day) by gavage for 3 consecutive weeks. After treatment, a chronic manganese exposure model was established by subcutaneous injection of manganese chloride tetrahydrate. A normal control group (without manganese exposure, administered CMC-Na) was also established. After modeling, the elevated cruciate maze and Y-maze tests were performed sequentially to assess anxiety-like behavior and spatial working memory in the mice, respectively. The results are shown in the table below. Figure 5 and Figure 6 .

[0045] The Y-maze was used to assess the short-term spatial working memory of mice, with the number of times they entered the new arm as the core indicator. Figure 5 Mice in the manganese exposure model group exhibited significantly fewer exploration attempts than the normal control group, suggesting impaired working memory. Treatment with compound AO showed a clear protective effect. The 25 mg / kg dose group showed the most significant effect, restoring the number and time of new arm exploration attempts in mice to normal levels, with a highly significant difference compared to the model group. The 1 and 5 mg / kg dose groups showed only a slight and unstable trend of improvement.

[0046] like Figure 6 As shown, compared with the normal control group, the percentage of mice entering the open arm in the manganese exposure model group was significantly reduced, exhibiting typical anxiety-like behavior. Treatment with the small molecule compound AO improved this behavioral deficit in a dose-dependent manner. Specifically, while the 5 mg / kg dose group showed a trend of improvement, the 25 mg / kg dose group significantly increased the number of mice entering the open arm, restoring the index to a level not significantly different from the normal control group, indicating that at this dose, the compound effectively alleviated the anxiety symptoms induced by manganese exposure.

[0047] A comprehensive behavioral assessment of cognitive impairment, including emotion and learning / memory testing, was conducted to systematically evaluate the effects of different doses of anti-acid (AO) intervention on cognitive function. The results showed a clear dose-response relationship in alleviating manganese exposure-induced cognitive impairment: the 5 mg / kg dose group may have provided mild relief for manganese exposure-induced impairment in learning / memory or emotion regulation, while the 25 mg / kg dose group showed significant improvement.

[0048] The preferred embodiments of the present invention disclosed above are merely illustrative of the invention. These preferred embodiments do not exhaustively describe all details, nor do they limit the invention to the specific implementations described. Clearly, many modifications and variations can be made based on the content of this specification. This specification selects and specifically describes these embodiments to better explain the principles and practical applications of the invention, thereby enabling those skilled in the art to better understand and utilize the invention. The invention is limited only by the claims and their full scope and equivalents.

Claims

1. Use of Cassia tora extract in the preparation of a high-affinity GLUT1 protein targeting inhibitor.

2. A high-affinity GLUT1 protein-targeting inhibitor, characterized in that, This includes the cassia seed extract as described in claim 1, and pharmaceutically acceptable excipients thereof.

3. The high-affinity GLUT1 protein-targeting inhibitor according to claim 2, characterized in that, The GLUT1 protein targeting inhibitor is available in tablet, powder, pill, capsule, or injection form.

4. Use of the cassia seed extract as described in claim 1 or the GLUT1 protein targeting inhibitor as described in claim 2 or 3 in the preparation of a medicament for alleviating neurotoxicity.

5. The use according to claim 4, characterized in that, The neurotoxicity is caused by astrocyte activation due to manganese exposure.

6. The use according to claim 4, characterized in that, The effective dose of cassia seed extract in the drug is 5-25 mg / kg.

7. A drug for alleviating neurotoxicity, characterized in that, This includes the cassia seed extract as described in claim 1, and pharmaceutically acceptable excipients thereof.

8. The medicament according to claim 7, characterized in that, The dosage form of the drug is tablets, powders, pills, capsules, or injections.

9. Use of the cassia seed extract of claim 1 or the GLUT1 protein targeting inhibitor of claim 2 or 3 in the preparation of a medicament for treating cognitive impairment.

10. The use according to claim 9, characterized in that, The effective dose of cassia seed extract in the drug is 5-25 mg / kg.

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