Organic small molecules for reducing copper overload toxicity and their applications

Abstract

This invention belongs to the field of pharmaceutical technology and discloses small organic molecules for reducing copper overload toxicity and their applications. Specifically, it discloses four small organic molecules for reducing copper overload toxicity and methods for doing so. These four small organic molecules are not copper chelators and do not reduce intracellular copper levels, thus not affecting the activity of copper-related enzymes. Furthermore, these four small organic molecules are not copper ion transporters and do not transport copper into cells, thus not exacerbating the cytotoxicity of copper overload.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and more specifically to small molecules with Cu ion-binding activity and their use in diseases associated with copper overload in humans or other mammals. Background Technology

[0002] Copper is a cofactor for enzymes involved in respiration (COX), activation of neuroendocrine peptide (PAM) pigmentation (tyrosinase), catecholamine synthesis and clearance (DBH), free radical defense (SOD1 and SOD3), and many other cellular processes. Therefore, copper deficiency can lead to the inactivation of copper-related enzyme molecules, resulting in diseases such as Menkes syndrome. Copper is a double-edged sword; copper overload can also cause cell death, leading to copper overload-related tissue and organ lesions, such as Wilson's disease. Recent studies have shown that copper overload toxicity is caused by copper directly binding to the thioylated components of the tricarboxylic acid (TCA) cycle, leading to the aggregation of thioylated proteins and the loss of iron-sulfur proteins (Tsvetkov et al., Science, 2022, 375, 1254-1261).

[0003] Copper chelators, such as penicillamine, tricentine, and tetrathiomolybdate (TTM, drug code ALXN1840), can prevent cell death caused by copper overload. Multiple pieces of evidence suggest that the mechanism by which copper chelators prevent copper overload toxicity involves a reduction in intracellular copper levels. However, this treatment can affect the activity of copper-dependent enzymes, potentially leading to chronic disease in the long term; this side effect is one of the main reasons limiting the long-term use of these drugs. The mechanism by which cells regulate copper homeostasis involves various copper metal chaperones (ATOX1, COX17, and CCS) and intrinsic intracellular small molecules, such as glutathione (GSH) and metallothionein (MT), binding intracellular free copper to achieve copper storage and / or detoxification, and acting as exchangeable buffers for copper. This phenomenon proposes the hypothesis that copper-binding small molecules may be able to act as copper buffers, performing functions similar to GSH and MT, blocking the binding of copper to lipoxygenated proteins without reducing intracellular copper levels and thus not affecting the activity of copper-dependent enzymes.

[0004] Currently, penicillamine and trientazone are first-line drugs used clinically to treat Wilson's disease. Both are copper chelators and can cause iron deficiency anemia. Occasionally, adverse reactions such as heartburn, abdominal pain, acute gastritis, loss of appetite, rash, and myalgia may occur. Tetrathiomolybdate TTM (ALXN1840) is also a copper chelator. In June 2022, it completed a Phase 3 clinical trial with three times the efficacy of standard treatment. However, in April 2023, it was announced that its development as a drug for the rare disease Wilson's disease was abandoned.

[0005] Therefore, providing small organic molecules to reduce copper overload toxicity is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0006] In view of this, the present invention provides small organic molecules for reducing copper overload toxicity and their applications. The present invention screened four small molecules that can avoid cytotoxicity caused by copper overload. These four small molecules are not copper chelators and do not reduce intracellular copper levels, thus not affecting the activity of copper-related enzymes. Simultaneously, these four small molecules are not copper ion transporters and do not transport copper into cells, thus not exacerbating the cytotoxicity of copper overload. These four small molecules hold promise for treating or alleviating copper overload-related diseases, disorders, or symptoms, but are not limited to Wilson's disease.

[0007] The purpose of this invention is to discover four small molecules that provide a method to reduce copper overload toxicity without affecting the activity of copper-related enzymes. This method is expected to be used to replace penicillamine and trientine for the long-term treatment and relief of Wilson's disease.

[0008] One objective of this invention is to provide four small molecules, as shown in formulas (I-IV), with the following organic small molecule chemical structures for reducing copper overload toxicity:

[0009]

[0010] (I) Pyrogallol;

[0011]

[0012] (II) Kaempferol;

[0013]

[0014] (III) Eriodictyol;

[0015]

[0016] (IV) PQQ disodium pyrroloquinoline quinone.

[0017] The second objective of this invention is to utilize small organic molecules for reducing copper overload toxicity in the preparation of drugs for copper overload-related diseases in humans or other mammals.

[0018] Furthermore, the drug is an organic small molecule that produces therapeutic, alleviating, inhibiting, and regulating effects during the occurrence and development of copper overload-related diseases.

[0019] Preferably, the drug is a single formulation, a mixed formulation, or a composition containing an effective amount of the above four small organic molecule organic ligands, or a formulation loaded with a nanomedicine carrier.

[0020] Preferably, the drug is administered via one or more of the following methods: oral administration, intraperitoneal injection, intravenous injection, subcutaneous injection, intramuscular injection, local application, and transdermal absorption.

[0021] More preferably, the application method is as follows: first, prepare a dimethyl sulfoxide storage solution of any of the organic small molecules with a concentration of 1-100 mM, and dilute it with physiological saline, PBS or cell culture medium before use so that the final concentration of the organic small molecule is 1-100 μM, while the final concentration of dimethyl sulfoxide is not higher than 0.1%.

[0022] Furthermore, the four organic small molecules mentioned are not copper chelators and will not reduce intracellular copper levels; nor are they copper ion carriers and will not increase intracellular copper levels.

[0023] Furthermore, copper overload-related diseases are not limited to Wilson's disease, but also include cardiovascular diseases, liver diseases, and neuropathy caused by copper overload.

[0024] As can be seen from the above technical solution, compared with the prior art, the beneficial effects achieved by the present invention are as follows:

[0025] 1. The small molecules in this invention are not copper ion carriers and will not increase intracellular copper levels, nor are they copper chelators and will not decrease intracellular copper levels.

[0026] 2. The small molecules in this invention can shield the cytotoxicity of intracellular free copper without affecting the activity of copper-related enzymes, and are expected to be used for long-term treatment of copper overload-related diseases.

[0027] 3. The small molecules in this invention are expected to be used as alternatives to penicillamine and trientine for long-term treatment of Wilson's disease. Attached Figure Description

[0028] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0029] Figure 1 The alleviating effect of candidate small molecules on ilisimor-induced cytotoxicity;

[0030] Figure 2Ultraviolet absorption titration curves of ilisimo, TTM, and small molecules with copper ions and their fitted curves; (A, B) Ultraviolet absorption spectra of ilisimo with copper ions at different concentration ratios and their fitted curves of absorbance at 357 nm and concentration ratio; (C, D) Ultraviolet absorption spectra of TTM with copper ions at different concentration ratios and their fitted curves of absorbance at 338 nm and concentration ratio; (E) Ultraviolet absorption spectra of pyrogallol with copper ions at different concentration ratios; (F, G) Ultraviolet absorption spectra of kaempferol with copper ions at different concentration ratios and their fitted curves of absorbance at 300 nm and concentration ratio; (H, I) Ultraviolet absorption spectra of kaempferol with copper ions at different concentration ratios and their fitted curves of absorbance at 408 nm and concentration ratio; (J, K) Ultraviolet absorption spectra of disodium PQQ with copper ions at different concentration ratios and their fitted curves of absorbance at 390 nm.

[0031] Figure 3 CCK8 results of small molecule effects on copper overload-related cytotoxicity induced by ilismo; (A) rescue effect of different concentrations of TTM on copper overload toxicity induced by ilismo; (B) rescue effect of different concentrations of pyrogallol on copper overload toxicity induced by ilismo; (C) rescue effect of different concentrations of disodium PQQ on copper overload toxicity induced by ilismo; (D) rescue effect of different concentrations of kaempferol on copper overload toxicity induced by ilismo; (E) rescue effect of different concentrations of kaempferol on copper overload toxicity induced by ilismo.

[0032] Figure 4 Viable and inactive staining images of small molecule-induced copper overload-related cytotoxicity;

[0033] Figure 5 Quantification of live and dead cell staining results for copper overload-related cytotoxicity induced by small molecules on ilismo; (A) fluorescence intensity of live cell dye; (B) fluorescence intensity of dead cell dye PI;

[0034] Figure 6 Bright-field photographs of small molecules co-incubated with Hepa 1-6 cells;

[0035] Figure 7 ICP-MS was used to detect the effect of small molecule therapy on intracellular Cu content.

[0036] Figure 8 The effect of small molecule therapy on intracellular SOD activity in Hepa 1-6 cells;

[0037] Figure 9Effects of small molecule therapy on SOD1 expression in Hepa 1-6 cells; (A) Western Blot image of SOD1 protein; (B) Quantitative results of SOD1 protein level;

[0038] Figure 10 Effects of small molecule therapy on COX4 expression in Hepa 1-6 cells; (A) Western Blot image of COX4 protein in cells; (B) Quantitative results of COX4 protein level;

[0039] Figure 11 The effect of small molecule therapy on intracellular ATP content. Detailed Implementation

[0040] 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.

[0041] The technical solution of Example 1 is summarized as follows:

[0042] The application and mechanism of small molecules in copper overload-related diseases include the following steps:

[0043] 1) Identification of candidate small molecules:

[0044] The inventors selected suitable small molecules from a wide range of candidates to study the interaction between the candidate small molecules and copper ions. The inventors identified a total of 30 candidate small molecules, with the copper chelating agent tetrathiomolybdate TTM as a positive control, as shown in Table 1.

[0045] Table 1. Candidate small molecules for Cu 2+ Coordination ratio and dissociation constant

[0046]

[0047] Note: "―" indicates that the sample does not react with Cu under the test conditions. 2+ Coordination

[0048] Incubation with CuCl2 alone did not affect cell viability, but incubation with both the copper ion transporter elesclomol (1 μM) and CuCl2 significantly reduced cell viability. The effects of pre-incubation with the copper ion chelator TTM (10 μM) and various small molecules (10 μM) before adding elesclomol on alleviating copper overload-induced cytotoxicity were observed. The results showed that the copper ion chelator TTM significantly reduced elesclomol-induced copper overload-related toxicity. Four other small molecules, namely pyrogallol, kaempferol, eriodictyol, and disodium pyrroquinone (PQQ disodium), showed similar effects.

[0049] 2) A total of 4 small molecules were screened out:

[0050] The chemical structural formula of the organic small molecule is:

[0051]

[0052] (I) Pyrogallol;

[0053]

[0054] (II) Kaempferol;

[0055]

[0056] (III) Eriodictyol;

[0057]

[0058] (IV) PQQ disodium pyrroloquinoline quinone.

[0059] See Figure 2 UV absorption titration curves of ilismoxol, TTM, and four other small molecules with copper ions and their fitting:

[0060] The test method is as follows:

[0061] Titration curves of candidate small molecules with copper were obtained using a spectrophotometer. MOPS (50 mM, pH 7.2) was used as the buffer solution. The concentration of small molecule ligand L was fixed at [L], and CaCl2 solution was added to the solution of small molecule ligand L to change the concentration of copper ions [Cu]. 2+ ], obtaining copper ions and ligand L in different ratios [Cu 2+[[C]] solution, the solution was prepared within 1 minute, and scanned in the wavelength range of 200-600 nm, with wavelength as the abscissa, and the different [Cu] values ​​were recorded. 2+ The absorbance of the [Cu] / [L] solution. The absorbance at the characteristic absorption peak is plotted on the ordinate, and the absorbance at the [Cu] / [L] solution is plotted on the ordinate. 2+ The ratio of [L] to [L] is used as the x-axis. Plot the graph and fit the curve. The component concentration ratio corresponding to the inflection point of the curve is the composition ratio of the complex. Calculate its dissociation constant.

[0062] Depend on Figure 2 It can be seen that irismolybdate and tetrathiomolybdate TTM are related to Cu 2+ The molar ratio of the chelates was 1:1. K d They are 5.41×10 –7 M and 7.30×10 –7 M. That is, irismo and TTM for Cu 2+ The affinity is greater than that of the copper transporter Ctr1 on the cell membrane for Cu. + Affinity ( K d =1.0×10 –6 Therefore, oral administration of irismo and TTM can prevent the uptake of Cu by intestinal epithelial cells, thereby reducing the copper content in the body.

[0063] Meanwhile, kaempferol, kaempferol and disodium PQQ react with Cu 2+ The molar ratios of chelation were 1.0, 0.2, and 1.5, and their dissociation constants were all lower than those of irismol and TTM, indicating that the formed copper ion complexes were more stable. However, pyrogallol could not form a complex with copper ions.

[0064] If an isoabsorption point appears in the ultraviolet absorption titration curve, it indicates that a stable complex has formed between copper ions and small molecules. The absorbance value at the maximum absorption wavelength varies with the concentration of the small molecules. A fitted curve is obtained by plotting the absorption intensity against the molar ratio of the two components of the complex in solution. The molar ratio corresponding to the intersection point of the two straight lines on the X-axis is the stoichiometric ratio of the complex components [Cu]. 2+ [L]. If no isoabsorption point appears in the ultraviolet absorption titration curve (such as pyrogallol), it indicates that copper ions cannot form a stable complex with small molecules, or the formed complex is unstable and will dissociate immediately.

[0065] The above figure shows that eliximole can interact with Cu 2+ A stable complex is formed at a molar ratio of 1:1; TTM and Cu 2+ It also forms a stable complex in a 1:1 molar ratio; pyrogallol and Cu 2+The resulting complex is unstable; PQQ disodium reacts with Cu 2+ It forms a stable complex with a molar ratio of 2:3; sennaol and Cu 2+ It forms a stable complex in a 1:1 molar ratio; kaempferol and Cu 2+ The complex is formed in a molar ratio of 5:1;

[0066] The stability of these complexes can be analyzed by examining their dissociation constants. The larger the dissociation constant, the higher the ion concentration in the solution, and the less stable the complex is.

[0067] See Figure 3 The alleviating effect of small molecules on copper overload-related cytotoxicity induced by ilismo, *and Cu 2+ Treatment ratio, # and Cu 2+ Compared with the combined treatment group of irismo:

[0068] The experimental method was as follows: Hepa 1-6 cells in good growth condition were counted, and the cell suspension density was adjusted to 1×10⁻⁶. 5 Cells were seeded at a concentration of 100 μL per well in 96-well plates. The concentrations of small molecule ligands were adjusted to 20, 10, 5, 2.5, 1.25, and 0 μM, and then 1 μM CuCl2 was added. After co-incubation for 24 h, ilisimo was added to a final concentration of 1 μM. Separate groups were set up: 1 μM CuCl2, 10 μM small molecule ligand, and 1 μM CuCl2. After co-incubation for 24 h, the supernatant was discarded, and 100 μL of CCK-8 solution diluted in 10% FBS medium was added to each well. The plates were then incubated for another 40 min. The absorbance at 450 nm was measured using a microplate reader, and cell viability was calculated using the following formula.

[0069] Cell viability % = (OD preparation group - OD blank group) / (OD control group - OD blank group) × 100%

[0070] Depend on Figure 3 It can be seen that CuCl2 alone does not cause cytotoxicity, but when CuCl2 and ilipin are co-incubated with cells, significant copper overload-related cytotoxicity is induced. TTM at concentrations greater than 5 μM significantly alleviates ilipin-induced copper overload cytotoxicity. The minimum doses of the four small molecules that alleviate copper overload cytotoxicity vary slightly: pyrogallol and kaempferol at concentrations greater than 10 μM, disodium PQQ at concentrations greater than 5 μM, and sennaol at concentrations greater than 2.5 μM all significantly alleviate ilipin-induced copper overload cytotoxicity.

[0071] See Figure 4 and Figure 5Live and dead staining of small molecules against copper overload-related cytotoxicity induced by ilismo:

[0072] The experimental method was as follows: Hepa 1-6 cells in good growth condition were counted, and the cell suspension density was adjusted to 1×10⁻⁶. 5 Cells were seeded at a rate of 100 μL / well in 96-well plates, and then incubated for 24 h to allow cell adhesion. The cell supernatant was discarded, and 200 μL of a 10 μM small molecule ligand diluted with whole culture medium and 1 μM CuCl2 were added to each well. After co-incubation for 24 h, irismol was added to a final concentration of 1 μM. All groups were configured with three replicates. Cells were stained using the Calcein-AM / PI live / dead cell double staining kit, and observed and photographed under an inverted fluorescence microscope. Figure 4 ).right Figure 4 The quantitative results of the fluorescence intensity of Calcein-AM and PI dyes are shown in the figure. Figure 5 ).

[0073] and Figure 3 Similar results were observed in the control group. Compared to the CuCl2-only treatment group, incubation of cells with both CuCl2 and ilimosin for 24 h induced significant copper overload-related cytotoxicity, resulting in a significant increase in PI fluorescence intensity. Treatment with the copper ion chelator TTM significantly reduced the copper overload-related toxicity induced by ilimosin, exhibiting lower PI fluorescence intensity. Similar results were observed with the other four small molecules.

[0074] See Figure 6 Bright-field images of small molecules co-incubated with Hepa 1-6 cells at 0h, 12h, and 24h:

[0075] The experimental method was as follows: Hepa 1-6 cells in good growth condition were counted, and the cell suspension density was adjusted to 1×10⁻⁶. 5 Cells were seeded at a density of 100 μL per well in a 96-well plate and then incubated for 24 h to allow cell adhesion. The cell supernatant was discarded, and 200 μL of a 10 μM small molecule ligand diluted with whole culture medium, 1 μM CuCl2, and 1 μM irismol were added to each well. After incubation for 24 h, the cells were observed and photographed under an inverted microscope.

[0076] and Figure 3 and Figure 4 Consistent with the results, when CuCl2 and irismo were incubated with cells for 24 hours, significant copper overload-related cytotoxicity was induced. As can be seen from the bright-field images, some cells had died and were floating in the culture medium.

[0077] When cells were pretreated with CuCl2 for 24 hours using copper ion chelator TTM and four small molecules, and then co-incubated with irismo for another 24 hours, the number of floating cells was significantly less than that in the group treated with CuCl2 and irismo in combination.

[0078] See Figure 7 ICP-MS detection of the effect of small molecule therapy on intracellular Cu content:

[0079] The experimental method was as follows: Hepa1-6 cells were cultured at a rate of 2 × 10⁻⁶. 6 Cells / dish were cultured at a density of 6 cm in culturing dishes and allowed to grow for 24 hours. The cell supernatant was discarded, and 5 mL of 1 μM Cu(II) diluted with complete culture medium and 10 μM small molecule ligand were added. After 24 h, 1 μM ilismo was added and incubated for another 24 h. The cell supernatant was then discarded, and the cells were washed three times with PBS to thoroughly remove Cu adsorbed on the cell membrane. 2+ Cells were then collected by digestion with trypsin and counted. After the cell pellet was freeze-dried, the intracellular copper content was determined by ICP-MS.

[0080] CuCl2 alone does not increase the intracellular copper content, but when ilismo and CuCl2 are present together, the intracellular copper content increases by 6.0 times.

[0081] When cells were pretreated with TTM and CuCl2 for 24 hours before the addition of irismol, the intracellular copper content decreased to 0.67 times, significantly lower than that in the normal group. This indicates that TTM alleviates copper overload-related cytotoxicity by reducing intracellular Cu content. The intracellular Cu content of the four small molecules pretreated were 5.9 times (pyrogallol), 6.9 times (disodium PQQ), 4.6 times (kaempferol), and 6.0 times (kaempferol) of the normal group, respectively. These results demonstrate that these four small molecules, unlike the copper chelator TTM, do not alleviate copper overload cytotoxicity by reducing intracellular Cu content.

[0082] See Figure 8 Effects of small molecule therapy on intracellular SOD activity in Hepa 1-6 cells:

[0083] The experimental method was as follows: Hepa1-6 cells were cultured at a rate of 2 × 10⁻⁶. 5 Cells / dish were cultured at a density of 1 / 2 well in 6-well plates and allowed to grow for 24 hours. The cell supernatant was discarded, and 5 mL of 1 μM Cu diluted with complete culture medium was added. 2+Add 10 μM small molecule ligand. After 24 h, add 1 μM ilisimor and co-incubate for another 24 h. Then discard the cell supernatant, wash three times with PBS, and lyse the cells on ice for 30 min using RIPA lysis buffer. Collect the lysis buffer and centrifuge at 10000 rpm for 10 min at 4 °C. Use the supernatant for subsequent assays. SOD activity in the supernatant was measured strictly according to the kit instructions. Protein quantification was performed using the Beyotime BCA protein concentration assay kit.

[0084] In most eukaryotic cells, two forms of intracellular superoxide dismutase (SOD) exist: SOD2, containing Mn, is found only in the mitochondrial matrix; and SOD1, containing Cu and Zu, is found almost everywhere else, including the mitochondrial intermembrane space. SOD1 is an intracellular metalloenzyme that scavenge superoxide anion free radicals and plays a crucial role in maintaining normal cellular life activities. SOD1 possesses important physiological functions for maintaining normal cellular activity, including antioxidant and anti-aging effects, prevention of DNA damage in the cell nucleus, and regulation of oxygen and glucose signal transduction.

[0085] TTM treatment significantly reduced intracellular SOD enzyme activity, which is attributed to the reduction of intracellular Cu content by TTM. The four screened small molecules did not reduce intracellular Cu content, and therefore had no significant effect on SOD enzyme activity.

[0086] See Figure 9 Effects of small molecule therapy on SOD1 expression in Hepa 1-6 cells:

[0087] The experimental method was as follows: 1) Hepa1-6 cells were inoculated at a rate of 2×10⁻⁶. 5 Cells / dish were cultured at a density of 24 hours in 6-well plates. The cell supernatant was discarded, and 5 mL of 1 μM Cu diluted with complete culture medium was added. 2+Add 10 μM small molecule ligand. After 24 h, add 1 μM irismo and incubate for another 24 h. Then discard the cell supernatant, wash three times with PBS, collect the cells from each group, add pre-prepared PMSF:RIRA (1:100) cell lysis buffer, lyse on ice for 30 min, centrifuge at 12000 rpm for 10 min at 4℃ and collect the supernatant. Use Beyotime BCA protein concentration assay kit to determine the total protein concentration of the samples, and dilute the protein to 2 mg / mL using the prepared cell lysis buffer. 2) Use GenScript SurePAGE 4-12% gradient precast gel, peel off the blue tape at the bottom of the gel plate, slowly pull out the comb, fix the precast gel in the electrophoresis tank, fill the inner tank with electrophoresis buffer, and add liquid to the outer tank to be 1 / 3 higher than the height of the electrophoresis tank. Use a pipette to draw electrophoresis buffer and gently blow it into the sample wells to remove residual storage buffer and impurities. Mix the protein sample and 5X SDS-PAGE protein loading buffer at a ratio of 1 μL of 5x SDS-PAGE protein loading buffer per 4 μL of protein sample. Heat in a metal bath at 95°C for 5 min to fully denature the protein, then cool to room temperature. Load 20 μg of protein into each sample well, and load protein markers into the first and last wells. Turn on the power, set the parameters to 130V, and run for 60 min. After electrophoresis, remove the gel for subsequent transfer experiments. 3) Transfer and blocking: Equilibrate the gel in pre-chilled transfer buffer for 5 min. Soak a 0.45 μm PVDF membrane in pure methanol for 2 min, then incubate in pre-chilled transfer buffer. Assemble the transfer clamps in the correct order and add transfer buffer. Turn on the power, set the parameters to 300 mA, and run for 60 min. After transfer, wash the membrane 2-3 times with TBST, 5 min each time. 4) Antibody incubation: Place the membrane and primary antibody (at the recommended dilution for product application) in 5 mL of primary antibody dilution buffer and incubate overnight at 4ºC on a shaker. Recover the primary antibody and wash the membrane three times with 5 mL TBST for 15 min each time to remove residual primary antibody. Then place the membrane and secondary antibody (at the recommended dilution for product application) in 10 mL of blocking buffer and incubate at room temperature for 2 hours; 5) Development: Remove the membrane with flat-tipped forceps and place it on filter paper to drain the washing solution, ensuring the membrane is not completely dry. Immerse the membrane completely in the luminescent working solution prepared by the Beyotime luminescence kit, ensuring full contact with the luminescent working solution, and incubate at room temperature for 3 min. Then place it in a Tanon chemiluminescence imager for color development in the dark and perform grayscale quantitative analysis using ImageJ software.

[0088] The inventors further used Western blot analysis to determine the expression level of SOD1 in cells. TTM also reduced the expression level of SOD1 in cells, while the four small molecules screened out did not reduce the expression level of SOD1 in cells.

[0089] See Figure 10 Effects of small molecule therapy on COX4 expression in Hepa 1-6 cells:

[0090] The test method is as follows: with Figure 9 Similarly, simply replace the SOD1 antibody with the COX4 antibody.

[0091] The inventors further analyzed the expression level of COX4 in cells using Western blot. The results showed that TTM reduced the expression level of COX4 in cells, while the four small molecules screened out did not reduce the expression level of COX4 in cells.

[0092] See Figure 11 The effect of small molecule therapy on intracellular ATP levels:

[0093] The experimental method was as follows: Hepa1-6 cells were cultured at a rate of 2 × 10⁻⁶. 5 Cells / dish were cultured at a density of 6 cm in culturing dishes and allowed to grow for 24 hours. The cell supernatant was discarded, and 5 mL of 1 μM Cu diluted with complete culture medium was added. 2+ Add 10 μM small molecule ligand. After 24 h, add 1 μM ilismo and co-incubate for another 24 h. Then discard the cell supernatant, wash three times with PBS, and measure the cellular ATP level according to the kit instructions.

[0094] The activity of cytochrome c oxidase subunit 4 (COX4) directly affects intracellular ATP production. Figure 10 The results from Western blotting were consistent with those from Western blotting. The intracellular ATP content in the TTM-treated group was significantly lower than that in the Normal group. This is because TTM treatment reduced intracellular COX4 levels, thus affecting ATP production. The four screened small molecules did not reduce intracellular COX4 levels and therefore did not affect ATP production. Conversely, the four screened small molecules significantly increased intracellular ATP content.

[0095] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. The application of an organic small molecule in the preparation of a drug that produces therapeutic, alleviating, inhibiting, and regulating effects in the occurrence and development of Wilson's disease, characterized in that, The organic small molecule is pyrogallol, sennaol, or disodium pyrroloquinoline quinone.

2. The application as described in claim 1, characterized in that, The drug is a single formulation, a mixed formulation, or a composition containing an effective amount of the formulation ingredient, or a formulation loaded with a nanomedicine carrier.

3. The application as described in claim 1, characterized in that, The drug can be administered orally, via intraperitoneal injection, intravenous injection, subcutaneous injection, intramuscular injection, or transdermal absorption, or by one or more of these methods.

4. The application as described in claim 3, characterized in that, The specific application method is as follows: First, prepare a dimethyl sulfoxide storage solution of any of the organic small molecules described in claim 1, with a concentration of 1-100 mM. Before use, dilute it with physiological saline, PBS or cell culture medium so that the final concentration of the organic small molecule is 1-100 μM, and the final concentration of dimethyl sulfoxide is not higher than 0.1%.

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