Organic small molecule for reducing copper overload toxicity and application thereof

Abstract

The invention belongs to the technical field of medicines, and discloses an organic small molecule for reducing copper overload toxicity and application thereof. The invention specifically discloses four small organic molecules for reducing the overload toxicity of copper and a method for reducing the overload toxicity of copper. The four small organic molecules are not copper chelating agents and cannot reduce the copper level in cells, so that the copper-related enzyme activity cannot be influenced. Meanwhile, the four small organic molecules are not copper ion transporters and cannot transport copper into cells, so that the cytotoxicity of copper overload cannot be aggravated.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of medicine, more particularly, to small molecules with Cu ion binding activity and their use in human or other mammals with copper overload related diseases. BACKGROUND

[0002] Copper is a cofactor for enzymes involved in respiration (COX), activation of neuroendocrine peptides (PAM), pigmentation (tyrosinase), catecholamine synthesis and clearance (DBH), free radical defense (SOD1 and SOD3), and many other cellular processes. Therefore, copper deficiency can cause copper-dependent enzymes to lose activity, leading to diseases such as Menkes syndrome; copper is a double-edged sword, and 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 sulfuryl component of the tricarboxylic acid (TCA) cycle, leading to sulfuryl protein aggregation and loss of iron-sulfur proteins (Tsvetkov et al., Science, 2022, 375, 1254-1261).

[0003] Copper chelators, such as penicillamine, trientine, and tetrathiomolybdate (TTM, drug code ALXN1840), can prevent cell death caused by copper overload. Various evidence suggests that the mechanism by which copper chelators prevent copper overload toxicity involves a decrease in intracellular copper levels. However, this treatment can affect the activity of copper-dependent enzymes, which can lead to chronic diseases in the long run, and this side effect is one of the main reasons for limiting the long-term use of drugs. The mechanism by which cells regulate copper homeostasis is to bind free copper in cells through various copper metal chaperones (ATOX1, COX17, and CCS) and intrinsic small molecules in cells, such as glutathione (GSH) and metallothionein (MT), to achieve copper storage and / or detoxification, and can act as an exchangeable buffer pool for copper. This phenomenon raises the hypothesis that copper-binding small molecules can act as a buffer pool for copper, play a similar role to GSH and MT, block copper binding to lipoacylated proteins, and not decrease intracellular copper levels, thereby not affecting the activity of copper-dependent enzymes.

[0004] Currently, penicillamine and trientine are the first-line drugs for the treatment of Wilson's disease in the clinic, both of which are copper chelators and can cause iron deficiency anemia, and occasionally cause adverse reactions such as heartburn, abdominal pain, acute gastritis, loss of appetite, skin rash, myalgia, etc. Tetrathiomolybdate TTM (ALXN1840) is also a copper chelator, and in June 2022, it completed a phase 3 clinical trial with 3 times the standard treatment effect, but in April 2023, it was announced that the development of the drug as a Wilson's rare disease drug was abandoned.

[0005] Therefore, it is an urgent problem for those skilled in the art to provide an organic small molecule for reducing copper overload toxicity. SUMMARY

[0006] Therefore, the present application provides an organic small molecule for reducing copper overload toxicity and its application. Four small molecules are screened out, which can avoid the cytotoxicity caused by copper overload. The four small molecules are not copper chelators, which will not reduce the copper level in cells, and thus will not affect the activity of copper-related enzymes. At the same time, the four small molecules are not copper ion transporters, which will not transport copper into cells, and thus will not exacerbate the cytotoxicity of copper overload. The four small molecules are expected to be used for treating or relieving copper overload related diseases, disorders or symptoms, but not limited to Wilson's disease.

[0007] The purpose of the present application is to find four small molecules, to provide a method for reducing copper overload toxicity, but not affecting the activity of copper-related enzymes, which is expected to be used for replacing penicillamine and trientine for long-term treatment and relief of Wilson's disease.

[0008] One of the purposes of the present application is to provide four small molecules as shown in formula (I-IV), which are organic small molecule chemical structural formula for reducing copper overload toxicity:

[0009] (I) Pyrogallol;

[0010] (II) Kaempferol;

[0011] (III) Eriodictyol;

[0012] (IV) PQQ disodium.

[0013] The second purpose of the present application is the use of the organic small molecule for reducing copper overload toxicity in the preparation of drugs for copper overload related diseases in humans or other mammals.

[0014] Further, the drug is an organic small molecule which produces treatment, relief, inhibition, regulation in the occurrence and development of copper overload related diseases.

[0015] Preferably, the drug is a single preparation, a mixed preparation or a composition containing an effective amount of preparation components of the above four organic small molecule organic ligands, or a preparation loaded with a nano-drug carrier.

[0016] Preferably, the application mode of the medicine is one or more of oral administration, intraperitoneal injection, intravenous injection, subcutaneous injection, intramuscular injection, topical application, and transdermal absorption.

[0017] Further preferably, the application mode is specifically as follows: a dimethyl sulfoxide storage solution of any of the organic small molecules is prepared first, with a concentration of 1-100 mM, and before use, the solution is diluted with normal saline, PBS or cell culture medium to make the final concentration of the organic small molecule 1-100 μM, while the final concentration of dimethyl sulfoxide is not higher than 0.1%.

[0018] Further, the four organic small molecules are neither copper chelators nor copper ion carriers, and thus will not reduce or increase the intracellular copper level.

[0019] Further, the copper overload-related diseases are not limited to Wilson's disease, but also include cardiovascular diseases, liver diseases, neuropathies and the like caused by copper overload.

[0020] According to the technical solutions described above, compared with the prior art, the present application has the following beneficial effects: 1. The small molecules in the present application are neither copper ion carriers nor copper chelators, and thus will not increase or decrease the intracellular copper level.

[0021] 2. The small molecules in the present application can shield the cytotoxicity of free copper in cells, and at the same time will not affect the activity of copper-related enzymes, and thus are expected to be used for long-term treatment of copper overload-related diseases.

[0022] 3. The small molecules in the present application are expected to be used for replacing penicillamine and trientine, and thus are expected to be used for long-term treatment of Wilson's disease. BRIEF DESCRIPTION OF DRAWINGS

[0023] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or the prior art description will be briefly introduced as follows. Obviously, the drawings in the following description are only embodiments of the present application, and for those skilled in the art, other drawings can also be obtained without creative labor on the basis of the provided drawings.

[0024] Figure 1 . The alleviating effect of the candidate small molecules on the cytotoxicity caused by elli sorb; Figure 2. UV absorption spectrum titration curves of Ilicicolin, TTM and small molecules with copper ions and their fitting curves; (A, B) UV absorption spectrum of Ilicicolin with different concentration ratios of copper ions and their fitting curves of absorbance value at 357 nm and concentration ratio; (C, D) UV absorption spectrum of TTM with different concentration ratios of copper ions and their fitting curves of absorbance value at 338 nm and concentration ratio; (E) UV absorption spectrum of pyrogallol with different concentration ratios of copper ions; (F, G) UV absorption spectrum of rutoside with different concentration ratios of copper ions and their fitting curves of absorbance value at 300 nm and concentration ratio; (H, I) UV absorption spectrum of kaempferol with different concentration ratios of copper ions and their fitting curves of absorbance value at 408 nm and concentration ratio; (J, K) UV absorption spectrum of PQQ disodium salt with different concentration ratios of copper ions and their fitting curves of absorbance value at 390 nm and concentration ratio; Figure 3 . CCK8 results of small molecules on copper overload-related cytotoxicity caused by Ilicicolin; (A) Rescue effect of different concentrations of TTM on copper overload toxicity caused by Ilicicolin; (B) Rescue effect of different concentrations of pyrogallol on copper overload toxicity caused by Ilicicolin; (C) Rescue effect of different concentrations of PQQ disodium salt on copper overload toxicity caused by Ilicicolin; (D) Rescue effect of different concentrations of rutoside on copper overload toxicity caused by Ilicicolin; (E) Rescue effect of different concentrations of kaempferol on copper overload toxicity caused by Ilicicolin; Figure 4 . Photos of dead and live staining of small molecules on copper overload-related cytotoxicity caused by Ilicicolin; Figure 5 . Quantitative results of dead and live staining of small molecules on copper overload-related cytotoxicity caused by Ilicicolin; (A) Fluorescence intensity of live cell dye; (B) Fluorescence intensity of dead cell dye PI; Figure 6 . Bright field photos of small molecules incubated with Hepa 1-6 cells; Figure 7 . ICP-MS detection of the effect of small molecule treatment on intracellular Cu content; Figure 8 . Effect of small molecule treatment on SOD activity in Hepa 1-6 cells; Figure 9 . Effect of small molecule treatment on SOD1 expression in Hepa 1-6 cells; (A) SOD1 protein Western Blot protein electrophoresis picture; (B) Quantitative results of SOD1 protein level; Figure 10The effect of small molecule treatment on the expression of COX4 in Hepa 1-6 cells; (A) Western Blot protein electrophoresis picture of COX4 protein in cells; (B) quantification result of COX4 protein level; Figure 11 The effect of small molecule treatment on the ATP content in cells. DETAILED DESCRIPTION

[0025] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative labor fall within the scope of protection of the present application.

[0026] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative labor fall within the scope of protection of the present application. The application of small molecules in copper overload related diseases and its mechanism includes the following steps: 1) Determination of candidate small molecules: The inventor selected suitable small molecules for research from a wide range of candidates, studied the interaction between the candidate small molecules and copper ions, and determined 30 candidate small molecules, with copper chelator tetrathiomolybdate TTM as a positive control, as shown in Table 1. Table 1. Coordination ratio and dissociation constant of candidate small molecules to Cu 2+

[0027] Note: “―” indicates that the sample does not coordinate with Cu 2+ Among them, CuCl2 incubation alone does not affect cell activity, but when copper ion transporter elesclomol (1 μM) and CuCl2 are added to the cell incubation, it significantly reduces cell activity. When copper ion chelator TTM (10 μM) and various small molecules (10 μM) are pre-incubated with cells, elesclomol is then added, and the effect of the small molecules on the alleviation of copper ion overload induced cell toxicity is observed. The results show that the copper ion chelator TTM can significantly reduce the copper overload related toxicity caused by elesclomol, and the other four small molecules also have similar effects, namely, pyrogallol, kaempferol, eriodictyol and PQQ disodium.

[0028] 2) A total of 4 small molecules are screened: The chemical structure of the organic small molecule is: ​​

[0029] (I) Pyrogallol;

[0030] (II) Kaempferol;

[0031] (III) Eriodictyol;

[0032] (IV) PQQ disodium.

[0033] See Figure 2 , UV absorption titration curves of Elsamir, TTM and four small molecules with copper ions and their fitting: The test method is as follows: The spectrophotometer is used to obtain the titration curve of the candidate small molecule and copper, MOPS (50 mM, pH = 7.2) is used as the buffer, the concentration of the small molecule ligand L is fixed as [L], and CaCl2 solution is added to the small molecule ligand L solution, the concentration of copper ions [Cu 2+ ] is changed, the solution of different ratios of copper ions to ligand L [Cu 2+ ] / [L] is obtained, the solution is prepared within 1 min, scanning is performed in the wavelength range of 200-600 nm, and the absorbance of different [Cu 2+ ] / [L] solutions is recorded with the wavelength as the abscissa. The absorbance value under the characteristic absorption peak is taken as the ordinate, the ratio of [Cu 2+ ] / [L] is taken as the abscissa, a graph is drawn and a curve is fitted, the composition ratio corresponding to the turning point of the curve is the composition ratio of the complex, and the dissociation constant is calculated.

[0034] As can be seen from Figure 2 , the molar ratio of Elsamir and TTM chelated with Cu 2+ is 1:1, K d 5.41 x 10 –7 M and 7.30 x 10 –7 M, respectively. That is, the affinity of Elsamir and TTM for Cu 2+ is greater than the affinity of the copper transporter Ctr1 on the cell membrane for Cu + ( K d = 1.0 x 10 –6 M), so that after oral administration of Elsamir and TTM, the uptake of Cu by the intestinal epithelial cells can be avoided, thereby reducing the copper content in the body.

[0035] Meanwhile, the Cu 2+ The molar ratios of chelation are 1.0, 0.2 and 1.5 respectively, and their dissociation constants are all less than that of elli sorb and TTM, which indicates that the formed copper ion complexes are more stable. However, pyrogallol cannot form complex with copper ion.

[0036] If there is an isosbestic point in the UV absorption spectrum titration curve, it indicates that stable complex is formed between copper ion and small molecule. The absorbance value at the maximum absorption wavelength changes with the concentration of small molecule. The molar ratio of the two components in the complex is plotted with the absorbance intensity, and the stoichiometric ratio of the complex components is obtained at the intersection point of the two straight lines on the X axis, which is [Cu 2+ ] / [L]. If there is no isosbestic point in the UV absorption spectrum titration curve (such as pyrogallol), it indicates that stable complex cannot be formed between copper ion and small molecule, or the formed complex is unstable and will immediately dissociate.

[0037] The above figure shows that elli sorb can form stable complex with Cu 2+ with a molar ratio of 1:1; TTM also forms stable complex with Cu 2+ with a molar ratio of 1:1; pyrogallol forms unstable complex with Cu 2+ ; PQQ disodium forms stable complex with Cu 2+ with a molar ratio of 2:3; eriodictyol forms stable complex with Cu 2+ with a molar ratio of 1:1; kaempferol forms complex with Cu 2+ with a molar ratio of 5:1; The stability of these complexes can be analyzed from the dissociation constant. The larger the dissociation constant, the higher the ion concentration in the solution, and the more unstable the complex.

[0038] See Figure 3 , the alleviating effect of small molecules on the copper overload-related cytotoxicity caused by elli sorb, * compared with the Cu 2+ treatment group, # compared with the Cu 2+ and elli sorb combined treatment group: The test method is as follows: count the well-grown Hepa 1-6 cells, adjust the cell suspension density to 1×10 5Hepa 1-6 cells were seeded at a density of 1 x 104 cell / mL in 96-well plates with 100 μL cell suspension per well. The concentration of the small molecule ligand was adjusted to 20, 10, 5, 2.5, 1.25, 0 μM, and 1 μM CuCl2 was added. After 24 h of co-incubation, 1 μM ursolic acid was added. A group with 1 μM CuCl2 alone, a group with 10 μM small molecule ligand and 1 μM CuCl2 were set. After 24 h of co-incubation, the supernatant was discarded, and 100 μL of CCK-8 solution diluted with 10% FBS complete medium was added to each well, and the plates were incubated for another 40 min. The plates were removed, and the absorbance at 450 nm was measured using a microplate reader. The cell viability was calculated according to the following formula.

[0039] Cell viability% = (OD preparation group - OD blank group) / (OD control group - OD blank group) x 100% From Figure 3 It can be seen that CuCl2 alone does not cause cytotoxicity, but when CuCl2 and ursolic acid are co-incubated with cells, it can cause significant copper overload-related cytotoxicity. When TTM is at a concentration greater than 5 μM, it can significantly alleviate the copper overload cytotoxicity caused by ursolic acid. The minimum dose of the four small molecules to alleviate copper overload cytotoxicity is slightly different. Pyrogallol and kaempferol at a concentration greater than 10 μM, PQQ disodium at a concentration greater than 5 μM, and rutin at a concentration greater than 2.5 μM can significantly alleviate the copper overload cytotoxicity caused by ursolic acid.

[0040] See Figure 4 and Figure 5 , live and dead staining of small molecules on ursolic acid-induced copper overload-related cytotoxicity: The test method was as follows: Hepa 1-6 cells in good growth condition were counted, and the cell suspension density was adjusted to 1 x 104 5 cell / mL, and 100 μL of cell suspension was seeded in a 96-well plate, which was then placed in an incubator for 24 h to allow the cells to adhere. The cell supernatant was discarded, and 200 μL of small molecule ligand at a concentration of 10 μM and 1 μM CuCl2 diluted with complete medium were added, respectively. After 24 h of co-incubation, 1 μM ursolic acid was added. Three replicate wells were set for all groups. Calcein-AM / PI live and dead cell double staining kit was used for staining, and the photographs were taken under an inverted fluorescence microscope. Figure 4 ). The quantification results of the fluorescence intensity of Calcein-AM and PI dyes in Figure 4 are shown in the graph Figure 5 .

[0041] and Figure 3Similar 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.

[0042] See Figure 6 Bright-field images of small molecules co-incubated with Hepa 1-6 cells at 0h, 12h, and 24h: 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.

[0043] 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.

[0044] 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.

[0045] See Figure 7 ICP-MS detection of the effect of small molecule therapy on intracellular Cu content: 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.

[0046] 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.

[0047] 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.

[0048] See Figure 8 Effects of small molecule therapy on intracellular SOD activity in Hepa 1-6 cells: 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.

[0049] 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.

[0050] 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.

[0051] See Figure 9Effects of small molecule therapy on SOD1 expression in Hepa 1-6 cells: 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.

[0052] 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.

[0053] See Figure 10 Effects of small molecule therapy on COX4 expression in Hepa 1-6 cells: The test method is as follows: with Figure 9 Similarly, simply replace the SOD1 antibody with the COX4 antibody.

[0054] 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.

[0055] See Figure 11 The effect of small molecule therapy on intracellular ATP levels: 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.

[0056] 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.

[0057] 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 drugs for copper overload-related diseases, characterized in that, The organic small molecule is pyrogallol, kaempferol, sennaol, or disodium pyrroloquinoline quinone.

2. The application as described in claim 1, characterized in that, The drug is an organic small molecule that exerts therapeutic, alleviating, inhibiting, and regulating effects in the occurrence and development of copper overload-related diseases.

3. The application as described in claim 2, 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.

4. The application as described in claim 2, characterized in that, The drug can be administered orally, via intraperitoneal injection, intravenous injection, subcutaneous injection, intramuscular injection, local application, or transdermal absorption, or by one or more of these methods.

5. The application as described in claim 4, 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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