Application of chlorothiophenol as GST (glutathione S-transferase) inhibitor in tumor treatment

Abstract

The invention discloses application of chlorothiophenol as a GST inhibitor in tumor treatment, and belongs to the technical field of biological medicine. The invention finds a new function of thiophenol serving as the GST inhibitor, and the thiophenol can take GST protein as a target spot, increase ROS level, reduce ATP level, stop cell growth and induce mixed cell death, so that the effect of inhibiting tumors is achieved. In addition, the sensitivity of tumor cells to chemotherapeutic drugs can be remarkably improved, and the treatment effect of the chemotherapeutic drugs is enhanced.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to the use of thiochlorophenol as a GST inhibitor in treating tumors. Background Art

[0002] Reactive oxygen species (ROS) are continuously produced by living cells as byproducts of aerobic respiration and play a variety of roles in cellular processes, ranging from regulating cell signaling to antimicrobial immunity. However, excessive ROS can damage cellular components, including DNA and mitochondria, activate the JNK signaling pathway, and, at high concentrations, induce apoptosis. To combat elevated ROS levels, cells employ a variety of defense mechanisms, particularly the glutathione S-transferase (GST) family of proteins (divided into seven subfamilies), which neutralize ROS by catalyzing the conjugation of ROS to glutathione (GSH). In addition to their detoxification effects, overexpression of GSTs in cancer cells often promotes cell proliferation and mediates drug resistance. Consequently, GST inhibitors have been actively explored in preclinical studies and clinical trials for oncology.

[0003] Existing research reports indicate that the antiparasitic drug thiochlorophenol (BT) sensitizes a panel of cancer cells to anticancer drugs, including venetoclax, cisplatin, and paclitaxel, by generating reactive oxygen species (ROS) and inducing apoptosis. Although BT has been reported to bind to human soluble adenylate cyclase (sAC), this interaction does not adequately explain these observations. However, whether BT inhibits human GST enzymes remains unclear. Summary of the Invention

[0004] In view of the above-mentioned deficiencies in the prior art, the present invention provides a use of thiochlorophenol as a GST inhibitor in treating tumors.

[0005] To achieve the above-mentioned purpose, the technical solution adopted by the present invention to solve the technical problem is: The present invention aims to provide use of thiochlorophenol (BT) as a GST inhibitor.

[0006] Another object of the present invention is to provide a GST inhibitor comprising thiochlorophenol as an active ingredient and a pharmaceutically acceptable adjuvant.

[0007] Another object of the present invention is to provide the use of the above-mentioned thiochlorophenol or GST inhibitor in the preparation of a preparation for improving the efficacy of chemotherapy drugs.

[0008] Furthermore, the chemotherapy drug is cisplatin or doxorubicin.

[0009] Another object of the present invention is to provide a combination drug comprising the above-mentioned thiochlorophenol or GST inhibitor and a chemotherapy drug.

[0010] Furthermore, the chemotherapy drug is cisplatin or doxorubicin.

[0011] Another object of the present invention is to provide the use of the above-mentioned thiochlorophenol, GST inhibitor or combination drug in the preparation of drugs for treating tumors.

[0012] Furthermore, the tumor is gastric cancer or lung adenocarcinoma.

[0013] Furthermore, thiochlorophenol, GST inhibitors, or combined drugs target GST, increase ROS levels, decrease ATP levels, induce cell death, and growth arrest.

[0014] Beneficial effects of the present invention: The present invention discovered a novel functional role for thiochlorophenol as a GST inhibitor. It can target GST proteins, increase ROS levels, reduce ATP levels, arrest cell growth, and induce mixed cell death, thereby inhibiting tumors. Furthermore, it can significantly increase the sensitivity of tumor cells to chemotherapy drugs, enhancing the therapeutic efficacy of chemotherapy drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 is the inhibition of BT binding to different GST isoforms; Figure 2 For BT, the Vmax of GSTM1 and A1 to GSH was reduced; Figure 3 It is the specific binding site of BT and GSTM1; Figure 4 BT induces ROS production and causes mixed cell death; Figure 5 BT reduces ATP / GSH levels and induces cell growth arrest; Figure 6 It is the synergistic effect of BT and chemotherapy drugs. DETAILED DESCRIPTION

[0016] The specific embodiments of the present invention are described below to facilitate understanding of the present invention by those skilled in the art. However, it should be clear that the present invention is not limited to the scope of the specific embodiments. For those skilled in the art, as long as various changes are within the spirit and scope of the present invention as defined and determined by the appended claims, these changes are obvious, and all inventions and creations utilizing the concepts of the present invention are protected.

[0017] Example 1 Inhibition of GST by BT 1. Detect the inhibition of BT on GST using the following method (1) Traction test GST protein and MBP were immobilized on GST beads and MBP beads, respectively, and then mixed. 500 μL of the mixed bead suspension was incubated with various concentrations of BT at 4°C for 1.5 hours. The beads were then washed with pulling buffer (20 mM Tris-HCl, pH 8.0, 200 mM NaCl, 10% glycerol, 2 mM MgCl2, 0.005% Triton X-100, 1 mM DTT). Bound proteins were eluted, separated by SDS-PAGE, and visualized with Coomassie Brilliant Blue staining.

[0018] (2) DSF method Various concentrations of BT were incubated with GST (1.5 μM) and a fluorescent dye (SSYPRO Orange, S5692-50UL) in DSF buffer (30 mM Tris-HCl, pH 8.0, 150 mM NaCl). Samples were analyzed using a real-time PCR system with a pre-set temperature gradient (25–95°C, 0.5°C / min). Fluorescence intensity was monitored in real time to detect thermal denaturation of the protein.

[0019] According to the pulling experiment, BT's inhibition of GST is specific, because BT does not inhibit the binding of MBP to MBP beads ( Figure 1 A). This inhibitory effect is much stronger than that of GSH, the natural substrate of GST protein ( Figure 1 A). Differential scanning fluorimetry (DSF) revealed that low micromolar BT increased the Tm of the GST tag in a concentration-dependent manner, but 150 μM GSH did not ( Figure 1 B). These results suggest that BT may be a new GST inhibitor.

[0020] Sequence BLAST searches revealed that the GST tag used shared greater than 40% identity with the human GST Mu isoform and less than 30% identity with other GST family proteins. To investigate BT binding to human GST homologs, we incubated GST beads with HeLa cell lysate in the presence or absence of BT and identified bound proteins using mass spectrometry (MS). In the absence of BT, GSTM3 and GSTP1 showed the highest binding (Table 1). Incubation with BT slightly reduced GSTP1 levels and completely abolished GSTM3 binding to GST beads (Table 1).

[0021] Table 1 GST protein peptides identified by MS in the absence or presence of BT

[0022] In addition to M3 and P1, two well-characterized GST proteins, M1 and A1, were purified from E. coli to test their interaction with BT. Pull-on experiments showed that BT effectively inhibited the binding of GST isoforms A1, M1, and M3, and moderately inhibited the binding of GSTP1 ( Figure 1 C). DSF showed that 10 μM BT could reduce the Tm of A1, M1, M3 and P1 by 3.2℃, 6.4℃, 2.1℃ and 1.5℃, respectively ( Figure 1 D), confirming their direct interaction. These results suggest that BT binds to different human GST members with varying strengths and may bind to more GST isoforms in cells besides M3 and P1.

[0023] 2. Enzyme inhibition test The reaction mixture (total volume of 100 μL) containing different concentrations of BT and reduced glutathione (GSH), 1.0 mM 1-chloro-2,4-dinitrobenzene (CDNB), and 0.1 μM purified GST enzyme was loaded into a transparent 96-well plate in triplicate. The reaction was initiated by the addition of CDNB and immediately monitored at 340 nm for 5 minutes at 25°C with 10-second intervals using a microplate reader. The detection buffer consisted of 30 mMTris pH 8.0 and 150 mM NaCl. The initial reaction velocity (V0) was calculated from the linear phase of the absorbance change, and the IC was determined by nonlinear regression analysis. 50 Value, see the result Figure 2 .

[0024] Figure A) Concentration-dependent inhibition of GSTA1 (0.1 μM) enzymatic activity by BT. The concentrations of GSH and CDNB were 2.5 mM and 0.5 mM, respectively. Data are the mean ± SD of three biological replicates. B) Concentration-dependent inhibition of GSTM1 (0.1 μM) enzymatic activity by BT. The concentrations of GSH and CDNB were 2.5 mM and 0.5 mM, respectively. C) Michaelis-Menten analysis of the reaction catalyzed by GSTA1 (0.1 μM) in the presence or absence of 0.3 μM BT. The CDNB concentration was 1.0 mM. D) Michaelis-Menten analysis of the reaction catalyzed by GSTM1 (0.1 μM) in the presence or absence of 5 μM BT. The CDNB concentration was 1.0 mM.

[0025] like Figure 2 As shown, BT can inhibit the enzyme reactions catalyzed by A1 and M1, and the measured IC 50 The values ​​were 0.24 μM and 5.7 μM ( Figure 2A, B) Pulling experiments suggest that BT may compete with GSH, but enzyme kinetics show a different effect: 0.3 μM BT slightly reduces the Km of GSH for GSTA1 ( Figure 2 C, 1.90 mM to 1.07 mM), while 5 μM BT induced a marginal increase in the Km of GSTM1 ( Figure 2 (D, 0.74 mM to 1.05 mM). Notably, BT reduced the Vmax of both proteins. Overall, BT inhibited GSTA1 and GSTM1 primarily by reducing Vmax.

[0026] 3. BT and GSTM1 binding sites To investigate the binding mode of BT, we obtained the co-crystal structure of BT and GSTM1 in the absence or presence of GSH to elucidate the binding site of BT and GSTM1. Figure 3 .

[0027] like Figure 3 As shown in A and B, the complex bound to BT was crystallized in the P21 space group with a resolution of 2.49 Å; the GSH / BT ternary complex was crystallized in the P2 (1) 2(1)2(1) The BT residues in both crystals occupy the same xenobiotic binding H site and bind to the same amino acid residue ( Figure 3 C). The observation that BT does not occupy the GSH binding site mechanistically explains its primary effect on Vmax rather than Km and plausibly explains the pulling results showing that BT disrupts GST-bead binding.

[0028] like Figure 3 As shown in Figure B, one BT loop inserts into a hydrophobic pocket formed by residues F209, M109, H108, G12, Y7, and I10. In both crystals, the other BT loop is partially solvent-exposed and interacts with M113, Y116, and M212 through van der Waals interactions. Due to the relatively high mobility of this loop, the position of the hydroxyl group within this loop cannot be determined. In the GSH / BT complex, BT forms a hydrogen bond (2.8 Å) with the thiol group of GSH. This observation suggests that variations in the position of the bound BT in different GST isoforms can directly modulate GSH affinity, thereby affecting its Km value for cleavage.

[0029] The residues interacting with BT are poorly conserved among different GST proteins ( Figure 3D). To validate the structural findings and explain isoform selectivity, we mutated each of these GSTM1 residues to the equivalent residues in P1. Mutation F209N rendered GSTM1 enzymatically inactive and was not further analyzed. Two mutants (I10V and M113S) were predicted to worsen hydrophobic packing around BT and exhibited reduced inhibition at the same BT concentration ( Figure 3 E). In contrast, the H108R mutation enhanced BT's potency, likely due to the relief of a steric clash with the ligand's chlorine atom (3.0 Å in both structures). Mutations M109C and M212G had no significant effect on BT's potency, consistent with their peripheral interactions within the binding pocket. Together, these mutagenesis data validate the structural model and explain BT's selectivity for M1 over P1.

[0030] Example 2 BT induces cell death and growth arrest Cells were rinsed twice with prewarmed PBS and then incubated with 10 µM 2',7'-dichlorofluorescin diacetate (Solarbio, D6470) in serum-free DMEM for 30 minutes at 37°C in a 5% CO2 incubator. After three washes with PBS, cells were trypsinized and resuspended in serum-free DMEM. Fluorescence was immediately measured using a microplate reader (BioTek, Synergy H1) at an excitation wavelength of 488 nm and an emission wavelength of 525 nm, and background was subtracted. Each treatment was performed in triplicate, and data are presented as mean ± SD (n = 3) and normalized to the corresponding cell number (by MTT assay) and untreated control cells. Cellular ATP levels were measured using a firefly luciferase-based ATP assay kit (YEASON, 40210ES10) according to the manufacturer's instructions. LDH release was measured using the LDH assay kit WST-8 (Beyotime, C0018) according to the manufacturer's instructions.

[0031] Figure 4Middle, A) Changes in ROS levels in A549, MGC-803, and MDA-MB-453 cell lines after 48-hour treatment with different concentrations of BT (0, 10, 20, and 40 μM). B) Relative ROS levels in BT-treated A549 and MGC-803 cells overexpressing empty vector (EV) or GSTM1 for 48 hours. C) Flow cytometric analysis of A549, MGC-803, and MDA-MB-453 cells treated with or without 20 μM BT for 48 hours, stained with Annexin V-FITC and propidium iodide (PI). D) Changes in the percentages of apoptotic and necrotic cells before and after BT treatment (lower right and upper right quadrants). E) Changes in the percentage of early apoptotic cells (lower right quadrant) before and after BT treatment. F) Immunoblot analysis of p-JNK, PARP, c-PARP (cleaved PARP), and GAPDH protein expression after 48-hour treatment with different concentrations of BT (0 and 20 μM). Compared with the control group, *p<0.05, **p<0.01, ***p<0.001. G) Relative LDH release after 48 h of treatment with different concentrations of BT. H) LDH release after 48 h of overexpression of empty vector or GSTM1 in A549 and MGC-803 cell lines.

[0032] Figure 5 A) Loss of mitochondrial membrane potential in the presence of 20 μM BT for 48 hours, as detected by TMRE (tetramethylrhodamine methyl ester). B) Relative ATP levels in cells treated with various concentrations of BT for 48 hours. *p<0.05, **p<0.01, ***p<0.001 compared to the control group. C) ATP levels in A549 and MGC-803 cells overexpressing empty vector or GSTM1 after 48-hour BT treatment. D) Immunoblot analysis of p-AMPK (phosphorylated AMP-activated protein kinase), p-ERK1 / 2 (phosphorylated extracellular signal-regulated kinase), and T-ERK (total ERK1 / 2) in A549, MGC-803, and MDA-MB-453 cells treated with 0 or 20 μM BT for 48 hours. E) Relative GSH levels after 48-hour treatment with various concentrations of BT. F) Schematic diagram of the anti-cancer mechanism of BT. BT inhibits GST by binding to the xenobiotic-binding H site, disrupting its ROS detoxification function and leading to oxidative stress. The resulting ROS overload induces mitochondrial damage, leading to ATP / GSH depletion. Changes in ROS and ATP trigger phosphorylation of JNK, AMPK, and ERK1 / 2, and release of LDH, ultimately leading to cell growth arrest and death.

[0033] like Figure 4As shown in Figure A, BT may target GST family proteins in cells and increase ROS levels. In fact, BT significantly increased ROS levels in A549 and MGC-803 cells, but had a smaller effect on MDA-MB-453 (breast cancer) cells. Overexpression of wild-type GSTM1 (WT) significantly reduced the ROS increase induced by BT, while the GSTM1 M113S mutant was impaired in its ability to bind to BT, significantly reducing its protective ability ( Figure 4 B). These results mechanistically link the ROS-inducing effect of BT to its direct targeting of GST proteins.

[0034] Consistent with the elevated ROS levels, flow cytometry analysis showed that BT significantly increased the proportion of apoptotic and necrotic cells ( Figure 4 C and 4D), which was most obvious in A549 cells (increased from 12.1% to 58.7%) and MGC-803 cells (increased from 9.3% to 33.9%). The increase in cells in the lower right quadrant suggests that some of these cells underwent apoptosis ( Figure 4 E). Western blot analysis confirmed enhanced JNK phosphorylation in A549 and MGC-803 cells ( Figure 4 F), which is consistent with the reported role of ROS stress in activating this signaling pathway. However, the extent of PARP cleavage in these cells was relatively mild ( Figure 4 F), suggesting that BT may induce other types of cell death.

[0035] Given that high concentrations of reactive oxygen species (ROS) may trigger necrosis and ferroptosis, we investigated this possibility by measuring the release of lactate dehydrogenase (LDH), a marker of membrane damage in these cell death pathways. Indeed, BT concentration-dependently increased the release of LDH into the culture medium in all three cell lines, particularly in A549 and MGC-803 cells ( Figure 4 G). Transfection of GSTM1 WT almost completely abolished LDH release, whereas the M113S mutant only partially reduced LDH release ( Figure 4 H), indicating that GSTM1 inhibition mediates BT-induced LDH release. Taken together, these results indicate that BT induces ROS generation and leads to mixed cell death in cancer cells.

[0036] As shown in Figures A and B, BT treatment led to a collapse of the mitochondrial membrane potential (ΔΨm) and ATP depletion. ATP levels were fully restored after overexpression of wild-type GSTM1 and partially restored after overexpression of the M113S mutant ( Figure 5 C). Consistent with ATP depletion, Western blot analysis showed that BT treatment induced AMPK hyperphosphorylation and ERK1 / 2 dephosphorylation ( Figure 5D), reflecting growth arrest and possible enhanced autophagy. Meanwhile, BT reduced intracellular GSH levels ( Figure 5 E), a finding consistent with ATP-dependent GSH biosynthesis. Since GSH is an important cosubstrate for GST-mediated ROS detoxification, this depletion may exacerbate ROS accumulation and thus exacerbate oxidative stress in BT-treated cells. Together, these findings suggest that BT targets GST, increases ROS and depletes ATP / GSH, inducing growth arrest and ultimately cell death ( Figure 5 F).

[0037] Example 3 Effect of BT on Chemotherapeutic Drug Sensitivity Cells were pretreated with BT for 12 hours and then co-treated with CDDP or DOX for 48 hours. Cell viability was assessed by MTT, and the combination index (CI) value was calculated using CompuSyn software (version 1.0). A CI < 0.8 indicated synergistic effect. Each condition was tested in triplicate (n = 3), and data were analyzed as mean ± SD. Figure 6 .

[0038] Figure 6 Middle, A) Cell viability curves of A549, MGC-803, and MDA-MB-453 cells treated with different concentrations of BT for 72 h, showing IC 50 Values. B) Synergistic effect of different concentrations of BT and DOX on A549 and MGC-803 cells. *p<0.05, **p<0.01, ***p<0.001 compared with the control group. C) ROS levels in A549 and MGC-803 cells treated with DOX (500 nM) or the combination of DOX and BT (20 μM). D) Synergistic effect of different concentrations of BT and CDDP in A549 and MGC-803 cells. E) ROS levels in A549 and MGC-803 cells treated with CDDP (3 μM) or the combination of CDDP and BT (20 μM).

[0039] like Figure 6 As shown in A, BT inhibited the growth of A549 and MGC-803 cells more effectively than MDA-MB-453 cells ( Figure 6 A). Cell viability assays showed that BT and DOX exhibited only moderate synergy in A549 cells, whereas significant synergy was observed in MGC-803 cells ( Figure 6 B). Mechanistic analysis showed that BT increased ROS production in DOX-treated A549 cells and caused a strong synergistic ROS upregulation in MGC-803 cells ( Figure 6C). Similarly, combined with CDDP showed stronger synergistic effect in MGC-803 cells ( Figure 6 D), which is consistent with the higher synergistic ROS generation effect in these cells ( Figure 6 E). These results indicate that different ROS responses underlie the different efficacy of the combination, suggesting that BT can enhance the cytotoxicity of DOX in MGC-803 cells by amplifying oxidative stress.

[0040] The present invention discovered that BT can mediate anticancer activity by targeting GST family proteins (such as GSTA1 and GSTM1). By occupying the H site, BT blocks the binding of GSH to ROS, thereby impairing the cell's ROS scavenging function and leading to elevated ROS. The resulting ROS stress activates the JNK signaling pathway, reduces mitochondrial membrane potential, and depletes ATP and GSH levels. The combined effects of elevated ROS and ATP / GSH depletion may trigger autophagy, cell growth arrest, and mixed cell death with features of necrosis / ferroptosis. Furthermore, it can increase the sensitivity of tumor cells to chemotherapeutic drugs, enhancing the therapeutic efficacy of chemotherapeutic drugs.

[0041] Finally, it should be noted that the above specific implementation methods are only used to illustrate the technical solutions of the present invention and are not limiting. Although the present invention has been described in detail with reference to examples, those skilled in the art should understand that the technical solutions of the present invention can be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the claims of the present invention.

Claims

1. Use of thiochlorophenol as a GST inhibitor.

2. A GST inhibitor, characterized in that The GST inhibitor uses thiochlorophenol as an active ingredient and also includes a pharmaceutically acceptable adjuvant.

3. Use of the thiochlorophenol according to claim 1 or the GST inhibitor according to claim 2 in the preparation of a preparation for improving the efficacy of a chemotherapeutic drug.

4. The use according to claim 3, characterized in that The chemotherapy drug is cisplatin or doxorubicin.

5. A combination drug, characterized in that: The invention comprises the thiochlorophenol according to claim 1 or the GST inhibitor according to claim 2, and a chemotherapeutic drug.

6. The combination drug according to claim 5, characterized in that The chemotherapy drug is cisplatin or doxorubicin.

7. Use of the thiochlorophenol according to claim 1, the GST inhibitor according to claim 2, or the combined drug according to claim 5 in the preparation of a drug for treating tumors.

8. The use according to claim 7, characterized in that The tumor is gastric cancer or lung adenocarcinoma.

9. The use according to claim 7 or 8, characterized in that Thiochlorophenol, GST inhibitors, or combined drugs target GST, increase ROS levels, reduce ATP levels, induce cell death, and growth arrest.

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