Iridium complex probe, preparation method and application in PPI inhibition monomer screening
By using an iridium complex probe to competitively bind to the Keap1 protein, the problem of interference with existing fluorescent molecular probes was solved, and the accuracy and efficiency of PPI-inhibited monomer screening were achieved.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-11-20
- Publication Date
- 2026-03-27
AI Technical Summary
Existing fluorescent molecular probes are easily affected by the autofluorescence of the test compound and the background fluorescence of the cell in PPI-inhibited monomer screening, leading to frequent false positive and false negative results, and are especially unsuitable for large-scale screening of compounds containing conjugated groups.
Iridium complex probes were prepared using 6-(2-pyridyl)-1H-benzimidazole-1-ylhexylamine, oleanolic acid, and cyclometalated iridium complex dimers. These probes were used to competitively bind to the Keap1 protein, reducing the probe's optical signal. Combined with high-content screening imaging, cell activity was observed, and regulators of target-target interactions were screened out.
Iridium complex probes have a long and stable fluorescence lifetime and a large Stokes shift, which can effectively reduce the occurrence of false negative and false positive results, and improve the accuracy and reliability of screening.
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Figure CN121736037A_ABST
Abstract
Description
Technical Field
[0001] This application relates to the field of drug screening, and more particularly to an iridium complex probe, its preparation method, and its application in screening PPI inhibitory monomers. Background Technology
[0002] Protein-protein interactions (PPIs) refer to the process by which two or more proteins with their own physiological functions exert their functions through transient or permanent binding. Nuclear transcription-associated factor 2 (Nrf2) is an important protein for maintaining redox homeostasis in the body. Under normal circumstances, Nrf2 interacts with Kelch-like epichlorohydrin-associated protein 1 (Keap1) in the cytoplasm via a PPI. Keap1 targets and links polyubiquitin chains to Nrf2, leading to proteasomal degradation and maintaining a low basal level of Nrf2. Decreased Keap1 activity in cells leads to upregulation of Nrf2 levels and stimulates the expression of downstream antioxidant genes, such as NAD(P)H:quinone oxidoreductase 1 (NQO1) and heme oxygenase-1 (HO-1). Based on this, upregulating Nrf2 levels by disrupting the Keap1-Nrf2 PPI, thereby activating antioxidant genes in cells and downregulating inflammatory mediators, is a classic therapeutic target for anti-inflammation. Therefore, it is necessary to screen Keap1-Nrf2 PPI inhibitory monomers to obtain molecularly targeted drugs. Currently, the mainstream screening methods for Keap1-Nrf2PPI inhibitor monomers are also mainly based on fluorescence signals, such as bimolecular fluorescence complementarity, fluorescence correlation spectroscopy, fluorescence resonance energy transfer, and fluorescence polarization.
[0003] Existing methods employ high-content screening (HCS) technology, using fluorescent molecules as "signal reporter" units. These molecules suffer from short fluorescence lifetimes, small Stokes shifts, and poor photostability, making them susceptible to interference from the intrinsic fluorescence of the test compound and background fluorescence of cells. This leads to frequent false positive and false negative results, and they are particularly unsuitable for large-scale screening of monomers containing conjugated inhibitory groups. Summary of the Invention
[0004] The main objective of this application is to provide an iridium complex probe, its preparation method, and its application in screening PPI-inhibiting monomers, aiming to solve the problem that existing probes are susceptible to interference from the autofluorescence of the analyte compound and the background fluorescence of the cell.
[0005] To achieve the above objectives, this application provides an iridium complex probe, the structural formula of which is: .
[0006] Optionally, the iridium complex probe is prepared by 6-(2-pyridyl)-1H-benzimidazole-1-ylhexylamine, oleanolic acid, and a cyclometalated iridium complex dimer; wherein, during the preparation process, 6-(2-pyridyl)-1H-benzimidazole-1-ylhexylamine and oleanolic acid undergo a condensation reaction to obtain an oleanolic acid-containing ligand; the oleanolic acid-containing ligand undergoes a metal coordination reaction with the cyclometalated iridium complex dimer to obtain the iridium complex probe.
[0007] To achieve the above objectives, this application also provides a method for preparing an iridium complex probe, comprising: mixing 2-(2-pyridyl)benzimidazole, potassium hydroxide, and tert-butyl(6-bromohexyl)carbamate in a first solvent, and carrying out a nucleophilic substitution reaction to obtain tert-butyl[6-(2-pyridyl)-1H-benzimidazole-1-yl]hexylcarbamate; and further mixing tert-butyl[6-(2-pyridyl)-1H-benzimidazole-1-yl]hexylcarbamate. The ester was placed in an acidic second solvent and subjected to an acid-catalyzed reaction to obtain 6-(2-pyridyl)-1H-benzimidazole-1-ylhexylamine; 6-(2-pyridyl)-1H-benzimidazole-1-ylhexylamine was mixed with oleanolic acid and subjected to a condensation reaction to obtain an oleanolic acid-containing ligand; the oleanolic acid-containing ligand was mixed with a cyclometalated iridium complex dimer in a third solvent and subjected to a metal coordination reaction, and ammonium hexafluorophosphate was added to obtain an iridium complex probe.
[0008] Optionally, the molar ratio of 2-(2-pyridyl)benzimidazole, potassium hydroxide, and tert-butyl(6-bromohexyl)carbamate is 1:(1-2):(1-2); the molar ratio of 6-(2-pyridyl)-1H-benzimidazole-1-ylhexylamine to oleanolic acid is 0.8-1.5:1; and the molar ratio of the oleanolic acid ligand to the cyclometalated iridium complex dimer is 1-5:1.
[0009] Optionally, the first solvent is acetone, acetonitrile, DMSO or DMF, the second solvent is 1,4-dioxane containing HCl, and the third solvent is a mixture of DCM and MeOH, or a mixture of DCM and EtOH.
[0010] Optionally, the preparation method of the oleanolic acid ligand is as follows: oleanolic acid is dissolved in a fourth solvent, and EDCI and HOBt are added to it to obtain an intermediate product; 6-(2-pyridyl)-1H-benzimidazol-1-ylhexylamine is dissolved in DIPEA and added to the intermediate product to obtain the oleanolic acid ligand.
[0011] Optionally, the molar ratio of oleanolic acid, EDCI and HOBt is (0.2-1):1:1; the molar ratio of 6-(2-pyridyl)-1H-benzimidazole-1-ylhexylamine and DIPEA is 1:1-10; and the fourth solvent is DCM.
[0012] Optionally, the cyclometalated iridium complex dimer is Ir2[dfpq]4Cl2. The preparation method of the cyclometalated iridium complex dimer is as follows: IrCl3·xH2O and dfpq are mixed in a fifth solvent to undergo a metal activation reaction to obtain Ir2[dfpq]4Cl2.
[0013] To achieve the above objectives, this application also provides an application of an iridium complex probe in screening PPI inhibitor monomers.
[0014] Optionally, the PPI is Keap1-Nrf2 PPI, and TEPP-46 is used as a negative control reagent. The screening method includes: incubating cells for the first time with culture media containing the negative control reagent and the inhibitory monomer to be screened, respectively; adding iridium complex probes to culture media containing the negative control reagent and the inhibitory monomer to be screened for the second time; performing confocal cell imaging on the results of the second incubation using a high-content screening instrument; comparing the confocal cell imaging results corresponding to the negative control reagent with the confocal cell imaging results corresponding to the inhibitory monomer to be screened, and selecting the inhibitory monomer to be screened with reduced optical signal of iridium complex probes as the PPI inhibitory monomer.
[0015] Compared with the prior art, the beneficial effects of this application are as follows: The iridium complex probe of this invention is prepared by using 6-(2-pyridyl)-1H-benzimidazol-1-ylhexylamine, oleanolic acid, and a cyclic metallized iridium complex dimer. After incubation with cells, it exhibits low cytotoxicity, and the cyclic metallized iridium complex dimer provides fluorescence properties for the probe, making it suitable for use as a live-cell imaging probe. When the iridium complex probe of this invention is used for screening PPI inhibitor monomers, the inhibitor competitively binds to the Keap1 protein, reducing the probe's optical signal. Combined with high-content screening imaging to observe cell activity, this eliminates some overly toxic monomers, further accurately screening for regulators of target-target interactions. The iridium metal probe has a long and stable fluorescence lifetime, a large Stokes shift, is easy to synthesize, and is convenient to design for binding with the biomolecule oleanolic acid to obtain an iridium complex probe, effectively solving the current problems faced by "fluorescent molecules," thus greatly reducing false negative and false positive results. Attached Figure Description
[0016] Figure 1 This is a reaction mechanism diagram of a method for preparing an iridium complex probe according to this application; Figure 2 This is a diagram illustrating the PPI-inhibiting monomer screening process of an iridium complex probe according to this application; Figure 3This is a cytotoxicity detection diagram of an iridium complex probe and methylbardoxolone according to this application; Figure 4 The image shows the probe results of HaCaT cells against the iridium complex; Figure 5 This is a time-dependent fluorescence imaging result of an iridium complex probe according to this application; Figure 6 This is a dose-dependent fluorescence imaging result of an iridium complex probe according to this application; Figure 7 This is a fluorescence imaging result of an iridium complex probe of this application competing with oleanolic acid for protein binding in HaCaT cells; Figure 8 The image shows the cellular thermal migration results of an iridium complex probe and oleanolic acid for Keap1 and Nrf2 proteins in HaCaT cells, as described in this application. Figure 9 This is an immunofluorescence imaging result of an iridium complex probe and Keap1 antibody in HaCaT cells according to this application; Figure 10 This is an immunoprecipitation verification diagram of an iridium complex probe that disrupts Keap1-Nrf2 PPI in HaCaT cells according to this application; Figure 11 This image shows the effect of an iridium complex probe of this application on the activation of Nrf2 protein levels in HaCaT cells. Figure 12 This image shows the effect of a competitive luminescent high-content screening platform constructed using an iridium complex probe according to this application.
[0017] The realization of the purpose, functional features and advantages of this application will be further explained in conjunction with the embodiments and with reference to the accompanying drawings. Detailed Implementation
[0018] To make the objectives, technical solutions, and advantages of this application clearer, the technical solutions of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.
[0019] The first embodiment of the present invention provides an iridium complex probe, the structural formula of which is: (abbreviated as LT-8).
[0020] The iridium complex probe was prepared by 6-(2-pyridyl)-1H-benzimidazol-1-ylhexylamine (hereinafter referred to as compound S2), oleanolic acid, and cyclometalated iridium complex dimer. During the preparation process, compound S2 and oleanolic acid underwent a condensation reaction to obtain an oleanolic acid-containing ligand, hereinafter referred to as compound S3. Compound S3 underwent a metal coordination reaction with the cyclometalated iridium complex dimer to obtain the iridium complex probe.
[0021] A second embodiment of the present invention provides a method for preparing an iridium complex probe, such as... Figure 1 As shown, the specific steps include: In step S10, 2-(2-pyridyl)benzimidazole, potassium hydroxide, and tert-butyl(6-bromohexyl)carbamate are mixed in a first solvent to carry out a nucleophilic substitution reaction to obtain compound S1; the first solvent is acetone, acetonitrile, DMSO, or DMF. The molar ratio of 2-(2-pyridyl)benzimidazole, potassium hydroxide, and tert-butyl(6-bromohexyl)carbamate is 1:(1-2):(1-2).
[0022] Specifically, to ensure a higher yield, 2-(2-pyridyl)benzimidazole and potassium hydroxide were first dissolved in acetone and stirred at 60-65°C for 0.5-1.5 h. Then, tert-butyl (6-bromohexyl) carbamate was added and stirred overnight. Under the catalysis of potassium hydroxide, 2-(2-pyridyl)benzimidazole and tert-butyl (6-bromohexyl) carbamate underwent a nucleophilic substitution reaction. The product was cooled and directly purified by silica gel column chromatography to obtain tert-butyl[6-(2-pyridyl)-1H-benzimidazole-1-yl]hexyl carbamate, hereinafter referred to as compound S1.
[0023] In step S20, compound S1 is placed in an acidic second solvent to carry out an acid-catalyzed reaction to obtain compound S2; wherein the second solvent is 1,4-dioxane containing HCl.
[0024] Specifically, compound S1 was placed in an excess of 1,4-dioxane solution containing HCl and stirred at room temperature for 3.5-4.5 h. The product was monitored in real time by thin-layer chromatography. During this process, compound S1 removed the Boc group under the acid catalysis of 1,4-dioxane in HCl, and the solvent was directly evaporated from the product to obtain compound S2.
[0025] In step S30, compound S2 is mixed with oleanolic acid and subjected to a condensation reaction to obtain compound S3; wherein the molar ratio of compound S2 to oleanolic acid is 0.8-1.5:1.
[0026] Specifically, oleanolic acid is dissolved in a fourth solvent, and EDCI (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) and HOBt (1-hydroxybenzotriazole) are added to it to obtain an intermediate product; compound S2 is dissolved in DIPEA (N,N-diisopropylethylamine) and added to the intermediate product to obtain compound S3. The fourth solvent is DCM. The molar ratio of oleanolic acid, EDCI, and HOBt is (0.2-1):1:1. The molar ratio of compound S2 to DIPEA is 1:1-10.
[0027] In this embodiment, EDCI is the core condensing agent, HOBt is used to suppress side reactions, and DIPEA is used as a base to regulate the reaction environment. Under the action of the three, the amine bond of compound S2 condenses with the carboxylic acid bond of oleanolic acid to form an amide bond (-CONH-).
[0028] In step S40, compound S3 is mixed with the cyclometalated iridium complex dimer in a third solvent to carry out a metal coordination reaction, and excess ammonium hexafluorophosphate (NH4PF6) is added to obtain the iridium complex probe. The molar ratio of compound S3 to the cyclometalated iridium complex dimer is 1-5:1. The third solvent is a mixture of DCM and MeOH, or a mixture of DCM and EtOH, with a volume ratio of 1:1. The cyclometalated iridium complex dimer is Ir2[dfpq]4Cl2.
[0029] Specifically, the cyclometalated iridium complex dimer is Ir2[dfpq]4Cl2, which is prepared by mixing IrCl3·xH2O with dfpq (6,7-difluoro-2-methyl-3-phenylquinoxaline) in a fifth solvent and carrying out a metal activation reaction overnight under N2 to obtain the cyclometalated iridium complex dimer, Ir2[dfpq]4Cl2. The fifth solvent is a mixture of 2-methoxyethanol and H2O in a 3:1 volume ratio.
[0030] A third embodiment of the present invention provides an application of an iridium complex probe in the screening of PPI inhibitor monomers. The PPI is Keap1-Nrf2 PPI, and TEPP-46 is used as a negative control reagent to establish a competitive screening platform based on the LT-8 probe in the HCS system. The screening method is as follows: Figure 2As shown, cells were incubated for the first time using culture media containing negative control reagent and the inhibitory monomer to be screened, respectively; iridium complex probes were added to the culture media containing negative control reagent and the inhibitory monomer to be screened for the second incubation, respectively; confocal cell imaging was performed on the results of the second incubation using a high-content screening instrument; the confocal cell imaging results corresponding to the negative control reagent were compared with the confocal cell imaging results corresponding to the inhibitory monomer to be screened, and the inhibitory monomer to be screened with reduced optical signal of iridium complex probe was identified as the PPI inhibitory monomer.
[0031] In this embodiment, the basic principle of the competitive luminescence screening method is as follows: the hydroxyl group at the C3 position of the oleanolic acid moiety of the iridium complex probe binds to the cysteine residue of the Keap1 protein via hydrogen bonds, affecting the interaction between the target and the Nrf2 protein. This binding changes the microenvironment of the iridium complex probe, the "luminescent element," from an aqueous environment to a relatively hydrophobic environment, resulting in highly efficient luminescence. 3 MLCT generates a fluorescent signal. When an active molecule that inhibits PPI is present, the active molecule can competitively bind to the Keap1 protein, thereby reducing the probe's optical signal. The high-content screening instrument can output a signal through changes in fluorescence intensity and can visually observe cell activity, thus preliminarily screening out regulators of target-target interactions.
[0032] Example 1 Preparation of LT-8 Step 1: 8.0 mmol of 2-(2-pyridyl)benzimidazole and 16 mmol of potassium hydroxide were mixed in 40 mL of acetone and stirred at 63 °C for 1 h. 12 mmol of tert-butyl (6-bromohexyl) carbamate was added and stirred at 63 °C overnight. The product was cooled and purified directly by silica gel column chromatography to obtain S1.
[0033] Step 2: Dissolve 1.0 mmol of compound S1 in 5 mL of 1,4-dioxane containing 4.0 M HCl, then stir at room temperature for 4 h, and monitor the product in real time by thin-layer chromatography. After completely removing the Boc group, completely evaporate the solvent to obtain compound S2.
[0034] Step 3: Dissolve 1.2 mmol of oleanolic acid in 30 mL of LDCM, and add 2.0 mmol of EDCI and 2.0 mmol of HOBt to obtain a mixture; mix 1 mmol of compound S2 and 5 mmol of DIPEA, add the mixture to the above mixture, stir overnight at room temperature, and then purify by silica gel column to obtain compound S3. Step 4: IrCl3·xH2O and 2.1 equivalents of dfpq were mixed at 130°C in a mixture of 2-methoxyethanol and H2O at a volume ratio of 3:1, and reacted overnight under N2. After the product was cooled to room temperature, the mixture was filtered and washed three times with deionized water and three times with diethyl ether to obtain the cyclometalated dichlorobridged dimer (Ir2[dfpq]4Cl2). 0.08 mmol of Ir2[dfpq]4Cl2 and compound S3 (0.16 mmol) were mixed in 6 mL of a 1:1 DCM:MeOH suspension and stirred overnight at room temperature. Excess ammonium hexafluorophosphate was added, and the mixture was stirred for another 0.5 h. The crude product was purified by column chromatography to obtain the iridium metal probe, LT-8.
[0035] Example 2 Step 1: Harvest well-grown human keratinocytes (HaCaT) and seed them into 96-well clear flat-bottom plates, 5 × 10⁶ cells per well. 3 Cells. The culture medium used was a complete medium: high glucose DMEM, 10% fetal bovine serum and 1% penicillin-dextrose antibody, and the culture conditions were 5% carbon dioxide and 37°C. Step 2: After 24 hours of adhesion, the culture medium was replaced with a medium containing different concentrations (100 μM, 30 μM, 10 μM, 3 μM, 1 μM, 0.3 μM, 0.1 μM, 0.03 μM, 0.01 μM, 0 μM) of iridium complex probes LT-7, LT-8 and CDDO-Me (methylbardoxone); and incubated at 5% carbon dioxide and 37°C for 72 hours. Step 3: Replace the culture medium with 100 μL MTT (3-(4,5-dimethyl-2-thiazole)-2,5-diphenyltetrazolium bromide thiazole blue) reagent (1 mg / mL). After incubation for 4 h, replace with 100 μL LDMSO and measure using a 570 nm microplate reader.
[0036] Experimental results are as follows Figure 3 As shown, the 72hIC50 values of iridium complex probes LT-7 and LT-8 are 0.67 μM and 0.87 μM, respectively, while the 72hIC50 value of CDDO-Me reaches 0.08 μM. This indicates that CDDO-Me has high cytotoxicity and is not suitable for use as a live-cell imaging probe when conjugated with iridium (III). The iridium complex probes LT-7 and LT-8 obtained in Examples 1 and 2 can be used as live-cell imaging probes.
[0037] The structural formula of LT-7 is: .
[0038] Example 3 uses inductively coupled plasma mass spectrometry to detect the uptake of probes LT-7 and LT-8 by cells.
[0039] Take well-grown HaCaT cells and use 2×10 6 Cells were seeded at a depth of 25 cm. 2 In cell culture dishes; after 24 hours of cell adhesion, add culture medium containing 10 μMLT-7 or 10 μMLT-8 to the cell culture dishes and incubate for 90 min; add 1 mL of 0.25% trypsin to digest the cells, collect the cells in a 15 mL centrifuge tube, centrifuge the cells at 1000 rpm for 3 min, and discard the supernatant; add concentrated nitric acid to the cells and digest overnight; add 2% nitric acid solution to dilute to 5 mL, and use an inductively coupled plasma mass spectrometer to detect the iridium content.
[0040] Experimental results are as follows Figure 4 As shown in the figure, the experimental results indicate that cells treated with probes LT-7 and LT-8 exhibited higher iridium content, suggesting that probes LT-7 and LT-8 have good cell permeability.
[0041] Example 4 Take well-grown HaCaT cells and use 2×10 4 Cells were seeded in PerkinElmer 96-well glass plates; after 24 hours of adhesion, culture medium containing 10 μM LT-7 or LT-8 was added to the 96-well plates and incubated for 30 min, 40 min, 50 min, and 60 min; the cells were washed three times with PBS buffer; and 63x confocal cell imaging was performed using a High-Content Screening System. Fluorescence intensity analysis.
[0042] Experimental results are as follows Figure 5 As shown in the figure. The experimental results show that after 1 hour of treatment with LT-7 and LT-8, clearly visible fluorescence appeared in the cells. Therefore, a treatment time of 1 hour is a suitable imaging time parameter.
[0043] Example 5 Well-grown HaCaT cells were seeded at a rate of 2 × 10⁴ cells into 96-well PerkinElmer glass-bottomed plates. After 24 h of adhesion, culture medium containing 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM LT-7 or LT-8 was added to the 96-well plates and incubated for 60 min. The cells were washed three times with PBS buffer and then subjected to 63x confocal cell imaging using a High-Content Screening System.
[0044] Fluorescence intensity analysis was performed, and the experimental results are as follows: Figure 6As shown in the figure, the experimental results show that clearly visible fluorescence appeared after treatment with 8 μM (LT-7) and 7 μM (LT-8) for 1 h, and the fluorescence intensity was the highest. 8 μM (LT-7) and 7 μM (LT-8) are suitable probe imaging concentration parameters, further indicating that these two probes have ideal fluorescence performance as screening probes.
[0045] Example 6 Well-grown HaCaT cells were seeded at a rate of 2 × 10⁴ cells per well in PerkinElmer 96-well plates. After 24 hours of adhesion, medium containing 10 μM, 30 μM, 100 μM, or 300 μM oleanolic acid was added to the 96-well plates and incubated for 240 min. Medium containing 8 μM LT-7 or 7 μM LT-8 was then added to the 96-well plates and incubated for 60 min. Cells were washed three times with PBS (phosphate-buffered saline). 63x confocal cell imaging was performed using a High-Content Screening System.
[0046] Fluorescence intensity analysis was performed, and the experimental results are as follows: Figure 7 As shown in the figure. The experimental results show that the luminescence intensity of both LT-7 and LT-8 decreased after oleanolic acid pretreatment. Moreover, after 30 μM oleanolic acid pretreatment, the luminescence intensity of LT-8 was lower than that of LT-7, indicating that LT-8 is more suitable as a competitive screening probe.
[0047] Example 7: Cell thermal migration assay to study the ability of iridium complex probes to target Keap1 and Nrf2 in the cellular environment.
[0048] Step 1: Obtain well-grown HaCaT cells at a volume of 5 × 10⁶ cells. 6 Add cell lysis buffer containing protease inhibitors and phosphatase inhibitors, and lyse on ice; Step 2: Incubate HaCaT cell lysate with 20 μM LT-8 and dimethyl sulfoxide at room temperature for 60 min; incubate HaCaT cell lysate with dimethyl sulfoxide and oleanolic acid at room temperature for 60 min as controls. Step 3: The lysis buffer was aliquoted into 1.5 mL centrifuge tubes and heated individually at specified temperatures (25℃, 55℃, 60℃, 65℃, 70℃, 75℃). After centrifugation, the supernatant of the heated lysis buffer was collected and then analyzed by Western blot (WB) using Keap1, Nrf2, and β-actin antibodies.
[0049] Experimental results are as follows Figure 8 As shown in the figure. The experimental results show that... Figure 8Treatment of cell lysates with the LT-8 probe significantly increased the melting temperature of Keap1 from 55°C to 70°C, but had no effect on the stability of Nrf2 or β-actin. Figure 8 The data shows that modifying oleanolic acid with an iridium metal group alters its binding to the protein, confirming that the drug binds to the Keap1 protein rather than Nrf2. These results demonstrate that even in complex cellular environments, the probe LT-8 can bind to Keap1 and disrupt the Keap1-Nrf2 protein interaction.
[0050] Example 8: Immunofluorescence assay to assist in verifying the ability of iridium complex probes to target Keap1 protein in the cellular environment.
[0051] Step 1: Take well-grown HaCaT cells and use 3×10 4 Cells were seeded in PerkinElmer 96-well glass plates; after 24 hours of adhesion, 7 μM LT-8 was added to the 96-well plates and incubated for 4 hours. The supernatant was discarded and the cells were washed three times with PBS buffer for 5 minutes each time. Step 2: Fix cells with 4% paraformaldehyde as a fixative. Fix cells with 100 μL of 4% paraformaldehyde for 15 min, and wash three times with PBS buffer for 5 min each time. Step 3: Permeabilize cells with 0.5% Triton™ X-100, fix cells with 100 μL of 0.5% Triton™ X-100 for 15 min, and wash three times with PBS buffer for 5 min each time; Step 4: Use PBS as a solvent to prepare 5% BSA as a blocking solution. Block cells with 100 μL of 5% BSA for 30 min, and wash three times with PBS buffer for 5 min each time. Step 5: Prepare a primary antibody solution using 5% BSA at a ratio of 1:300, and incubate the cells obtained in Step 4 in the primary antibody solution at 4°C overnight; then wash three times with PBS buffer, each time for 5 min. Step 6: Prepare a secondary antibody solution at a ratio of 1:500 using 5% BSA as the solvent. Incubate the cells obtained in Step 5 in the secondary antibody solution at room temperature in the dark for 1 hour. Rinse three times with PBS buffer, each time for 5 minutes. Observe the imaging results using a High-Content Screening System.
[0052] Step 7: Open the captured Anti-Keap1 and LT-8 images in ImageJ software. Then, hold down the Ctrl key (Windows) or Command key (Mac) to select the Anti-Keap1 and LT-8 images as the channels to be merged. Under the "Image" menu, select "Color" -> "Make Composite". Select the "Red" channel for the LT-8 image and the "Green" channel for the Anti-Keap1 image. ImageJ will automatically create a composite image, merging the selected images, with each channel displayed as a different color. Save the merged composite image by selecting the desired format through "File" -> "Save As".
[0053] Experimental results are as follows Figure 9 As shown in the figure. The results indicate that the live-cell fluorescence imaging distribution of probe LT-8 is roughly the same as the distribution of Keap1 protein in cells, proving that the binding site of LT-8 in live cells is Keap1 protein.
[0054] Example 9 Step 1: Obtain well-grown HaCaT cells, with a cell volume of 2 × 10⁶ cells. 6 10 μM LT-8 and oleanolic acid were added and treated for 4 h respectively; Step 2: Rinse adherent cells 2-3 times with PBS buffer; add an appropriate volume of IP lysis buffer to the culture plate / flask and lyse for 3-5 min. Use a cell scraper to scrape off the cells and reagents and collect them into a 1.5 mL centrifuge tube. Incubate on ice for 30 min to ensure complete cell lysis. Centrifuge at 12000 rpm for 15 min at 4℃, collect the supernatant, and use 1 / 10 of the supernatant as input. Use the remaining sample for immunoprecipitation of cell lysis buffer. Step 3: Use the BCA protein concentration assay kit to determine the protein concentration of the sample in the immunoprecipitation cell lysate to ensure consistent loading volume; Step 4: Take the supernatant of the cell lysate from the above immunoprecipitation into a 1.5 ml centrifuge tube, add 2.5 μL (1.5 μg) of Nrf2 antibody (add the same amount of ordinary IgG of the same species as the antibody used for precipitation as a negative control, negative control experiment), and then incubate at 4 °C for 2 h; Step 5: Add 20 μl of magnetic beads to the cell lysis buffer obtained in step 4 (mix thoroughly before use), gently tap with your finger to mix, and incubate overnight at 4°C with shaking. Step 6: Place the centrifuge tube on a magnetic rack and let it stand for 5-10 seconds, then aspirate the supernatant (avoid contact with the magnetic beads as much as possible). Add 1 mL of washing buffer to the centrifuge tube, place it on a vertical shaker and wash for 3 minutes. Then, place the centrifuge tube on a magnetic rack and let it stand for 5-10 seconds, then aspirate the supernatant (avoid contact with the magnetic beads as much as possible). Repeat this process 3 times. Step 7: After discarding the supernatant, add an appropriate volume of 2×SDS loading buffer containing mercaptoethanol, boil in water for 10 min, and store the sample at -20℃ to obtain the IP sample.
[0055] Step 8: Perform Western blotting (WB) analysis on the Input samples using Keap1, Nrf2, and β-actin antibodies, respectively. For the IP samples, perform WB analysis using Keap1. The main purpose of WB analysis with β-actin antibody is to correct for differences in sample loading amounts, correct and standardize errors generated during the experiment, and ensure the accuracy and comparability of quantitative results.
[0056] Experimental results are as follows Figure 10 As shown in the figure. The experimental results show that LT-8 can effectively disrupt Keap1-Nrf2 protein-protein interactions in living cells.
[0057] Example 10 Step 1: Take well-grown HaCaT cells and use 2×10⁻⁶ cells. 6 Cells were seeded at a density in six-well plates; Step 2: Harvest HaCaT cell lysates after incubation with 0 μM, 5 μM, 10 μM, 20 μM LT-8 and 20 μM oleanolic acid for 8 h. Step 3: Prepare WB samples, separate HaCaT cell lysates using 10% SDS-PAGE gel, and transfer them to a PVDF (polyvinylidene fluoride) membrane. Step 4: Incubate the membrane overnight with Keap1, Nrf2, and β-actin antibodies. Wash the membrane three times with washing buffer. Add secondary antibody (1:1000) to the membrane, incubate for 2 hours, then detect protein bands using an enhanced chemiluminescence reagent and analyze using ImageLab.
[0058] Experimental results are as follows Figure 11 As shown in the figure. The experimental results show that when the probe LT-8 concentration is 10 μM, treatment of HaCaT cells for 8 h can significantly activate the intracellular Nrf2 protein level.
[0059] Example 11 TEPP-46 is a classic small molecule activator of pyruvate kinase M2, which can induce the conversion of intracellular PKM2 dimers to tetramers. In this example, TEPP-46 is used as a negative control reagent to establish a competitive screening platform based on the LT-8 probe in the HCS system.
[0060] Step 1: Take well-grown HaCaT cells and use 2×10⁻⁶ cells. 4 Cells were seeded in PerkinElmer glass-bottomed 96-well plates; Step 2: After 24 hours of adhesion, add culture medium containing 10 μM, 30 μM, 100 μM, or 300 μM of TEPP-46 or CDDO-Me to a 96-well plate and incubate for 240 min. Step 3: Add the culture medium containing 7 μMLT-8 to the 96-well plate after incubation in Step 2, and incubate for 60 min; wash three times with PBS buffer. Step 4: Perform 63x confocal cell imaging using the High-Content Screening System; analyze the fluorescence intensity of the cells.
[0061] Experimental results are as follows Figure 12 As shown in the figure. The experimental results show that, compared to the LT-8 group (without TEPP-46 treatment), the fluorescence intensity of LT-8 did not change with the concentration of TEPP-46, indicating that the fluorescence intensity of the TEPP-46 pretreated group was persistent. However, the fluorescence intensity of LT-8 decreased significantly with the concentration of CDDO-Me, indicating that the fluorescence reduction only occurred when treated with the Keap1-Nrf2 PPI inhibitor. Furthermore, LT-8 competitively binds to the Keap1 protein with CDDO-Me, signifying that the iridium complex probe LT-8 of this invention can achieve high-content PPI inhibitory monomer screening through competitive luminescence.
[0062] The above are merely preferred embodiments of this application and do not limit the patent scope of this application. Any equivalent structural or procedural transformations made using the content of this application's specification and drawings, or direct or indirect applications in other related technical fields, are similarly included within the patent protection scope of this application.
Claims
1. An iridium complex probe characterized by, The structural formula of the iridium complex probe is: 。 2. The iridium complex probe according to claim 1, wherein The iridium complex probe is prepared by 6-(2-pyridyl)-1H-benzimidazol-1-yl hexylamine, oleanolic acid and cyclometalated iridium complex dimer. In the preparation process, the 6-(2-pyridyl)-1H-benzimidazol-1-yl hexylamine and the oleanolic acid are subjected to condensation reaction to obtain an oleanolic acid-containing ligand. The oleanolic acid-containing ligand is subjected to metal coordination reaction with the cyclometalated iridium complex dimer to obtain the iridium complex probe.
3. A method of preparing the iridium complex probe according to claim 1 or 2, characterized by, It comprises: 2-(2-pyridyl) benzimidazole, potassium hydroxide and tert-butyl (6-bromohexyl) carbamate are mixed in a first solvent to perform nucleophilic substitution reaction to obtain tert-butyl [6-(2-pyridyl)-1H-benzimidazol-1-yl] hexyl carbamate; The tert-butyl [6-(2-pyridyl)-1H-benzimidazol-1-yl] hexyl carbamate is placed in an acidic second solvent to perform acid catalysis reaction to obtain 6-(2-pyridyl)-1H-benzimidazol-1-yl hexylamine; The 6-(2-pyridyl)-1H-benzimidazol-1-yl hexylamine is mixed with the oleanolic acid to perform condensation reaction to obtain an oleanolic acid-containing ligand; The oleanolic acid-containing ligand is mixed with the cyclometalated iridium complex dimer in a third solvent to perform metal coordination reaction, and ammonium hexafluorophosphate is added to obtain the iridium complex probe.
4. The method for preparing the iridium complex probe according to claim 3, characterized in that, The mass molar ratio of the 2-(2-pyridyl) benzimidazole, potassium hydroxide and tert-butyl (6-bromohexyl) carbamate is 1:(1-2):(1-2); The mass molar ratio of the 6-(2-pyridyl)-1H-benzimidazol-1-yl hexylamine and the oleanolic acid is 0.8-1.5:1; The mass molar ratio of the oleanolic acid-containing ligand and the cyclometalated iridium complex dimer is 1-5:
1.
5. The method for preparing the iridium complex probe according to claim 3, characterized in that, The first solvent is acetone, acetonitrile, DMSO or DMF, the second solvent is 1,4-dioxane containing HCl, and the third solvent is a mixture of DCM and MeOH, or a mixture of DCM and EtOH.
6. The method for preparing the iridium complex probe according to claim 3, characterized in that, The preparation method of the oleanolic acid-containing ligand is: The oleanolic acid is dissolved in a fourth solvent, and EDCI and HOBt are added to obtain an intermediate product; the 6-(2-pyridyl)-1H-benzimidazol-1-yl hexylamine is dissolved in DIPEA, and the intermediate product is added to obtain the oleanolic acid-containing ligand.
7. The method for preparing the iridium complex probe according to claim 6, characterized in that, The mass molar ratio of the oleanolic acid, EDCI and HOBt is (0.2-1):1:1; the mass molar ratio of the 6-(2-pyridyl)-1H-benzimidazol-1-yl hexylamine and DIPEA is 1:1-10; and the fourth solvent is DCM.
8. The method for preparing the iridium complex probe according to claim 3, characterized in that, The cyclometalated iridium complex dimer is Ir2[dfpq]4Cl2, and the preparation method of the cyclometalated iridium complex dimer is: IrCl3·xH2O is mixed with dfpq in a fifth solvent to perform metal activation reaction to obtain Ir2[dfpq]4Cl2.
9. Use of the iridium complex probe of claim 1 or 2 in screening of PPI inhibiting monomers.
10. Use of an iridium complex probe according to claim 9 in PPI inhibiting monomer screening, characterized in that, The PPI is Keap1-Nrf2 PPI, TEPP-46 is used as a negative control reagent, and the screening method comprises the following steps: First incubation of cells with culture medium containing a negative control reagent and a to-be-screened inhibiting monomer respectively; Second incubation of the culture medium containing the negative control reagent and the to-be-screened inhibiting monomer with the iridium complex probe respectively; Confocal cell imaging of the second incubation result by a high-content screening instrument; Comparison of the confocal cell imaging result corresponding to the negative control reagent with the confocal cell imaging result corresponding to the to-be-screened inhibiting monomer, and the to-be-screened inhibiting monomer with reduced optical signal of the iridium complex probe is used as a PPI inhibiting monomer.