Use of ibrutinib in the preparation of mdmx protein inhibitors and antitumor drugs

Abstract

The application discloses an application of icotinopapa in preparation of MdmX protein inhibitors and antitumor drugs, and belongs to the technical field of biological medicines. △F19 The polypeptide synergistically enhances the stability of the ternary complex, and plays an antitumor role through three mechanisms: blocking MdmX-p53 interaction to release p53, up-regulating p21 / PUMA to start cycle arrest and apoptosis; degrading MdmX through the ubiquitin-proteasome pathway; and down-regulating SLC7A11 to weaken the defense of ferroptosis. Icotinopapa is an already-marketed drug, and the application finds a new application of icotinopapa in MdmX overexpression type colorectal adenocarcinoma, breast cancer and non-small cell lung cancer, and can support the rapid clinical transformation of a new indication of MdmX targeted antitumor drugs.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the second pharmaceutical use (drug repurposing) of the known marketed drug eltrombopag (trade name Promacta® / Revolade®), namely, the use of eltrombopag as an MdmX (Murine Double Minute X, also known as MDM4, HDMX) protein inhibitor and anti-tumor drug. Background Technology

[0002] MdmX (also known as Mdm4) is one of the most crucial negative feedback regulators in the p53 tumor suppressor pathway. The p53 protein, hailed as the "guardian of the genome," is responsible for sensing various endogenous and exogenous stress signals in normal cells, such as DNA damage, oncogene activation, and hypoxic stress. It maintains genome stability by coordinating the transcriptional programs of downstream effector genes, including inducing G1 / S phase cell cycle arrest via the p21 / WAF1 / CDKN1A pathway, initiating mitochondrial apoptosis via PUMA / BBC3 and Bax, and weakening cellular antioxidant defenses and promoting ferroptosis by transcriptionally inhibiting SLC7A11 expression. In approximately 50% of malignant tumors that retain the wild-type TP53 gene, the p53 protein itself is not mutated or missing, but its tumor suppressor function is functionally silenced by the aberrantly overexpressed Mdm2 and MdmX. Mdm2 primarily exerts its E3 ubiquitin ligase activity through its C-terminal RING domain, catalyzing the polyubiquitination of lysine residues such as K48 in the p53 protein and targeting it for degradation by the 26S proteasome. While MdmX exhibits significantly weaker inherent E3 ubiquitin ligase activity than Mdm2 due to sequence differences in key catalytic residues within its RING domain, it can directly occupy the transcriptional activation surface of p53 through its N-terminal p53-binding domain (with higher affinity than Mdm2), thereby efficiently blocking the trans-transcriptional activation function of p53. Furthermore, MdmX can form a functional heterodimer with Mdm2 through its C-terminal RING domain, further enhancing the ubiquitination and degradation efficiency of p53 by Mdm2. Mouse models with knockout of both Mdm2 and MdmX genes exhibit a p53-dependent early embryonic lethal phenotype, fully demonstrating their non-substitutability in the p53 signaling regulatory network from a classical genetic perspective.

[0003] Currently, single-target small molecule inhibitors of Mdm2, such as Roche's Idasanutlin (RG7388) and Novartis' Siremadlin (HDM201), have advanced to the clinical trial stage. However, subgroup analyses and pharmacodynamic biomarker monitoring data from multiple clinical studies have revealed that compensatory feedback modulation of MdmX affects the durability of Mdm2 inhibitor efficacy. After pharmacological inhibition of Mdm2, intracellular p53 protein levels rapidly accumulate. Activated p53 tetramers enter the nucleus and bind to the p53 response element (p53RE) in the MdmX gene promoter region, transactivating MdmX gene transcription. This leads to compensatory upregulation of MdmX protein levels, continuously inhibiting the transcriptional activity of residual and newly added p53 proteins. Therefore, directly targeting MdmX is not only a key strategy to overcome compensatory resistance to Mdm2 inhibitors but also provides a targeted therapeutic pathway for MdmX-overexpressing malignancies. However, MdmX presents significant structural biology challenges as a drug target: its p53 binding interface is shallower and flatter than that of Mdm2; the aromatic ring side chain of the key entry residue Tyr99 is rigidly locked into a "closed" conformation by neighboring residues (Leu104 and Phe93); and it lacks the dynamic conformation of the Mdm2 homolog Tyr100, which can be flipped outward to an "open" conformation under ligand induction. This makes it difficult for Nutlin inhibitors (Nutlin-3a, Idasanutlin, etc.) designed using the deep hydrophobic cracks of Mdm2 as templates to be effectively adapted to MdmX. Currently, no small molecule inhibitors targeting MdmX have entered clinical trials globally.

[0004] Eltrombopag is an orally bioavailable small molecule thrombopoietin (TPO) receptor agonist with the chemical name 3'-[(2Z)-[1-(3,4-dimethylphenyl)-1,5-dihydro-3-methyl-5-oxo-4H-pyrazol-4-yl]hydrazino]-2'-hydroxy-[1,1'-biphenyl]-3-carboxylic acid, and the molecular formula C 25 H 22 N4O4, with a molecular weight of 442.47 g / mol, was approved by the US FDA in 2008 for chronic immune thrombocytopenia (ITP) and for monitoring protein thermal unfolding with hydrophobic dyes. Its indications were subsequently expanded to severe aplastic anemia (SAA) and hepatitis-related thrombocytopenia. Oral formulations (trade names Promacta® / Revolade®) are available in doses ranging from 12.5 mg to 75 mg, administered once daily. Its oral bioavailability is approximately 52%-70%, plasma protein binding is >99%, and its half-life is approximately 21-32 hours. Its only known pharmacological target is a non-canonical allosteric site on the TPO-R / c-Mpl transmembrane domain. Eltrombopag has never been reported to possess MdmX binding activity or antitumor activity in any published literature and patents. Summary of the Invention

[0005] To address the technical challenges of the scarcity of small molecules targeting MdmX, the tendency of Mdm2 inhibitors to develop resistance due to compensatory upregulation of MdmX, and the lack of rapidly convertible targeted drugs for p53 wild-type tumors, this invention provides the use of eltrombopag in the preparation of MdmX protein inhibitors and antitumor drugs. Eltrombopag binds to the hydrophobic subpocket of MdmX F19 via a molecular wedge mechanism, with a KD of 2.47 μM. It exerts its antitumor activity through a triple mechanism of blocking MdmX-p53 interaction, upregulating p21 / PUMA, degrading MdmX, and downregulating SLC7A11. It is effective against IC50 in HCT116 and MCF-7 cells. 50 The concentrations are 1.94 μM and 3.69 μM, respectively. This drug is an already marketed oral formulation, and existing clinical safety data can be used to rapidly advance the translation of MdmX into new indications for targeted anti-tumor therapy.

[0006] The technical solution adopted in this invention is as follows:

[0007] This invention provides the application of eltrombopag (CAS No.: 496775-61-2) in the preparation of MdmX protein inhibitors.

[0008] The MdmX protein inhibitor is used to prepare anti-tumor drugs.

[0009] The eltrombopag binds to the F19 hydrophobic subpocket of the MdmX protein in a molecular wedge manner, along with p53p. △F19 Peptides synergistically enhance the thermodynamic stability of ternary complexes.

[0010] The tumor is a p53 wild-type tumor.

[0011] The tumor is selected from at least one of colon cancer, breast cancer, and non-small cell lung cancer.

[0012] The drug is used in combination with an Mdm2 protein inhibitor.

[0013] Application of eltrombopag in the preparation of ferroptosis inducers or ferroptosis sensitizers.

[0014] This invention is based on the following key discovery: Eltrombopag was discovered by the inventors from approximately 12,000 commercially available molecules through large-scale high-throughput virtual screening and stepwise biochemical validation. △F19 -N-MdmX targeting inhibitor. Eltrombopag precisely embeds itself in the F19 hydrophobic subpocket exposed by the deletion of p53 Phe19 residues and adjacent residues in the MdmX protein in a molecular wedge manner, and interacts with p53p △F19The peptides occupy different subregions of the p53 binding domain pocket, generating synergistic effects through overall protein conformation stabilization.

[0015] Eltrombopag exerts its antitumor effect through a unique triple pharmacological mechanism: (a) it occupies the F19 hydrophobic subpocket to block MdmX-p53 protein-protein interactions, releases p53 transcriptional activity, restores the expression of p53 downstream factors p21 and PUMA protein, and simultaneously initiates cell cycle arrest and mitochondrial apoptosis programs; (b) it achieves deep clearance of MdmX target proteins through the classic ubiquitin-proteasome negative feedback pathway, and the released p53 protein further transactivates the transcription of downstream Mdm2 gene. Mdm2, as an E3 ubiquitin ligase, specifically mediates the K48 polyubiquitination degradation of MdmX, achieving near-complete clearance of MdmX in the 5-20 μM range; (c) it downregulates SLC7A11 expression through the transcriptional repression function of p53, weakens tumor cell ferroptosis defense, and achieves near-complete inhibition of SLC7A11 at 20 μM.

[0016] All-atom molecular dynamics (100 ns) simulations further revealed that eltrombopag has a unique N-terminal locking effect on the flexible region (residues 13-18) of MdmX, which sharply reduces the RMSF of this region from 0.3-0.9 nm in other candidate complexes to about 0.1 nm, and the average RMSF of the whole protein Cα atom is only 0.0743 nm.

[0017] Beneficial effects:

[0018] (1) A novel pharmacological activity of eltrombopag, an FDA-approved drug, as a targeted inhibitor of MdmX was discovered for the first time. Eltrombopag precisely occupies the hydrophobic subpocket of the MdmX protein F19 in a molecular wedge manner, with a KD of 2.47 × 10⁻⁶. -6 M, with affinity leading among reported non-peptide MdmX small molecule ligands, is superior to the positive control Nutlin-3a (KD=2.23×10). -4 M) is increased by approximately 90 times, and exhibits typical kd-driven slow dissociation binding characteristics (kd=9.99×10). -3 s -1 ), combined with excellent durability.

[0019] (2) Eltrombopag exhibits potent low-molecular-weight inhibitory activity against proliferation in multiple p53 wild-type tumor cell lines (HCT116 colon cancer IC50). 50 =1.94μM, MCF-7 breast cancer IC 50 =3.69μM), and achieved dual synergistic activation of the p53 apoptosis pathway and ferroptosis pathway.

[0020] (3) Eltrombopag achieves deep clearance of MdmX target proteins (nearly complete clearance at 5-20 μM) through the ubiquitin-proteasome pathway and significantly downregulates SLC7A11 in a dose-dependent manner (nearly complete inhibition at 20 μM), filling the research gap of MdmX target molecules in the direction of ferroptosis mechanism.

[0021] (4) Molecular dynamics simulations revealed the N-terminal locking effect, which reduced the N-terminal flexible region RMSF to about 0.1 nm and the total protein RMSF to only 0.0743 nm, elucidating the dynamic basis of the optimal structural stability of the eltrombopag complex at the atomic level. Attached Figure Description

[0022] Figure 1 : The chemical structural formula of eltrombopag.

[0023] Figure 2 : Etruppa and p53p △F19 Differential scanning fluorescence (DSF) thermal drift results of the -N-MdmX fusion protein.

[0024] Figure 3 Figure: Results of competitive DSF validation experiments for eltrombopag, N-MdmX protein, and mutant peptides.

[0025] Figure 4 : Etruppa and p53p △F19 Multi-concentration kinetic binding sensing map of the -N-MdmX fusion protein with surface plasmon resonance (SPR).

[0026] Figure 5 Eltrombopag in four cell lines with different p53 genetic backgrounds (MCF-7, HCT116, H1299, and H1299) R213X CCK-8 proliferation inhibition curve.

[0027] Figure 6 Western blot analysis of p53, MdmX, p21, PUMA and SLC7A11 proteins after treatment of MCF-7 cells with eltrombopag (0-20 μM, 24 h).

[0028] Figure 7 : Etruppa and p53p △F19 A comprehensive overview of the 100 ns all-atom molecular dynamics simulation analysis of the -N-MdmX fusion protein ternary complex, including... Figure 7 'a' represents the root square fluctuation of the residue level (RMSF). Figure 7 b is the time evolution curve of the radius of gyration (Rg). Figure 7 c represents the time evolution curve of the root mean square deviation (RMSD) of Cα atoms. Figure 7d is the time evolution heatmap of the secondary structure (DSSP). Figure 7 e represents the time evolution curve of the number of protein-ligand hydrogen bonds. Detailed Implementation

[0029] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments, but the scope of protection of the present invention is not limited to the following embodiments.

[0030] This invention revolves around the core objective of "discovery and validation of MdmX-targeting inhibitors," and proceeds with research following a progressive logic: virtual screening discovery → biochemical binding validation → cellular activity evaluation → mechanism of action analysis → structural dynamics elucidation. Firstly, targeting the shallow binding interface and poor drug-likeness of wild-type MdmX protein p53, a p53p inhibitor was constructed. △F19 The pocket was reshaped to expose the deep F19 hydrophobic subpocket, and this was used as a target for high-throughput virtual screening of a library of approximately 12,000 commercially available APE and MCE small molecules, resulting in the candidate molecule eltrombopag. Subsequently, DSF thermal drift, mutant peptide competitive complementation, and SPR kinetics were used to confirm its direct binding to MdmX and its binding mode at the biochemical level. Furthermore, its proliferation inhibitory activity was evaluated in various tumor cell lines with different p53 genetic backgrounds. Western blot was used to verify its triple action mechanism at the protein level, which involves blocking MdmX-p53 interaction, upregulating p21 / PUMA, degrading MdmX, and downregulating SLC7A11. Finally, 100 ns all-atom molecular dynamics simulations were used to elucidate the structural basis of eltrombopag's locking effect on the N-terminal flexible region of MdmX and the high stability of the complex at the atomic level.

[0031] Example 1: The discovery of eltrombopag, based on p53p △F19 -N-MdmX Reshapes Pocket High-Throughput Virtual Screening

[0032] The purpose of this embodiment is to construct a hydrophobic subpocket target model for MdmX F19 and to discover potential small molecule inhibitors targeting MdmX from a compound library through high-throughput virtual screening. Operationally, using the MdmX crystal structure (PDB ID: 3DAB) as a template, the p53 peptide Phe19 residue was deleted to expose the deep hydrophobic subpocket. AutoDock Vina was used to perform large-scale molecular docking on approximately 12,000 commercially available small molecules from APE and MCE. Through a four-stage progressive screening process—binding energy threshold screening, artificial conformation review, pharmacophore cross-validation, and final availability review—113 candidate molecules were ultimately obtained, including eltrombopag.

[0033] MdmX protein surface p53p △F19The interface was used as the target (PDB ID: 3DAB, resolution 1.9 Å). The PyMOLMutagenesis tool was used to delete the Phe19 residue and adjacent amino acid residues of the p53 peptide, exposing the F19 hydrophobic subpocket (DoGSiteScorer assessment: DrugScore = 0.66, volume 487.36 Å). 3 Surface area 769.1 Å 2 The small molecule compound library comprises approximately 12,000 commercially available molecules, including FDA-approved drugs, clinical investigational molecules, and known bioactive compounds. After Open Babel 3D conformation generation and MMFF94 force field energy minimization, large-scale molecular docking (Grid Box 16.123×16.709×18.112Å) was performed using AutoDock Vina. 3 (exhaustiveness=16). After reviewing the binding energy of Vina (≤-7.0 kcal / mol), the artificial visual conformation in Discovery Studio, the dual 3D pharmacophore cross-validation in LigandScout / PyMOL, and the final review for commercial availability, 113 candidate molecules were finally selected. Among them, eltrombopagine has a binding free energy of -8.7 kcal / mol, excellent docking conformation, is a marketed oral drug, and has mature clinical safety data, which can support rapid translation. Therefore, it was selected as the key research target.

[0034] Eltrombopag is derived from APE's commercial drug library APE-F02, with the following structural formula: Figure 1 As shown, the English name is Eltrombopag, CAS number: 496775-61-2, Vina binding free energy is -8.7 kcal / mol, and HPLC purity is ≥98%.

[0035] Example 2: In vitro biochemical experiments to verify the relationship between eltrombopag and p53p △F19 Targeted binding of -N-MdmX

[0036] The purpose of this embodiment is to systematically verify the specific binding of eltrombopag to MdmX protein at the biochemical level. Operationally, three complementary methods were employed in a step-by-step manner: first, differential scanning fluoroscopy (DSF) was used to thermodynamically verify the thermostability of eltrombopag to MdmX protein; second, a DSF experiment using a mutant peptide was used to confirm that the binding site of eltrombopag is the F19 hydrophobic subpocket; finally, surface plasmon resonance (SPR) was used to quantitatively determine the binding kinetic parameters and equilibrium dissociation constant, clarifying its affinity level and slow dissociation binding characteristics.

[0037] 1. Differential scanning fluorescence (DSF) was used to verify the thermostabilizing effect of eltrombopag on MdmX protein.

[0038] To rapidly determine whether eltrombopag and MdmX protein have a direct interaction, differential scanning fluorometry was first used for qualitative verification at the thermodynamic level. This method reflects the effect of ligand binding on protein thermal stability by detecting the fluorescence changes of hydrophobic dyes during protein thermal denaturation.

[0039] The thermal unfolding of proteins was monitored using SYPRO Orange hydrophobic dye. A total reaction system of 25 μL was constructed in a micro-volume 96-well PCR plate, with three replicates. The 25 μL reaction system contained 100 μM p53p △F19 -N-MdmX, 100μM small molecule eltrombopag (HPLC purity ≥98%, stock solution prepared with anhydrous DMSO) and 62.5×SYPRO Orange fluorescent folding probe dye were used. The temperature was increased at a uniform gradient of 25-95℃ (1.8℃ / min) to capture the folding exposure signal in real time (excitation wavelength 498 nm / emission wavelength 580 nm). The inflection point peak was defined as the melting transition midpoint temperature (Tm) by calculating the first negative derivative of temperature (-dRFU / dT) from the collected fluorescence intensity values.

[0040] The results are as follows Figure 2 As shown, with the solvent DMSO group as a blank control (ΔTm=0), the apparent transition temperature of the target protein showed a strong positive thermodynamic drift after the addition of eltrombopag compared to the solvent DMSO group. The measured ΔTm of eltrombopag was +3.16±0.28℃, which is much higher than the significance threshold of |ΔTm|>1.5℃ (dashed line in the figure), indicating that it is a positive stabilizer. This confirms that it acts as a highly efficient structural stabilizer from an independent thermodynamic steady-state level.

[0041] 2. Competitive DSF verification of binding sites by mutant peptides

[0042] To further clarify whether it specifically binds to the hydrophobic subpocket exposed after F19 deletion, a mutant peptide competitive complementation assay was used to confirm the binding site.

[0043] Artificial solid-phase synthesis of the mutant peptide p53p, which removes the key Phe19 amino acid and adjacent amino acid residues. △F19 (Sequence: Ac-WKLLPE-NH2, purity >95%) and PMI △F19 Mutant polypeptide (sequence: Ac-WNLLSP-NH2, purity >95%). The unlabeled wild-type full-length receptor protein N-MdmX was used. 23-110 The mixture was co-incubated with the mutant peptide and eltrombopag in a ternary complex. The Tm value was determined under a completely consistent DSF gradient temperature-controlled denaturation program.

[0044] The results are as follows Figure 3As shown, in the ternary complex system where small molecules and mutant peptides coexist, the peak temperature of its thermal denaturation transition is significantly higher than that of the binary state where only peptides are bound, and a significant additive energy accumulation synergy is generated in the high-temperature range. This thermodynamic backfilling phenomenon confirms that eltrombopag precisely acts as a spatial molecular wedge, filling the deep hydrophobic gap vacated by the loss of F19 in the mutant peptide, and exerting a synergistic stabilizing effect with the p53-derived peptide.

[0045] 3. Quantitative determination of binding kinetics using surface plasmon resonance (SPR)

[0046] After qualitatively confirming the binding activity and binding sites, surface plasmon resonance (SPR) technology was further used to perform quantitative kinetic analysis on the binding of eltrombopag and MdmX, and to accurately determine its affinity and binding dissociation characteristics.

[0047] Using the Biacore 1K interaction system, a standard amine coupling method was employed. The carboxyl groups on the CM5 sensor chip surface were activated using an EDC / NHS mixture, and high-purity p53p... △F19 -N-MdmX protein was diluted in 10 mM sodium acetate buffer (pH 5.0) and flowed through experimental channel 2 at a flow rate of 20 μL / min, with the coupling amount controlled at approximately 6000-8000 response units (RU) to circumvent the mass transfer effect. Residual active sites on the channel surface were then blocked by flow of 1 M ethanolamine hydrochloride (pH 8.5). Control channel 1 (Reference channel) underwent only activation and blocking treatment, without protein coupling.

[0048] The purchased eltrombopag was serially diluted with running buffer PBS (containing 1% DMSO to increase compound solubility) to prepare six concentration gradients (0, 1.5625, 3.125, 6.25, 12.5, 25 μM). These were sequentially injected into the chip surface at a flow rate of 30 μL / min, with association time set to 60–120 s and dissociation time set to 120–300 s. Rigorous DMSO solvent multi-concentration calibration curves were performed before and after the reaction to eliminate refractive index signal interference caused by bulk kinetic fluctuations.

[0049] Sensor images and fitted data as follows Figure 4 As shown, the binding curve exhibits a concentration-dependent stepwise waveform. A standard 1:1 Langmuir binding model was used for global kinetic fitting, with the eltrombopag binding rate constant ka = 4.04 × 10³ M. -1 s -1However, its dissociation rate constant kd decreases by an order of magnitude, to kd = 9.99 × 10⁻⁶. -3 s -1 Finally, its apparent equilibrium dissociation constant KD = 2.47 × 10⁻⁶ was obtained through the ratio (KD = kd / ka). −6 M (i.e., 2.47 μM). This constant performs excellently for non-peptide small molecule ligands; the concentration curves in the sensing plot show a clear binding-dissociation trend, with no superstoichiometric binding or non-specific adsorption observed. The KD value is significantly higher than that of the positive control Nutlin-3a (2.23 × 10⁻⁶). -4 M) is increased by about 90 times, exhibiting typical kd-driven slow dissociation characteristics.

[0050] Example 3: Validation of the in vitro antitumor activity and mechanism of eltrombopag

[0051] The purpose of this embodiment is to evaluate the antitumor proliferative activity of eltrombopag at the cellular level and to preliminarily verify its mechanism of action at the protein level. The procedure consists of two parts: first, the CCK-8 assay was used to determine the 72-hour half-maximal inhibitory concentration (IC50) of eltrombopag against four tumor cell lines with different p53 genetic backgrounds, evaluating its proliferative inhibitory activity and p53 pathway dependence; second, MCF-7 cells were selected for concentration gradient administration, and Western blot was used to detect changes in the expression of key proteins such as p53, MdmX, p21, PUMA, and SLC7A11, verifying its multiple mechanisms of action.

[0052] 1. Cell Culture

[0053] The following human tumor cell lines were used in this experiment: MCF-7 (human breast cancer cells), H1299 / H1299 R213X (p53 deletion / normal expression, human non-small cell lung cancer cells), HCT116 (human colon cancer cells). H1299 / H1299 R213X(p53-deficient / normal expression, human non-small cell lung cancer cells) were cultured in RPMI-1640 medium containing 10% FBS and 1% triple antibodies, while the remaining cells were cultured in DMEM medium containing 10% FBS and 1% triple antibodies (D-glucose 1000.0 mg / L, sodium pyruvate 110.0 mg / L, phenol red 15.0 mg / L; inorganic salts: sodium chloride 6400.0 mg / L, sodium bicarbonate 3700.0 mg / L, potassium chloride 400.0 mg / L, anhydrous calcium chloride 200.0 mg / L, sodium dihydrogen phosphate monohydrate 125.0 mg / L, anhydrous magnesium sulfate 97.67 mg / L, ferric nitrate nonahydrate 0.1 mg / L; 15 amino acids: L-glutamine 584.0 mg / L, L-lysine hydrochloride 146.0 mg / L, L-isoleucine 105.0 mg / L, L-leucine 105.0 mg / L). The following substances were present: L-tyrosine disodium dihydrate 104.0 mg / L, L-threonine 95.0 mg / L, L-valine 94.0 mg / L, L-arginine hydrochloride 84.0 mg / L, L-phenylalanine 66.0 mg / L, L-biscysteine ​​dihydrochloride 63.0 mg / L, L-histidine hydrochloride monohydrate 42.0 mg / L, L-serine 42.0 mg / L, glycine 30.0 mg / L, L-methionine 30.0 mg / L, L-tryptophan 16.0 mg / L; and eight B vitamins: i-inositol 7.2 mg / L, choline chloride 4.0 mg / L, D-calcium pantothenate 4.0 mg / L, folic acid 4.0 mg / L, nicotinamide 4.0 mg / L, pyridoxine hydrochloride 4.0 mg / L, thiamine hydrochloride 4.0 mg / L, and riboflavin 0.4 mg / L. Cells were cultured at a concentration of mg / L, with the medium changed every 2-3 days. All cells were placed in an incubator at 37°C, 5% CO2, and saturated humidity for static culture.

[0054] 2. CCK-8 cell proliferation inhibition experiment

[0055] Take cells in logarithmic growth phase at 1×10 4Seeds were planted at a density of 100 μL / well in 96-well plates, with 200 μL of PBS added to the outer wells to prevent evaporation. Incubation was carried out at 37°C and 5% CO2 for 24 h until complete adhesion. The old medium was discarded, and serum-free medium containing eltrombopag was added (concentration gradient: 0, 0.781, 1.563, 3.125, 6.25, 12.5, 25, 50 μM, final DMSO concentration <0.1%, 6 replicates per concentration). Experimental drug administration group, solvent control group (negative control containing an equal amount of DMSO), and cell-free blank group were included. After 72 h of continuous drug administration, 10 μL of CCK-8 reagent was added in the dark, and incubation was carried out at 37°C for 1-2 h. Absorbance was measured at 450 nm using a microplate reader. Inhibition rate = [(OD...] 对照 -OD 给药 ) / (OD 对照 -OD 空白 []×100%. IC50 for each cell line over 72 h. 50 The values ​​are shown in Table 1.

[0056] Table 1. 72-h IC50 of eltrombopag in different cell lines 50 value

[0057]

[0058] Experimental results are as follows Figure 5 As shown, eltrombopag has an IC50 effect on three types of p53 wild-type or p53-normal-expressing tumor cells. 50 Both were in the low micromolar range of 1.94-3.79 μM and showed a dose-dependent effect. H1299 and H1299 R213X IC between 50 The ratio was approximately 1.2, indicating that eltrombopag retained some inhibitory activity in p53-deficient cells, suggesting that it may have a p53-independent helper mechanism.

[0059] 3. Western blot analysis of the MdmX-p53 and SLC7A11 axis mechanisms.

[0060] MCF-7 cells were selected at a ratio of 3 × 10⁻⁶. 5 Inoculate 1 cell per well into a 6-well plate and when the cells adhere to the wall to a density of approximately 90%, add eltrombopag at different concentrations (0, 0.1, 0.5, 1, 5, 10, 20 μM) for 24 h.

[0061] Discard the culture medium, wash twice with cold PBS, add RIPA high-efficiency lysis buffer containing 0.1% PMSF and protein phosphatase inhibitor (approximately 60-100 μL / well), and lyse on ice for 30 min (shaking once every 10 min). Collect the lysis buffer with a cell scraper, centrifuge at 12000 rpm for 15 min at 4°C, and collect the supernatant.

[0062] BCA kit for measuring OD 562 Quantitative protein concentration (BSA standard curve y=0.001415x+0.1521, R) 2 =0.9956), after adjusting each group to equal protein levels, add 5×SDS-PAGE loading buffer (1:4 dilution), and boil in a 95℃ metal bath for 10 min for denaturation.

[0063] Equal amounts of protein (10 μg / well) were loaded onto a 12% SDS-PAGE separating gel (5% stacking gel). Electrophoresis was performed at 80 V until bromophenol blue entered the separating gel, then the V was switched to 120 V until the dye reached the bottom of the gel. Wet transfer was performed at a constant current of 200 mA for 120 min to a 0.45 μm PVDF membrane (pre-activated with methanol). Blocking was performed at room temperature for 1 h with 5% skim milk (prepared with TBST).

[0064] The membrane was incubated overnight at 4°C with primary antibodies (antibody dilution ratios: rabbit anti-p53 polyclonal antibody 1:5000, rabbit anti-MdmX polyclonal antibody 1:6000, rabbit anti-p21 polyclonal antibody 1:1000, rabbit anti-PUMA monoclonal antibody 1:2000, rabbit anti-SLC7A11 polyclonal antibody 1:2000, rabbit anti-β-actin monoclonal antibody 1:5000). The membrane was washed with TBST 3 × 10⁻⁶ min, incubated with HRP-goat anti-rabbit IgG secondary antibody (1:10000) at room temperature for 1 h, and washed again with TBST 3 × 10⁻⁶ min. The membrane surface was covered with ECL chemiluminescent buffer (solution A:solution B = 1:1), and signals were acquired using a CHEMIDOC XRS stain-free electrophoresis imaging system.

[0065] ImageJ grayscale extraction and analysis, such as Figure 6 As shown, the internal control bands were uniform. With increasing eltrombopag concentration, significant p53 accumulation was observed at low concentrations of 0.5-1 μM, peaking at 10-20 μM, confirming the effectiveness of MdmX-p53 blockade. Downstream functional executive proteins p21 and PUMA showed consistent stepwise upregulation, indicating that classical p53 transcriptional activity was fully restored, simultaneously initiating cell cycle arrest and apoptosis. With increasing eltrombopag concentration, MdmX protein was significantly downregulated in a dose-dependent manner. Within the medium-to-high dosage range of 5-20 μM, the intracellular direct target MdmX protein band underwent ubiquitination and degradation. This degradation blocked the continuous source of p53 inhibitory signals, resulting in a positively amplified antitumor effect. Simultaneously, the core negative regulator of ferroptosis, SLC7A11, was significantly inhibited with increasing eltrombopag concentration, reaching near-complete inhibition at 20 μM. The above data indicate that eltrombopag can simultaneously activate the p53 apoptosis pathway and weaken ferroptosis defense in MCF-7 cells at a low micromolar concentration.

[0066] Example 4: Eltrombopag-p53p △F19 100 ns all-atom molecular dynamics simulation of the -N-MdmX complex

[0067] The purpose of this embodiment is to quantitatively analyze the binding stability and structural characteristics of eltrombopag and the MdmX remodeling pocket at the atomic level under dynamic conditions close to physiological environments. Operationally, a ternary complex all-atom simulation system was constructed using Gromacs software. After energy minimization, NVT isothermal equilibration, and NPT isobaric equilibration, a 100 ns production simulation was performed. The overall stability, residue-level flexible distribution, and interaction characteristics of the complex were systematically evaluated using trajectory analysis indicators such as RMSD, radius of gyration, RMSF, secondary structure evolution, and the number of hydrogen bonds, revealing the unique N-terminal locking effect of eltrombopag.

[0068] A 100 ns all-atom molecular dynamics (MD) simulation was performed using Gromacs 2020.6 software. The production system used AMBER ff14SB force field parameterization for the protein backbone, the GAFF universal model to describe the eltrombopag ligand, and the atomic charges were assigned using the standard RESP method with two-dimensional constraint fitting.

[0069] The initial docking-preferred conformation was placed in a three-dimensional cubic TIP3P explicit solvent chamber, with a minimum water layer boundary spacing of 1.0 nm, and Na was added. + and Cl - Excess charge in the system was neutralized with ions and adjusted to a physiological ionic strength of 0.15 M. A three-dimensional periodic boundary condition (PBC) was introduced. The entire system contains 1532 protein atoms (the acceptor is p53). 13-18 -N-MdmX 23-109 The fusion protein contains 93 amino acids, and the ligand eltrombopag has 38 atoms, for a total of 1570 atoms in the protein and ligand.

[0070] Perform a two-stage energy minimization (steepest descent method + conjugate gradient method), converging until the maximum force is less than 1000 kJ·mol⁻¹. -1 ·nm -1 Subsequently, under positional constraints, thermodynamic fine-tuning equilibrium was alternately performed under the NVT ensemble (V-rescale thermostat, 100-300 K, 300 ps linear temperature control) and the NPT ensemble (Parrinello-Rahman pressure coupler, 1 bar, 1 ns pressure density equilibrium). Finally, the positional constraints of all spatial atoms and heavy atoms were released, and an isothermal and isobaric production stage simulation lasting 100 ns was performed with an integration step size of 2 fs. The trajectory was automatically output as one frame every 1 ps, and a total of 100,001 frames of continuous topology were processed.

[0071] Trajectory analysis results as follows Figure 7 As shown. According to Figure 7 The average RMSF of the 93 Cα residues in the whole protein was only 0.0743 nm. The RMSF of the N-terminal region (residues 13-18) was compressed to approximately 0.10 nm, with residue 13 (TRP) decreasing to 0.0985 nm and residue 14 (LYS) decreasing to 0.0986 nm. This ability to directionally immobilize N-terminal flexibility is a significant kinetic characteristic that distinguishes eltrombopag from other candidate molecules. The only moderately flexible region is loop 64-72 (residue 68 GLN RMSF = 0.2448 nm), located far from the ligand binding site. Figure 7 b shows an average Rg of 1.2574 nm during the production period, with a fluctuation range of 1.221–1.293 nm (only 0.072 nm), and a standard deviation of approximately 0.01 nm. The extremely low fluctuation in Rg indicates a significant locking effect on the overall protein conformation. RMSD analysis results (as shown...) Figure 7 c) The eltrombopag complex reached a stable plateau after approximately 5 ns. The RMSD during the 0-5 ns equilibrium period was 0.1694 nm; the RMSD during the 5-100 ns production period was 0.1955 nm, with a maximum of only 0.2662 nm. The RMSD remained in a very narrow and nearly flat range of 0.15-0.22 nm from 5 ns onwards, without any dissociation or long-range structural unfolding.

[0072] Secondary structure (DSSP) time evolution heatmap (e.g.) Figure 7 d) indicates that the α-helix has a persistence of 86-94%, and the β-fold has a persistence of 73-83%. Eltrombopag does not achieve stability by rigidly locking all secondary structures, but rather allows local secondary structure units to dynamically exchange between helices / folds and coils / loops to absorb thermal energy without conducting it to the global framework.

[0073] The average number of protein-ligand hydrogen bonds over the entire 100 ns trajectory was 0.51 per frame (truncation criteria: donor-acceptor distance ≤ 0.35 nm, hydrogen-donor-acceptor angle ≤ 30°), and the total number of hydrogen bonds (including intraprotein hydrogen bonds and protein-solvent hydrogen bonds) was 66.1 per frame. Figure 7 e).

Claims

1. Application of eltrombopag in the preparation of MdmX protein inhibitors.

2. The application according to claim 1, characterized in that, The MdmX protein inhibitor is used to prepare anti-tumor drugs.

3. The application according to claim 1, characterized in that, The eltrombopag binds to the F19 hydrophobic subpocket of the MdmX protein in a molecular wedge manner, along with p53p. △F19 Peptides synergistically enhance the thermodynamic stability of ternary complexes.

4. The application according to claim 2, characterized in that, The tumor is a p53 wild-type tumor.

5. The application according to claim 4, characterized in that, The tumor is selected from at least one of colon cancer, breast cancer, and non-small cell lung cancer.

6. The application according to claim 2, characterized in that, The drug is used in combination with an Mdm2 protein inhibitor.

7. Application of eltrombopag in the preparation of ferroptosis inducers or ferroptosis sensitizers.

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