Use of GCN2-IN-6 in preparation of MdmX protein inhibitor and antitumor drugs

Abstract

The application discloses a use of GCN2-IN-6 in preparation of MdmX protein inhibitors and antitumor drugs, and belongs to the technical field of biological medicines. The application finds that GCN2-IN-6 is a direct targeting small-molecule inhibitor of MdmX protein, can competitively bind to an MdmX N-terminal p53 binding pocket to displace a p53 polypeptide, destroy MdmX-p53 complex homeostasis, up-regulate intracellular p53, p21 and PUMA, and activate a tumor cell apoptosis pathway; in addition, the drug can inhibit an iron death defense pathway by down-regulating SLC7A11, and synergistically enhance cancer cell inhibition effect. GCN2-IN-6 regulates eIF2alpha phosphorylation in integrated stress response (ISR) by inhibiting GCN2 kinase activity. The application finds a use of GCN2-IN-6 in three kinds of MdmX overexpression type cancers, and provides a novel drug intervention strategy for MdmX overexpression related tumors.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a new pharmaceutical use of the known bioactive compound GCN2-IN-6 (CAS No.: 2183470-09-7, screening number MCE-C06), namely, GCN2-IN-6 as a small molecule inhibitor that directly targets the MdmX (Murine DoubleMinute X, also known as MDM4, HDMX) protein and its use in the preparation of antitumor drugs. Background Technology

[0002] The tumor suppressor protein p53 maintains cellular genome stability by coordinating the transcriptional processes of downstream effector genes such as p21, PUMA, and SLC7A11. In approximately 50% of malignant tumors that retain the wild-type TP53 gene, the tumor suppressor function of p53 is functionally silenced by the overexpression of the negative feedback regulators Mdm2 and MdmX. MdmX, through its N-terminal p53-binding domain, occupies the p53 transcriptional activation surface with a higher affinity than Mdm2, effectively blocking the trans-transcriptional activation of p53.

[0003] Clinical trials of Mdm2 single-target inhibitors (such as Roche's Idasanutlin) have revealed that Mdm2 inhibition leads to p53 accumulation, which in turn transactivates MdmX transcription, promoting compensatory upregulation of MdmX. The sustained inhibition of p53 activity by MdmX and this compensatory upregulation of MdmX is a key bottleneck limiting the clinical efficacy of Mdm2 inhibitors. Therefore, directly targeting MdmX is not only a core strategy for overcoming Mdm2 inhibitor resistance but also an independent therapeutic pathway for MdmX-overexpressing tumors.

[0004] However, the p53 binding interface of MdmX is shallower and flatter than that of Mdm2. Tyr99 at the entrance is rigidly locked into a closed conformation by neighboring residues, lacking ligand-induced dynamic plasticity. This makes it difficult for Nutlin-like molecules designed using the deep hydrophobic cracks of Mdm2 as templates to be effectively adapted to MdmX.

[0005] GCN2-IN-6 (CAS No.: 2183470-09-7) is a known GCN2 (general control nonderepressible 2, i.e., eIF2AK4) kinase inhibitor. Literature reports that it regulates eIF2α phosphorylation in the integrated stress response (ISR) by inhibiting GCN2 kinase activity. GCN2 is a key serine / threonine kinase in the integrated stress response (ISR), sensing amino acid deficiencies and phosphorylating eIF2α to regulate protein translation. In all published literature and patents, GCN2-IN-6 has never been reported to possess MdmX protein-binding activity, antitumor activity, or function in regulating the ferroptosis pathway.

[0006] Current technologies lack a class of small molecule inhibitors that do not require p53 peptide synergy, can directly target MdmX, simultaneously synergistically activate apoptosis and ferroptosis pathways, and can avoid compensatory degradation of MdmX. There is an urgent clinical need to develop novel MdmX-targeting drugs. Summary of the Invention

[0007] The technical problem to be solved by this invention is to provide a small molecule compound that can directly target the MdmX protein and exert its inhibitory effect without relying on p53-derived peptides, so as to overcome the shortcomings of the prior art and provide an effective targeted therapy for p53 wild-type tumors.

[0008] To overcome this structural bottleneck, we used an innovative strategy to delete the Phe19 residue and adjacent amino acid residues in the p53 peptide, exposing the deep F19 hydrophobic subpocket (DrugScore=0.66, volume 487.36 ų) on the MdmX surface that was originally hidden. We then used this remodeled F19 pocket as a target for drug screening.

[0009] Based on this screening strategy, MdmX inhibitor candidate compounds in this field can be divided into two completely different types according to their binding modes:

[0010] 1. Ternary interface cooperative stable type:

[0011] The molecule precisely fills the depths of the F19 hydrophobic subpocket, remodeled by residue loss, using a molecular wedge-like mechanism, requiring p53p. △F19 Mutant peptides coexist to form a ternary complex. When p53p... △F19 When mutant peptides simultaneously anchor in adjacent subpockets, they occupy different regions of the receptor groove, and their steric hindrances do not exclude each other. Through interface stacking optimization and global conformational rigidity locking (such as the N-terminal locking effect), a strong thermodynamic synergistic effect is generated, exhibiting an extremely low off-target dissociation rate constant (k). d As low as 10 -3 s -1 (Level), forming a stable, high-affinity ternary complex. This class of compounds releases transcriptionally active free p53 protein by blocking the protein-protein interaction of MdmX-p53, thereby transactivating the transcription and expression of downstream Mdm2. The significantly upregulated Mdm2 acts as an E3 ubiquitin ligase to directionally mediate K48-linked polyubiquitination of MdmX and direct it to the proteasome, ultimately inducing a phenotypic characteristic of complete degradation and clearance of MdmX protein within the cell.

[0012] 2. Direct competition for destabilizing compounds

[0013] In systems without the synergistic effect of p53-derived peptides, it can be directly embedded into the N-terminal p53-binding domain of the MdmX protein, and through specific steric hindrance, it can forcefully squeeze out or replace the p53 peptides originally bound to the MdmX protein interface. △F19 The peptide fragments, with their core rigid scaffolds such as quinazoline and indole, forcefully expand the naturally closed network of residues (e.g., Tyr99) within the MdmX receptor protein through a strong inducible fit effect, inducing a significant negative denaturing thermal shift (destabilization phenotype) in the receptor protein thermodynamically. This binding mode forms a small molecule drug-protein inactivation complex that completely blocks the interaction between MdmX and p53 protein in physical space. Simultaneously, due to its specific inactivation conformation, it effectively protects the stably bound MdmX from endogenous ubiquitination and clearance, leading to specific loss of function of the target protein intracellularly, and subsequent accumulation. Such small molecule candidate compounds can reactivate the anti-tumor function of p53 and downregulate the expression of the ferroptosis core protein SLC7A11 without peptide synergy.

[0014] Based on the aforementioned F19 remodeling pocket screening strategy, this invention is the first to discover that GCN2-IN-6 is one of the very few small molecule ligands that can directly bind to the MdmX protein. GCN2-IN-6 is also the only ligand that simultaneously satisfies the requirement of not requiring p53p. △F19 Compounds that can independently stabilize N-MdmX protein with peptide synergy and whose N-terminal peptide RMSF is significantly increased (ortho-competitive substitution signal) as shown by MD simulation are classified as competitive substitution-type MdmX inhibitors, giving this known compound a novel MdmX-targeting pharmacological function.

[0015] After GCN2-IN-6 binds to MdmX, it forms a unique pharmacological phenotype of stable accumulation of MdmX protein. Unlike classical inhibitors that induce MdmX ubiquitination and degradation, GCN2-IN-6 protects MdmX from Mdm2-mediated ubiquitination and degradation. At the same time, after GCN2-IN-6 blocks the binding of MdmX-p53, it releases and activates p53. The activated p53 promotes MdmX gene transcription through the classical feedback regulatory network. Under the condition of inhibited protein degradation, it ultimately manifests as significant accumulation of MdmX protein.

[0016] GCN2-IN-6 exerts its antitumor effects through different pharmacological mechanisms: (a) it competitively binds to MdmX, blocking the MdmX-p53 protein-protein interaction, releasing p53 transcriptional activity, thereby promoting the expression of downstream p53 factors p21 and PUMA proteins, and simultaneously initiating cell cycle arrest and mitochondrial apoptosis programs; (b) it downregulates SLC7A11 expression through the transcriptional repression function of p53, weakening tumor cell ferroptosis defense, and achieving near-complete inhibition of SLC7A11 at 20 μM. This achieves dual synergistic activation of the classical p53 apoptosis pathway and the non-classical ferroptosis pathway.

[0017] All-atom molecular dynamics (100 ns) simulations further revealed that GCN2-IN-6 exhibits an elevated N-terminal RMSF. Specifically, the R14 and R15 RMSF of GCN2-IN-6 were increased by 48% and 84% respectively compared to the ligandless receptor protein, indicating that the tethered WKLLRE peptide was squeezed out of the N-MdmX pocket and allowed to swing freely. GCN2-IN-6 occupies this binding site and effectively stabilizes the β-sandwich fold of the N-MdmX domain (all four β-sheets are fully restored, with 96-98% persistence).

[0018] This invention provides the use of GCN2-IN-6 (CAS No.: 2183470-09-7) in the preparation of MdmX protein inhibitors, wherein the inhibitor directly binds to the N-terminal p53 binding pocket of the MdmX protein, competitively displacing the p53 protein bound to MdmX, thereby blocking the MdmX-p53 protein-protein interaction.

[0019] The inhibitor is used to prepare antitumor drugs.

[0020] The tumor is a p53 wild-type tumor.

[0021] The tumor is selected from at least one of colon cancer, breast cancer, or non-small cell lung cancer.

[0022] The inhibitor releases p53, which downregulates SLC7A11 protein expression via p53 transcriptional repression, thereby inhibiting SLC7A11-mediated ferroptosis defense, and can be used to prepare ferroptosis inducers or ferroptosis sensitizers.

[0023] The ferroptosis inducer or the ferroptosis sensitizer is used to prepare antitumor drugs.

[0024] Beneficial effects:

[0025] (1) The present invention discovered that GCN2-IN-6 (CAS: 2183470-09-7) can directly bind to MdmX protein and block MdmX-p53 interaction, giving this known GCN2 kinase inhibitor a novel MdmX-targeting pharmacological function.

[0026] (2) GCN2-IN-6 exhibits potent dose-dependent inhibitory activity against p53 wild-type tumor cells: H1299 R213X Cellular IC 50 =2.01 μM, IC50 in HCT116 cells 50 =4.11 μM, IC50 in MCF-7 cells 50 =4.95 μM; while p53-deficient H1299 cells IC50 50=10.93 μM, p53-dependent selectivity fold greater than 5-fold.

[0027] (3) GCN2-IN-6 achieves dual synergistic activation of the p53 apoptosis pathway and the ferroptosis pathway: on the one hand, it induces p53 accumulation and upregulates p21 and PUMA expression, and on the other hand, it significantly inhibits SLC7A11 (System Xc⁻ core subunit), almost completely inhibiting SLC7A11 protein expression at a concentration of 20 μM.

[0028] (4) All-atom MD simulation (100 ns) confirmed that GCN2-IN-6 and the N-terminal domain of MdmX form a highly conserved stable complex: the average RMSD during the production period (5-100 ns) is 0.2824 nm, the maximum RMSD is only 0.4209 nm, the α-helix persistence is 97-99%, the β-sheet persistence is 96-98%, and the secondary structure band in the DSSP heatmap is almost continuous throughout the 100 ns period. Attached Figure Description

[0029] Figure 1 The chemical structural formula of GCN2-IN-6.

[0030] Figure 2 GCN2-IN-6 and p53p ΔF19 Differential scanning fluorescence (DSF) thermal drift results of the -N-MdmX fusion protein.

[0031] Figure 3 Figure: Competitive DSF validation results of GCN2-IN-6, N-MdmX protein, and mutant peptide.

[0032] Figure 4 GCN2-IN-6 and p53p ΔF19 Multi-concentration kinetic binding sensing map of the -N-MdmX fusion protein with surface plasmon resonance (SPR).

[0033] Figure 5 GCN2-IN-6 in four cell lines with different p53 genetic backgrounds (H1299) R213X CCK-8 proliferation inhibition curves of H1299, MCF-7 and HCT116; among which Figure 5 (a) GCN2-IN-6 for H1299 R213X Cell viability inhibition curve of cells (G418-induced normal p53 expression); Figure 5 (b) shows the cell viability inhibition curve of GCN2-IN-6 on H1299 cells (p53-deficient type); Figure 5 (c) shows the cell viability inhibition curve of GCN2-IN-6 on MCF-7 cells (p53 wild type); Figure 5 (d) shows the cell viability inhibition curve of GCN2-IN-6 on HCT116 cells (p53 wild type).

[0034] Figure 6 Western blot analysis of p53, MdmX, p21, PUMA and SLC7A11 proteins after treatment of MCF-7 cells with concentration gradient GCN2-IN-6 (0-20 μM, 24 h).

[0035] Figure 7 GCN2-IN-6 and the fusion protein p53p △F19 100 ns all-atom molecular dynamics simulation of the root mean square (RMSF) distribution of residues in the -N-MdmX ternary complex.

[0036] Figure 8 GCN2-IN-6 and the fusion protein p53p △F19 The time evolution curve of the radius of gyration (Rg) of the -N-MdmX ternary complex in a 100 ns all-atom molecular dynamics simulation.

[0037] Figure 9 GCN2-IN-6 / p53p △F19 The 100 ns all-atom molecular dynamics simulation of the Cα atom root mean square deviation (RMSD) time evolution curve of the -N-MdmX ternary complex.

[0038] Figure 10 GCN2-IN-6 / p53p △F19 100 ns all-atom molecular dynamics simulation of protein secondary structure time evolution (DSSP) heatmap of the -N-MdmX ternary complex.

[0039] Figure 11 GCN2-IN-6 / p53p △F19 -N-MdmX ternary complex 100 ns whole atom molecular dynamics simulation of the time evolution of the number of protein-ligand hydrogen bonds. Detailed Implementation

[0040] 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.

[0041] To fully verify the technical effects of this invention's GCN2-IN-6 targeting MdmX protein, inhibiting tumor proliferation, and regulating apoptosis and ferroptosis pathways, this section presents four progressive experiments: Example 1 describes a high-throughput virtual screening process based on the F19 remodeled pocket, clarifying the screening source and initial screening advantages of GCN2-IN-6; Example 2 uses three in vitro biochemical methods—DSF thermal drift, peptide competition assay, and SPR kinetics assay—to visually verify that GCN2-IN-6 can independently bind to the MdmX N-terminal p53 binding pocket, replace p53-derived peptides, and induce protein destabilization; Example 3 uses CCK-8 cell viability assays and Western blot to evaluate the inhibitory activity of the compound on the proliferation of tumor cells with different p53 genotypes, and to clarify its cellular-level role in regulating p53 apoptosis and the SLC7A11 ferroptosis defense pathway; Example 4 uses all-atom molecular dynamics simulations to analyze the binding stability and N-terminal peptide replacement characteristics of the GCN2-IN-6-MdmX complex from the perspective of molecular dynamic conformation.

[0042] The experimental procedures, reagent parameters, and detection methods in the following embodiments are only preferred verification schemes of the present invention and are not intended to limit the scope of protection of the present invention. Those skilled in the art can equivalently replace and adjust the experimental conditions according to conventional experimental methods to achieve the same verification purpose.

[0043] Example 1: Discovery of GCN2-IN-6, based on p53p △F19 -N-MdmX Reshapes Pocket High-Throughput Virtual Screening

[0044] Existing small molecule screening methods targeting MdmX often employ natural closed pocket structures, making it difficult to screen for competitive destabilizers that do not require peptide synergy. This embodiment modifies the p53 binding interface to construct an F19-remodeled hydrophobic sub-pocket screening model. A library of tens of thousands of compounds was built to conduct virtual docking and multi-level screening validation. Ultimately, the target compound GCN2-IN-6 was obtained. The entire process of target structure modification, molecular library preprocessing, docking screening, and re-screening was fully documented, clarifying the screening criteria for the molecule's in vitro binding potential.

[0045] MdmX protein surface p53p △F19The interface was used as the target (PDB ID: 3DAB, resolution 1.9 Å). The PyMOL Mutagenesis tool was used to delete the Phe19 residue of the p53 peptide and adjacent amino acid residues, exposing the F19 hydrophobic subpocket (DoGSiteScorer assessment: DrugScore=0.66, volume 487.36 ų, surface area 769.1 Ų). The small molecule compound library comprised 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 was performed using AutoDock Vina (Grid Box 16.123×16.709×18.112 ų, exhaustiveness=16). After a rigorous review process including Vina binding energy ≤ -7.0 kcal / mol, visual conformation verification via Discovery Studio, cross-validation using LigandScout / PyMOL dual 3D pharmacophores, and final review for commercial viability, 113 candidate molecules were selected. Among them, GCN2-IN-6 was chosen as a key research focus due to its Vina binding free energy of -8.6 kcal / mol, excellent docking conformation, existing oral drug status, mature clinical safety data, and ability to support rapid translation. (GCN2-IN-6 structural formula shown below) Figure 1 (As shown) It was obtained from the MCE Commercial Drug Library (MCE-C06) and had an HPLC purity of ≥98%.

[0046] Example 2: In vitro biochemical experiments to verify the targeted binding of GCN2-IN-6 and N-MdmX

[0047] After initially identifying GCN2-IN-6 as a candidate molecule through virtual screening in Example 1, this example employs multi-level in vitro biochemical experiments to verify targeted binding: Differential scanning fluorescence (DSF) was used to detect changes in protein thermostability after the compound bound to MdmX, visually determining its protein destabilizing effect; a mutant peptide competitive DSF experiment was used to verify the binding competition relationship between the compound and the p53 peptide, clarifying the normal binding mode; and surface plasmon resonance (SPR) was used to quantitatively determine the binding kinetic parameters of the two, quantifying affinity and binding / dissociation characteristics. These three sets of experiments corroborate each other, comprehensively demonstrating from thermodynamic, competitive binding, and molecular dynamics perspectives that GCN2-IN-6 can independently and specifically bind to the N-terminal p53 binding pocket of the MdmX protein, forming a small molecule-protein inactivation complex without the need for p53-derived peptide synergy.

[0048] 1. Differential scanning fluorescence (DSF):

[0049] The thermal unfolding of proteins was monitored using SYPRO Orange hydrophobic dye. A total reaction volume of 25 μL was constructed in a micro-volume 96-well PCR plate, with three replicates. The 25 μL reaction volume contained 100 μM p53p △F19 -N-MdmX, 100 μM small molecule GCN2-IN-6 (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 (T0) by calculating the first negative derivative of temperature (-dRFU / dT) from the collected fluorescence intensity values. m ).

[0050] The results are as follows Figure 2 As shown, after the addition of GCN2-IN-6, the apparent transition temperature of the target protein exhibited a strong negative thermodynamic shift compared to the negative control (solvent DMSO group). The measured ΔT of GCN2-IN-6 was... m =-2.09±0.91℃, belonging to the category of negative destabilizers, higher than |ΔT m The significance threshold of 1.5℃ confirms its role as a highly efficient structural destabilizer from an independent thermodynamic steady-state level.

[0051] 2. Validation experiment of DSF compensatory addition of mutant peptides:

[0052] To demonstrate whether GCN2-IN-6 competitively occupies the N-terminal p53 binding domain of the MdmX protein, a mutant peptide p53p was artificially synthesized in a solid phase, in which the key Phe19 amino acid and adjacent amino acid residues were removed. △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 GCN2-IN-6 in a ternary complex, respectively. The Tg values ​​were measured under a completely consistent DSF gradient temperature-controlled denaturation program. m First derivative characteristic spectrum.

[0053] The results are as follows Figure 3 As shown. Figure 3The results clearly show that in the ternary complex system where the small molecule and the mutant peptide coexist, the peak temperature of the thermal denaturation transition is significantly lower than that of the binary state where only the peptide is bound. This thermodynamic destabilization phenomenon confirms that GCN2-IN-6 acts as a direct competitive inhibitor. This small molecule can embed into the N-terminal p53 binding domain of the MdmX protein without the need for exogenous peptide synergy. Through strong steric hindrance and induced fit effects, it dismantles the original hydrophobic interfacial network, repelling and displacing the p53 binding domain originally bound to N-MdmX. △F19 Peptide fragments. Acts as a direct-binding MdmX inhibitor.

[0054] 3. Surface plasmon resonance (SPR):

[0055] 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 avoid the mass transfer effect. Residual active sites on the channel surface were then blocked by flow through 1 M ethanolamine hydrochloride (pH 8.5). The control channel 1 (Reference channel) underwent only activation and blocking treatment, without protein coupling. Purchased GCN2-IN-6 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). The solution was sequentially injected into the chip surface at a flow rate of 30 μL / min, with the association time set to 60-120 s and the dissociation time set to 120-300 s. Rigorous multi-concentration calibration curves for DMSO solvent were applied before and after the process to eliminate refractive index signal interference caused by bulk kinetic fluctuations.

[0056] 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 GCN2-IN-6 binding rate constant k... a =1.35×10 3 M -1 s -1 Its dissociation rate constant k d It exhibits highly dynamic exchange characteristics, kd =4.25×10 -1 s -1 Finally, through the ratio (K) D =k d / k a The apparent equilibrium dissociation constant K is obtained. D =3.14×10 -4 M (i.e., 314 μM). The concentration curves in the sensor plots showed a clear binding-dissociation trend, with no superstoichiometric binding or non-specific adsorption observed. This apparent affinity value is similar to that of the positive control inhibitor Nutlin-3a (2.23 × 10⁻⁶). -4 M) is on the same order of magnitude, and the increase in k d The value response exhibits a dynamic displacement-type fast dissociation characteristic, reflecting the dynamic process of small molecules entering the F19 pocket and implementing steric repulsion.

[0057] Example 3: Preliminary study on the in vitro antitumor activity and mechanism of GCN2-IN-6

[0058] The purpose of this embodiment is to evaluate the antitumor proliferative activity of GCN2-IN-6 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 GCN2-IN-6 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 drug 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.

[0059] 1. Cell culture:

[0060] 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 in the following concentrations: 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-dicysteine ​​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: 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.

[0061] 2. CCK-8 cell proliferation inhibition experiment:

[0062] 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 GCN2-IN-6 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 = [(OD200...]2 ... 对照 -OD 给药 ) / (OD 对照 -OD 空白 )]×100%.

[0063] After cell drug administration and incubation and absorbance measurement, the cell proliferation inhibition rate was calculated based on the OD values ​​of each group. GraphPad Prism software was used for nonlinear regression fitting to calculate the half-maximal inhibitory concentration (IC50) of GCN2-IN-6 after 72 h of treatment in different cell lines. 50 The results are summarized in Table 1.

[0064] Table 1. 72 h IC50 of GCN2-IN-6 in different cell lines 50 value

[0065]

[0066] Experimental results are as follows Figure 5 As shown, GCN2-IN-6 exhibits potent low-molecular-weight killing activity against various tumor cell lines that retain wild-type p53, and shows high IC50 activity against human breast cancer MCF-7 cells. 50 The IC50 concentration was 4.95 μM, and its effect on human colorectal cancer HCT116 cells was [not specified]. 50 At 4.11 μM, it was effective against G418-induced p53 protein-expressing lung cancer cells H1299. R213X IC 50 The concentration was 2.01 μM, while H1299 lung cancer cells with p53 gene deletion in the same background showed significant tolerance (10.93 μM). The IC50 between p53-expressing cells and p53-deficient cells was [not specified in the original text]. 50 The ratio was approximately 5.4-fold, confirming that the antiproliferative activity of GCN2-IN-6 is highly dependent on the targeted activation of the p53 pathway.

[0067] 3. Western blot verification of the axis mechanism of MdmX-p53 and SLC7A11:

[0068] MCF-7 cells were selected at a ratio of 3 × 10⁻⁶. 5 Cells were seeded per well in 6-well plates. When the cells reached approximately 90% confluence, different concentrations (0, 0.1, 0.5, 1, 5, 10, 20 μM) of GCN2-IN-6 were added for 24 h. The culture medium was discarded, and the cells were washed twice with cold PBS. RIPA lysis buffer (approximately 60-100 μL / well) containing 0.1% PMSF and a protein phosphatase inhibitor was added, and the cells were lysed on ice for 30 min (shaking every 10 min). The lysis buffer was collected using a cell scraper, centrifuged at 12000 rpm for 15 min at 4 °C, and the supernatant was collected. OD was measured using a BCA kit. 562 Quantitative protein concentration (BSA standard curve y=0.001415x+0.1521, R) 2 =0.9956), after adjusting each group to an equal protein volume, add 5× SDS-PAGE loading buffer (1:4 dilution), and denature by boiling in a 95℃ metal bath for 10 min. Load an equal protein volume (10 μg / well) of sample onto a 12% SDS-PAGE separating gel (5% stacking gel), and electrophores at 80 V until bromophenol blue enters the separating gel. Switch to 120 V and continue electrophoresis until the dye reaches the bottom of the gel. Wet transfer at a constant current of 200 mA for 120 min to a 0.45 μm PVDF membrane (pre-activated with methanol). Block with 5% skim milk (prepared with TBST) at room temperature for 1 h. The membrane was incubated overnight at 4°C with primary antibodies (antibody dilution ratio: 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 three times with TBST for 10 min each time. It was then incubated with HRP-goat anti-rabbit IgG secondary antibody (1:10000) at room temperature for 1 h. The membrane was washed again three times with TBST for 10 min each time. 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.

[0069] ImageJ grayscale extraction and analysis results are as follows: Figure 6As shown, the internal control bands were uniform. Significant p53 accumulation was observed at drug concentrations of 0.5–1 μM, peaking at 10–20 μM, confirming the effectiveness of MdmX-p53 blockade. p21 showed a smooth, stepwise increase, and PUMA increased significantly at 5–10 μM, indicating full restoration of classical p53 transcriptional activity. After treatment with GCN2-IN-6, intracellular MdmX target protein levels showed a significant increase in accumulation. The small molecule directly embedded itself into the N-port pocket of MdmX, extruding the p53 peptide and immobilizing it in a stable state of an inactivated complex that lost its p53-inhibiting function but was simultaneously protected from endogenous ubiquitination degradation. Simultaneously, the ferroptosis core negative regulator protein SLC7A11 in the non-classical metabolic pathway was deeply downregulated with increasing GCN2-IN-6 concentration; at a 20 μM intervention, the band of this ferroptosis defense core transport subunit almost completely disappeared. The above data indicate that GCN2-IN-6 can simultaneously revive the p53-mediated classical apoptosis pathway in MCF-7 cells at a low micromolar concentration, and achieve tumor suppression by thoroughly downregulating and clearing SLC7A11 to weaken the tumor ferroptosis defense barrier.

[0070] Example 4: GCN2-IN-6 and p53p △F19 100 ns all-atom molecular dynamics simulation of the -N-MdmX complex

[0071] To quantitatively verify the dynamic binding stability of GCN2-IN-6 to the reconstructed subpocket under dynamic conditions simulating a near-realistic human physiological solvent environment, 100 ns all-atom molecular dynamics (MD) simulations were performed using Gromacs 2020.6 software. The production system employed AMBER ff14SB force field parameterization of the protein backbone, the GAFF universal model to describe the GCN2-IN-6 ligand, and two-dimensional constraint fitting allocation of atomic charges using the standard RESP method. 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 GCN2-IN-6 contains 37 atoms, for a total of 1569 atoms. A two-stage energy minimization method (steepest descent method + conjugate gradient method) was performed, converging until the maximum force was less than 1000 kJ·mol⁻¹. -1 ·nm -1Subsequently, under positional constraints, thermodynamic fine-tuning equilibrium was performed alternately 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 heavy atoms were released, and an isothermal and isobaric production stage simulation was performed for 100 ns. The integration step size was 2 fs, and the trajectory was automatically output as one frame every 1 ps, processing a total of 100,001 frames of continuous topology.

[0072] The complete trajectory output from the 100 ns molecular dynamics simulation was quantitatively analyzed, and the corresponding results are shown in Figures 7-11.

[0073] Figure 7 The results showed that the average RMSF of the Cα atoms of the entire protein was 0.1018 nm. The RMSF of the four α-helix core regions (α1: 31-40, α2: 49-63, α3: 80-85, α4: 96-105) were all below 0.1 nm, exhibiting consistent rigidity. The N-terminal region (residues 13-26) showed the highest conformational flexibility, with residue 14 (LYS) having an RMSF of 0.4723 nm and residue 15 (LEU) having an RMSF of 0.4424 nm, exhibiting a typical dynamic pattern of rigid core and flexible end.

[0074] like Figure 8 The average Rg during the production period was 1.2862 nm, with a fluctuation range of 1.223–1.341 nm and a standard deviation of 0.0128 nm. The low fluctuation of Rg indicates that the binding of GCN2-IN-6 maintains the compactness of the overall protein conformation.

[0075] The GCN2-IN-6 complex reached a stable plateau after 5 ns, with a production RMSD of 0.2824 nm and a maximum RMSD of 0.4209 nm (e.g., Figure 9 The stable trend of the RMSD curve indicates that GCN2-IN-6 and the N-terminal domain of MdmX form a stable and specific complex.

[0076] Following GCN2-IN-6 binding, the RMSD of the fusion protein during its production phase decreased from 0.3568 nm in the APO state to 0.2824 nm (a decrease of 20.9%), indicating that small molecule ligand binding significantly enhanced the conformational kinetics rigidity of the fusion protein at 298 K. By comparing the changes in the RMSF of the N-terminal residues in the APO and ligand-bound states, the N-terminal RMSF of MdmX increased upon GCN2-IN-6 binding. The WKLLRE peptide in the fusion protein is located in the N-terminal region (residues 13-18). In the APO state, the peptide occupies the N-MdmX pocket through intramolecular binding, resulting in conformational constraint and a relatively low RMSF. If the ligand squeezes the peptide out of the pocket through orthomeric competition, the peptide is only tethered by the linker and is in a free-moving state, and its RMSF should increase significantly. Conversely, if the ligand stabilizes the fusion protein through an allosteric mechanism without displacing the peptide, the N-terminal RMSF should remain unchanged or decrease. After GCN2-IN-6 is combined, the RMSF of R14 and R15 increases by 48% and 84% respectively compared to APO.

[0077] DSSP secondary structure analysis shows (e.g.) Figure 10 The α-helix exhibits a persistence of 97-99%, and the β-sheet exhibits a persistence of 96-98%. In the DSSP heatmap, the secondary structure bands are nearly continuous throughout the 100 ns timescale. The high conservatism of the DSSP is consistent with the stationarity of the RMSD, confirming that the N-terminal domain of the MdmX maintains a stable topology after the GCN2-IN-6 binding.

[0078] The average number of protein-ligand hydrogen bonds over the entire 100 ns trajectory was 0.75 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 65.6 per frame (e.g., Figure 11 ).

Claims

1. The use of GCN2-IN-6 in the preparation of MdmX protein inhibitors, characterized in that, The inhibitor directly binds to the N-terminal p53 binding pocket of the MdmX protein, competitively displacing the p53 protein bound to MdmX, thereby blocking the MdmX-p53 protein-protein interaction.

2. The use according to claim 1, characterized in that, The inhibitor is used to prepare antitumor drugs.

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

4. The use according to claim 3, characterized in that, The tumor is selected from at least one of colon cancer, breast cancer, or non-small cell lung cancer.

5. The use according to any one of claims 1-4, characterized in that, The inhibitor releases p53, which downregulates SLC7A11 protein expression via p53 transcriptional repression, thereby inhibiting SLC7A11-mediated ferroptosis defense, and can be used to prepare ferroptosis inducers or ferroptosis sensitizers.

6. The use according to claim 5, characterized in that, The ferroptosis inducer or the ferroptosis sensitizer is used to prepare antitumor drugs.

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