A mdm2 / mdmx polypeptide analog dual inhibitor and uses thereof

Abstract

The application provides a kind of Mdm2 / MdmX polypeptide analogue double inhibitor and its application, the structure of Mdm2 / MdmX polypeptide analogue double inhibitor is polypeptide-link-small molecule, wherein the sequence of polypeptide is NH2-Asp-Leu-Glu-Asn-Leu-Tyr-Phe-Gln-COOH.The application provides Mdm2 / MdmX polypeptide analogue double inhibitor, and the crystal structure analysis of N-MdmX compound by nutlin-3a and peptide shows that the peptide plays the role of patch, and the ligand binding pocket on MdmX is reconstructed as deep cavity, and nutlin-3a is closely docked.The double inhibitor provided by the application shows the activity of double target point low nanomolar concentration, and the values of Ki (MdmX) and Ki (Mdm2) are 0.75nM and 0.27nM respectively.

Description

Technical Field

[0001] This invention relates to the field of biology, and more specifically, to dual inhibitors of Mdm2 / MdmX peptide analogs and their applications. Background Technology

[0002] Aberrant protein-protein interactions (PPIs) can lead to many human diseases and are important drug targets. The shallow pockets characteristic of PPI interfaces present a significant challenge for drug design. Current advanced drug design strategies utilize high-throughput screening of combinatorial chemistry libraries and virtual compound libraries to obtain effective hits, followed by rational drug design to enhance the specificity of these hits. However, rational drug design has inherent limitations because it is difficult to predict conformational changes in the ligand-binding pocket to accommodate high-affinity ligand structures based on the rigid structure of the drug targeting the protein. This application proposes, at least in principle, to overcome these limitations of current drug design by using peptide molecules as “patching” to reshape the ligand-binding pocket and confine weak compounds within it. The patch peptides will bind not only to the ligand-binding pocket but also to the captured ligands.

[0003] Overexpressed dual microsomes Mdm2 and MdmX are a pair of cell signaling regulatory molecules with highly homologous amino acid sequences and structures. They reduce cellular p53 activity by binding their N-terminal domains (N-Mdm2 and N-MdmX) to the p53 transactivation domain (p53p). The discovery of highly efficient small-molecule inhibitors of Mdm2 targeting N-Mdm2 is one of the most successful examples of converting peptide molecules into small molecules. The crystal structure of the MdmX complex with nutlin-3a (a strong Mdm2 inhibitor but a weak inhibitor of MdmX) shows that three key amino acids of nutlin-3a mimic the p53 peptide and highly match the ligand-binding pocket of MdmX. In this work aimed at developing a universal peptide repair strategy to powerfully enhance weak ligand-binding affinity, this application treats nutlin-3a as a pseudo-hit to design high-affinity dual inhibitors of Mdm2 and MdmX. Summary of the Invention

[0004] This application provides a dual inhibitor of Mdm2 / MdmX peptide analogs, wherein the structure of the dual inhibitor is peptide-link-small molecule, and the sequence of the peptide is NH2-Asp-Leu-Glu-Asn-Leu-Tyr-Phe-Gln-COOH.

[0005] In some embodiments, the link is -Gly-Gly-Gly-Gly-.

[0006] In some embodiments, the link is -(CH2). n -, n=2-8.

[0007] In some embodiments, the link is -(C2H4). n -, n=2-8.

[0008] In some embodiments, the small molecule is: .

[0009] This application also provides the application of the above-mentioned Mdm2 / MdmX peptide analog dual inhibitor in the preparation of drugs for treating cancer.

[0010] The dual inhibitors provided in this application exhibit effective anticancer activity, with a Ki value of 0.75 nM. Attached Figure Description

[0011] Figures 1a to 1c The repair peptide patch for remodeling N-MdmX ligand binding pockets is shown. Figure 1a List of high-affinity peptide patches identified using phage display peptide library technology. Figure 1b Compared with PMI, the FP method is used to determine the nutlin-3 titer N-MdmX experiment in the presence or absence of TevP. Figure 1c The FP method was used to determine the N-MdmX titration of TevP in the presence or absence of nutlin-3a. n=3 independent experiments, and data are expressed as mean ± SD.

[0012] Figures 2a to 2c The 15N-1H HSQC NMR spectra of N-MdmX titrated with TevP and nutlin-3a are shown. Figure 2a The image represents a cartoon model of the N-MdmX secondary structure based on the N-MdmX / nutlin-3a complex crystal structure (7C3C.pdb). Figure 2b The 15N-1H HSQC NMR spectra of TevP and nutlin-3a bound to N-MdmX were compared. N-MdmX to ligand ratios: black, 1:0; red, 1:1.5 (nutlin-3a); and green, 1:1.5 (TevP). Figure 2c The 15N-1H HSQC cross-titration spectra are for the binding of TevP and nutlin-3a with N-MdmX. Ratios of N-MdmX, TevP, and nutlin-3a: black, 1:0:0; orange, 1:2:1.5; and cyan, 1:1.5:2.

[0013] Figures 3a to 3bThe TevP enhancement mechanism of nutlin-3a combined with N-MdmX is shown. Figure 3a The crystal structure of N-MdmX complexed with nutlin-3a and TevP is shown in the figure. The three sub-binding sites of the three key residues in p53p are marked in the figure. Figure 3b The reconstructed ligand binding bag forms a deep-pore structure and docks with nutlin-3a.

[0014] Figure 4 The diagram illustrates how TevP enhances nutlin-3a affinity through a mechanism distinct from p53p and PMI. a) Amino acid sequences of p53p, PMI, and TevP. Key residues in p53p and PMI are shown in red. b) Structures of p53p, PMI, and nutlin-3a. Key residues are represented using a ball-and-stick model. c) The three pharmacophores in nutlin-3a mimic three key residues in p53p, but lack the proline at position 28 (P28) found in PMI. d) Stacking of N-MdmX structures in four different complexes: N-MdmX / p53p complex, N-MdmX / nutlin-3a complex, MdmX / nutlin-3a / TevP complex, and N-MdmX / PMI complex. e) Comparison of the structures of the nutlin-3a molecules in the N-MdmX / nutlin-3a / TevP and N-MdmX / nutlin-3a complexes. f) Compare the structures of the N-MdmX / nutlin-3a / TevP complex and the N-MdmX / p53p complex. g) Compare the structures of the N-MdmX / nutlin-3a / TevP complex and the N-MdmX / PMI complex. h) The PMI peptide introduces a fourth key residue (p28) that binds to N-MdmX.

[0015] Figures 5a to 5c The molecular structure and binding force of Tn3a are shown. Figure 5a () represents the molecular structure of Tn3a. Figure 5b This study describes the titration experiments of N-MdmX to determine Tn3a, PMI, p53p, and nutlin-3a in FP. n=3 independent experiments, and data are expressed as mean ± SD. Figure 5c This study was conducted to determine Tn3a, PMI, p53p, and nutlin-3a in the titration of N-Mdm2 using FP. n = 3 independent experiments, and data are expressed as mean ± SD.

[0016] Figure 6The synthesis scheme of Tn3a is shown, wherein i) (Boc)2O, NaOH, dioxane / water mixture; ii) NH3, methanol; iii) TFA-CH2Cl2; iii) TevP, PyBOP, Et3N, CH2Cl2; iv) TFA-CH2Cl2; v) piperidine-CH2Cl2.

[0017] Figure 7a The mass spectrum of compound 2 is shown. Figure 7b The HPLC chromatogram of compound 2 is shown.

[0018] Figure 8a The mass spectrum of compound 3 is shown. Figure 8b The HPLC chromatogram of compound 3 is shown.

[0019] Figure 9a The mass spectrum of compound 4 is shown. Figure 9b The HPLC chromatogram of compound 4 is shown.

[0020] Figure 10 The HPLC chromatogram of compound 5 is shown.

[0021] Figure 11 The HPLC chromatogram of compound 6 is shown.

[0022] Figure 12a The HPLC chromatogram of compound 7 is shown. Figure 12b The mass spectrum of compound 7 is shown.

[0023] Figures 13a to 13e Used to illustrate the biological activity of Tn3a. The viability of H1299p53 cells overexpressing Mdm2 and / or MdmX, assessed by MTT assay, was significantly affected by Tn3a compared to nutlin-3a (positive control) and nutlin-3b (negative control). Empty bar: nutlin-3a (positive control of Mdm2 inhibitor); gray bar: nutlin-3b; and dark bar: Tn3a. H1299p53+ / Mdm2+ / MdmX+ cells (d); H1299p53+ / Mdm2+ / MdmX- cells (e); H1299p53+ / Mdm2- / MdmX+ cells (f) and H1299p53+ / Mdm2- / MdmX- cells (g). n = 3 independent experiments. Data are expressed as mean ± SD. (h). Tn3a treatment of cancer cells does not induce p53 gene expression, but it specifically induces p21 gene expression. Solid and hollow symbols represent p21 and p53 transcription levels, respectively. Triangle: HCT116 cells; Circle: H640 cells; Square: A549 cells. n = 3 independent experiments, data are expressed as mean ± SD. Detailed Implementation

[0024] The following embodiments are intended to enable those skilled in the art to more fully understand the present invention, but do not limit the invention in any way.

[0025] One of the most detrimental protein-protein interactions is that overexpression of double microsome 2 (Mdm2) and its homologue MdmX impairs the activity of the tumor suppressor p53 in many cancers. The discovery of Mdm2 inhibitors has been very successful. Despite the high structural similarity between MdmX and Mdm2, Mdm2 inhibitors exhibit weak affinity for MdmX, providing a potential hit for dual Mdm2 / MdmX inhibitors. How to transform weak Mdm2 inhibitors into high-affinity Mdm2 / MdmX dual inhibitors marks a common challenge in modern drug discovery, starting with the design and optimization of drugs from hits. Here, this application provides a peptide repair strategy to reshape the ligand-binding pocket on MdmX to enhance the affinity of the hit via a peptide-hit-conjugate (PHC) strategy. This application identified a short peptide that enhanced the binding affinity of nutlin-3a from 17.8 M to 2.5 nM by panning a phage library displaying the peptide with the N-MdmX / nutlin-3a complex. Nutlin-3a can be considered a hit in the design of MdmX inhibitors. The crystal structure of N-MdmX conjugated with nutlin-3a and the peptide showed that the peptide acts as a patch, reconstructing the ligand-binding pocket on MdmX into a deep-cavity structure, which tightly docks with nutlin-3a. Furthermore, by constructing a PHC inhibitor of MdmX with nutlin-3a and the peptide conjugate, PHC exhibited more potent anticancer activity with a Ki value of 0.75 nM. These results demonstrate an extended peptide-drug conjugate strategy that can easily improve the binding affinity of hits through various high-throughput screenings, representing a novel strategy in existing drug design.

[0026] Identification of supplementary peptides using a peptide library displayed by bacteriophages: A phage display peptide library of 12 amino acid peptide chains was panned against the N-MdmX / nutlin-3a complex using a natural N-MdmX-binding peptide (p53p, Kd ~ 420 nM) as a competitive agent. When the p53 concentration increased to 1:0.5 (N-MdmX:p53p), several identical sequences appeared at high frequency, and these sequences became more enriched with further increases in p53p concentration. Figure 1aThe selection of phage sequences for the complex was summarized. The amino acid sequence S1D2L3E4N5L6Y7F8Q9G10S11H12 (PP1) appeared 8 times in 20 samples at a ratio of 1:0.5 (N-MdmX:p53p), and 10 times in 20 samples at a ratio of 1:1 (N-MdmX:p53p). Another sequence, V1E2L3E4N5I6Y7F8N9G10A11D12 (i.e., PP2), also appeared. At higher p53p concentrations (N-MdmX:p53p = 1:2), the frequencies of the above sequences were 12 and 8 times in 20 samples, respectively. Therefore, a common sequence can be deduced: X1X2L3E4X5X6Y7F8X9X10X11X12, where X1, X10, and X11 are small hydrophobic residues; X2 and X12 are negatively charged residues; X5 and X9 are N or Q; and X6 is a large hydrophobic residue. Using bioinformatics methods, this application queried a protein database using PP1 and found that the amino acid sequence of PP1 is highly homologous to the amino acid sequence of the Tev protease cleavage site SQDLENLYFQG. Therefore, this application names this peptide TevP. Existing peptide drug design theories cannot accurately predict this patch peptide and can only rely on high-throughput screening.

[0027] TevP peptides synergistically enhance the binding affinity of nutlin-3a to N-MdmX. Evaluation using fluorescence polarization assays revealed that TevP significantly enhanced the binding of nutlin-3a to N-MdmX. Figure 1b As shown, in the absence of TevP in the assay mixture, nutlin-3a exhibited a very weak affinity for the inhibitory effect of the fluorescent probe (FITC-p53p) on the binding of N-MdmX, with a Ki value of 17.8 µM (Table 1), consistent with previous reports, while the positive control PMI showed a strong binding affinity for N-MdmX, with a Ki value of 2.1 nM (Table 1). At a TevP to N-MdmX mixing ratio of 1:1.1, the titration of nutlin-3a significantly replaced FITC-p53p, indicating an enhanced affinity of nutlin-3a for N-MdmX, with a Ki value of 2.5 nM (Table 1), comparable to the affinity of PMI. Conversely, the presence of TevP did not affect the binding affinity of PMI for N-MdmX. Figure 1c Table 1 shows the synergistic binding effect of TevP and nutlin-3a on MdmX.

[0028] Table 1

[0029] This application further examined whether nutlin-3a affects the binding of TevP to N-MdmX. For example... Figure 1c As shown, TevP exhibits a weak affinity for N-MdmX, with a Ki value of 220.1 mM. When the molar mixing ratio of nutlin-3a to N-MdmX in solution was measured to be 1:1.2 (protein:small molecule), the presence of nutlin-3a enhanced the binding of TevP to N-MdmX, with a Ki value of 25.3 mM. In conclusion, nutlin-3a and TevP can mutually enhance their binding affinity to N-MdmX.

[0030] To determine the mechanism by which TevP acts as a repair peptide to enhance the binding affinity of the spur compound nutlin-3a to the MdmX binding pocket, 15N-1H HSQC NMR titration was performed to monitor NMR spectral perturbations induced by TevP and nutlin-3a. N-MdmX consists of 89 amino acid residues, containing four α-helices and two β-sheet elements (…). Figure 2a In solution, 15N-labeled N-MdmX samples ( Figure 2b The 15N-1H HSQC NMR spectrum of nutlin-3 (black) produced fewer peaks than expected due to its dynamic conformation. Although nutlin-3 and TevP have weak binding affinity for N-MdmX, titration of nutlin-3a (… Figure 2b ) or TevP ( Figure 2b The titration of nutin-3a significantly perturbed the 15N-1H HSQC NMR peak. This application also notes that the titration of nutin-3a caused perturbation of the global NMR peak. Figure 2b ), while the titration of TevP only causes local perturbation of the nuclear magnetic resonance peak ( Figure 2b Specifically, such as Figure 2b As shown, the resonance peaks from M46, Y55, G57, and M101 are significantly perturbed by TevP, indicating that TevP may interact with helices a2 and a4 on N-MdmX. Figure 2a ).

[0031] This application further uses N-MdmX labeled with N15 ( Figure 2c (black) and TevP ( Figure 2c (cyan) and nutlin-3a ( Figure 2cCross-titer experiments were conducted on the orange sample. The TevP titration was performed under conditions where the molar ratio of N-MdmX to nutlin-3a was 1:1.5 (N-MdmX:nutlin-3a), and the nutlin-3a titration was performed under conditions where the molar ratio of N-MdmX to TevP was 1:1.5 (N-MdmX:TevP). Figure 2c As shown, in both cross-titer experiments, TevP and nutlin-3a significantly perturbed the N-MdmX NMR peaks. This application observed that, compared to a single titration, some resonance peaks were perturbed to the same degree, such as S91 and F90. Figure 2b However, this application also notes that TevP and nutlin-3a perturb many spectral peaks to varying degrees, such as H38, G57, G77, and G78. These observations suggest that TevP and nutlin-3a enhance each other's binding affinity in different ways, supporting... Figure 1c The data results shown are something that existing drug design theories cannot accurately predict.

[0032] Crystal structure of N-MdmX / TevP / nutlin-3a complex To visualize how the peptide sheet of TevP remodels the nutlin-3a binding pocket on N-MdmX, this application determined the X-ray crystal structure of the N-MdmX complex with TevP and nutlin-3a. The crystal of the complex exhibited a good diffraction pattern (1.93 Å). The crystal structure revealed a compact three-component complex (…). Figure 3a The N-MdmX structure in this three-component complex exhibits similar folding and surface features to that in the N-MdmX / nutlin-3a complex. TevP forms a helical structure linking helices a2 and a4 of N-MdmX, and acts as a patch across the L26 binding site. Figure 3a This transforms the originally open pocket into a deep-hole structure. Figure 3b In summary, 15N-HSQC NMR titration and X-ray crystal structure analysis indicate that the TevP patch plays a role in confining nutlin-3a within the N-MdmX pocket.

[0033] like Figure 4 As shown in a, the TevP peptide identified in this work is dissimilar to p53p and PMI. P53p and PMI form helical structures with N-MdmX via three or four key residues, respectively. Figure 4 (b) Nutline-3a molecules have a rigid conformation that mimics three key residues in the p53p peptide ( Figure 4c). When comparing the newly detected N-MdmX structure with those complexed with p53p (3DAB) or PMI or nutlin-3a (7C44)22, this application found that the N-MdmX backbone conformation in these complexes is globally folded in the same pattern ( Figure 4 (d) Compared to the structure of the N-MdmX / nutlin-3a complex, the binding of TevP in the N-MdmX / nutlin-3a / TevP complex does not affect the docking position of nutlin-3a. Figure 4 In both cases, nutlin-3a binds well into the ligand-binding pocket (e). Figure 4 The f in it mimics three key residues of p53p, namely F19. p53p W23 p53p and L26 p53p ( Figure 4 (b) Compared to the N-MdmX / PMI complex, the L3 residue of the TevP peptide is at the same position as the P28 residue of the PMI peptide ( Figure 4 In the g of the PMI peptide, the P28 residue is the main contributor to the high binding affinity of PMI, and the position of this amino acid residue is caused by the shift of the P26 residue of p53p. Figure 4 The h in the figure indicates that TevP, in addition to its function as a patch, partially mimics PMI. The degree of selection and defect linkage can only be determined through structural analysis or random testing.

[0034] Design of peptide-leader compound conjugates Inspired by the enhanced binding affinity of TevP patches for nutlin-3a and the compact structure of the N-MdmX / TevP / nutlin-3a complex, this application designs an MdmX inhibitor (PHC) of a peptide-sprout compound conjugate. To demonstrate this concept, an eight-residue TevP peptide is coupled with a nutlin-3a conjugate having a polyglycine chain; this PHC is abbreviated as Tn3a. Figure 6 The synthesis of Tn3a is carried out in five steps, including Boc protection of the piperazine group of nutlin-3a, alcoholysis of the Boc-protected piperazine group, deprotection of the Boc group, coupling of the protected TevP, and deprotection of all protecting groups in the N-terminus and side chain residues. Figures 7a to 12b ).

[0035] Compared with nutlin-3a, p53p, and PMI, this application evaluated the binding affinity of Tn3a for N-MdmX and N-Mdm2. The affinity was determined by fluorescence polarization titration. Figure 5bTn3a exhibits enhanced binding affinity for N-MdmX, with a Ki of 0.75 nM (Table 2). Data from this application also show that Tn3a enhances the binding affinity of nutline-3a for N-Mdm2. Figure 5c Ki is 0.27 nM (Table 2). Table 2 shows that Tn3a has a binding effect on both MdmX and Mdm2 compared with PMI, nutlin-3a and p53p.

[0036] Table 2 Tn3a inhibits the biological activity of cancer cells.

[0037] This application used the H1299 cell line (H1299 p53+) with p53 expression induced by G418 for MTT assay. Overexpression of Mdm2 and MdmX in H1299 cells was achieved via transient transfection with RFP-labeled MdmX and Mdm2 expression plasmids. Cell viability was measured after treatment with Tn3a, nutlin-3a (positive control), or nutlin-3b (negative control) for 48 hours. Results showed that Tn3a significantly inhibited cell viability in H1299 p53+ cells overexpressing Mdm2 and MdmX in a dose-dependent manner. A significant dose-dependent reduction in cell number was also observed in Tn3a-treated cells in the absence of Mdm2 or MdmX. In contrast, nutlin-3a showed a strong inhibitory effect on cells overexpressing Mdm2, while nutlin-3b did not show any inhibition of cell viability in any of the tested cells. Cells lacking Mdm2 and MdmX were largely unaffected by Tn3a treatment. These results suggest that Tn3a has a mechanism of action as a dual inhibitor of Mdm2 / MdmX.

[0038] Next, this application investigated whether Tn3a is specific for the p53-MdmX / Mdm2 signaling axis in vivo by inhibiting the binding of MdmX and Mdm2 to p53. This application treated three cancer cell lines expressing wild-type p53 (HCT116, A549, and H164) with Tn3a for 8 hours and measured the transcriptional levels of the p53 and p21 genes. p21 transcription increased in a dose-dependent manner in these cell lines, consistent with the accumulation of its transcriptional activator p53. Conversely, the transcription of the p53 gene itself was unaffected by Tn3a treatment. These observations indicate that the Tn3a-mediated reduction in cell viability is strictly dependent on p53. The data also show that simultaneously inhibiting both Mdm2 and MdmX in two overexpressed cancer cells is more effective in slowing or preventing cancer cell growth than inhibiting either protein alone.

[0039] Example 1: Protein Expression and Purification Human MdmX (N-MdmX, amino acid 22-110) and human Mdm2 (N-Mdm2, amino acid 22-110) were prepared according to the previously described protocol. Recombinant N-MdmX and N-Mdm2 proteins were prepared in *E. coli* BL21(DE3) cells using a histidine tag. The His tag was removed by cleavage with the Tev protease. The purified N-MdmX and N-Mdm2 proteins were concentrated to 0.4–0.8 mg / mL, freshly frozen in liquid nitrogen, and stored at -80°C.

[0040] Example 2: Screening of phage peptide libraries Biopanning of the M13 phage peptide library (Ph.D.-12, from New England Biolab, USA) was performed using the N-MdmX-nutlin-3a complex. His-labeled N-MdmX was attached to the wells of a Ni-NTA ELISA plate and coated overnight with BSA at 4°C. In the presence of nutlin-3a, the phage library was added to the wells containing N-MdmX to initiate the panning experiment. Unbound phages were removed by washing with TBS buffer containing 0.1% Tween-20, and bound phages were eluted with 2 M glycine-HCl (pH 2.2) solution, followed by immediate neutralization. The eluted phages were amplified and used for the next round of panning. The concentration of Tween-20 was progressively increased (i.e., 0.2%, 0.3%, 0.4%, 0.5%, 0.75%, and 1.0%), and the biopanning was repeated five to six times. Phage particles randomly selected from the final three panning steps were sequenced for DNA using universal primer 96gIII (NEB). The binding affinity of the selected peptides to the N-MdmX / nutlin-3a complex was assessed by ELISA using a monoclonal antibody against M13 protein VIII.

[0041] Example 3: 15N-1H NMR HSQC titration All NMR spectra were collected at 25°C on a Bruker Avance 600MHz or 800MHz spectrometer equipped with a triple resonance pulsed-field gradient probe. Protein samples were prepared by exchanging the NMR buffer. The concentration of the protein complex was 0.3–0.4 mM. 15N–1H HSQC NMR titration was performed by stepwise addition of ligands (high concentration) to 15N-labeled N-MdmX (typically 0.1–0.3 mM), with a final excess of 1–2 times. 15N–1H HSQC NMR spectra were recorded in States-TPPI mode for orthogonal detection. All NMR samples were prepared in a buffer containing 20 mM sodium phosphate, 200 mM NaCl, 2 mM DTT, and 95% H2O / 5% D2O (pH 6.8). All datasets were obtained using 2048 complex points in t2 and 128 complex points in t1, and processed using Topspin. The NMRViewJ software package was used for graph display and analysis.

[0042] Example 4: Protein Crystallization and Optimization Protein crystallization conditions were screened using a GRYPHON workstation (ARI, USA) against six common commercial kits, including Index™, PEGRx, and CrystalScreen™ (Hampton, USA), and AmSO4Suite, JCSG+ Suite, and PEGsSuite (Qiagen, USA). Diamond-like crystals appeared within two days under conditions containing 20% ​​(v / v) Tacsimate (pH 7.0), 0.1M HEPES (pH 7.5), and 2% (v / v) polyethylene glycol 200.

[0043] Protein crystallization was further optimized using the sitting drop method. Briefly, each 1 mL protein drop consisted of 0.5 mL of protein sample (10 mg / mL) and 0.5 mL of stock solution, and the drop was incubated at 18°C. Furthermore, new stock solution droplets were inoculated with needle-like crystals suspended in a slurry-like stock solution to prepare a large quantity of protein crystals; diamond-like crystals appeared after 2 days. The crystals were transferred to a cryoprotectant containing 50% (v / v) stock solution and 25% (v / v) glycerol, and rapidly cooled in liquid nitrogen.

[0044] Example 5: Data collection and refinement in protein crystallography X-ray diffraction data were collected by HKL2000 on BL17U1 of the Shanghai Synchrotron Radiation Facility (SSRF) using an EigerX16M detector. The dataset was integrated and scaled using the Aimless program. The data were integrated and integrated using the Xia2 and MOLREP programs, respectively. The MdmX structures in protein structures 6Q9W and 6V4F were used as templates for N-MdmX. The model was constructed using the molecular substitution-PHASER model in the CCP4i2 software package and improved using the REFMAC5 model in the CCP4i2 software package. The structure was manually evaluated using the COOT software.

[0045] Example 6: Fluorescence Polarization (FP) Measurement All fluorescence polarization assays were performed using black, low-protein-binding 96-well plates (Corning, New York), with a total volume of 100 μL per well containing 20 mM phosphate (pH 6.8), 200 mM NaCl, and 1 mM DTT. 10 nM fluorescein-p53p (fluorescein-GSGSSQETFSDLWKLLPEN, FITC-p53p) was pre-incubated with 10 μM N-MdmX or 1 nM 5'-fluorescein-pMI (5-fluorescein-GSGSSTSFAEYWALLSP, FAM-pMI) with 30 nM N-MdmX for 30 min, followed by the addition of inhibitors and incubation for another 30 min. FP readings were measured using a BioTek H1 multiplate reader equipped with Gen5 software, with excitation at 498 nm and emission at 517 nm. p53p peptide and nutlin-3a were used as positive controls.

[0046] All FP data were simulated using a dose-dependent pharmacology model in MicroCal Origin 2017 to obtain IC50 values. Ki values ​​were obtained using the formula IC50 = Ki(1 + [L] / Kd), where Kd is the binding affinity of FITC-p53p or FAM-PMI to N-MdmX or Mdm2, respectively.

[0047] Example 7: Synthesis of peptide-nutlin-3a conjugate Nutlin-3a was coupled with TevP peptide through the following steps ( Figure 6 The molecular weights of nutlin-3a analogs were determined using a Thermo Scientific LC-MS TSQ Altis™ Plus triple quadrupole mass spectrometer, and the molecular weights of peptide analogs were determined using a Bruker Autoflex U3000-Q-Exactive Speed ​​TOF / TOF mass spectrometer. HPLC analysis and purification were performed using a Waters 2459 HPLC system.

[0048] Preparation of Compound 2: 200 μl of nutlin-3a (1, 10 mM) was added to a 1:1 mixture of dioxane and water, and stirred thoroughly. The pH of the solution was adjusted to 9–10 with sodium hydroxide, and (Boc)₂O (0.6984 mg, 3.2 μmol) was added. The mixture was incubated at 100–150°C with stirring for 12 hours. The pH of the solution was calibrated to 9–10 with sodium hydroxide, and the solution was incubated with stirring for 24 hours until the reaction was complete. The solution was distilled under reduced pressure to evaporate the dioxane, and then the aqueous phase was extracted twice with ethyl acetate to purify Compound 2. The identity of Compound 2 was confirmed by mass spectrometry. Figure 7a The yield and purity of compound 2 were evaluated by HPLC. Figure 7b The yield was 94.22%.

[0049] Preparation of compound 3: Compound 2 was added to 2 mL of NH3 / MeOH solution at room temperature. The solution was stirred at 100–150 rpm for 3 hours. After the reaction was complete, the solvent was evaporated under reduced pressure. During evaporation, small amounts of dichloromethane (DCM) were added intermittently to co-evaporate the residual solvent, yielding compound 3. Compound 3 was confirmed by mass spectrometry. Figure 8a The yield and purity of compound 3 were evaluated by HPLC. Figure 8b The yield was 62.84%.

[0050] Preparation of compound 4: Compound 3 was dissolved in DCM (0.4 mL) and 1.6 mL of trifluoroacetic acid (TFA) was added. The mixture was stirred at room temperature for 3 hours. The solvent was evaporated to dryness and co-evaporated with DCM several times to remove residual TFA, yielding compound 4. Compound 4 was dissolved in acetonitrile and purified using a C18 column (acetonitrile:water = 9:1). Compound 4 was confirmed by mass spectrometry. Figure 9a The yield and purity of compound 4 were evaluated by HPLC. Figure 9b The yield was 45.8%.

[0051] Preparation of compound 5: Compound 4 was dissolved in 2 ml of DCM at room temperature, and 1.25 eq of PyBOP was added to the solution. Triethylamine (Et3N) was added dropwise until the pH of the solution became 9-10. The solution was stirred overnight to activate the carboxyl group of compound 4. Then, 1.25 eq of a side-chain protected TevP peptide analog was added to the solution and the reaction was carried out for 4 hours. The solvent was evaporated under reduced pressure to obtain compound 5 as the crude product. The yield and purity of compound 5 were evaluated by HPLC. Figure 10 The yield was 82.87%. The molecular weight of compound 5 was confirmed by mass spectrometry.

[0052] Preparation of compound 6: TFA (1.6 mL) was added to a solution of compound 5 in DCM (0.4 mL). The solution was stirred at room temperature for 3 hours to deprotect compound 5. The solvent was evaporated to dryness and co-evaporated with DCM several times to remove residual TFA, yielding compound 6. The yield and purity of compound 6 were evaluated by HPLC. Figure 11 The yield was 53.63%. The molecular weight of compound 6 was confirmed by mass spectrometry.

[0053] Preparation of compound 7: Piperidine (0.6 mL) was added to a solution of compound 6 in DCM (0.4 mL), and the reaction was carried out at room temperature for 3 hours to remove Fmoc. The solvent was evaporated to dryness, and the mixture was co-evaporated several times with DCM to remove residual piperidine. Compound 7 was then purified by preparative C18 reversed-phase column and Waters 2549 HPLC. The yield and purity of compound 7 were evaluated by HPLC. Figure 12a The yield was 65.27%, and the molecular weight of compound 7 was confirmed by mass spectrometry. Figure 12b ).

[0054] Preparation of TevP peptide analogs: Three types of peptide analogs, namely TevP-Gly-Gly-Gly-Gly-, TevP-Gly-Gly-Gly-Gly-COOH, and TevP-(CH2). n -COOH (n=2-8), TevP-(C2H4) n -COOH (n=2-8) was synthesized using conventional solid-phase chemical peptide synthesis technology with conventional side chain protection groups. The synthesis was outsourced to a third-party commercial company, with a purity greater than 98.% and a yield greater than 85%.

[0055] Example 8: Real-time RT-qPCR reaction Three types of cancer cells containing wild-type p53 (HCT116, A549, and H460) were treated with Tn3a for 8 hours. Changes in transcription levels were measured by quantitative PCR and compared with an untreated control. Total RNA was extracted using the Trizol method and the Qiagen (Texas, USA) RNeasy® Mini Kit. RNA concentration was measured using a Nanodrop 2000c. Real-time qPCR was performed using the BioRad CFX96 qPCR instrument (California, USA) with the Biorab (Beijing, China) BaldStar TaqMan One-Step RT-qPCR Kit. The primers and probes for qPCR were designed using Primer Express 3.0 software (Applied Biosystems, USA). The primer sequences for p21 were RT-p21-L1 (5'-CTTTGTCACCGAGACACCAC-3') and p21-R1 (5'-CAGGTCCACATGGTCTTCCT-3'), and the probe sequence for p21 was p21-P1 (5'-ACTCATCCCGGCCTCGCCGG-3'). The primer sequences for p53 were RT-p53-L1 (5'-GTCCAGATGAAGCTCCCAGA-3') and RT-p53-R1 (5'-CAAGAAGCCCAGACGGAAAC-3'), and the probe sequence for p53 was p53-P1. (5'-AGCTCCTACACCGGCGGCCC-3'), TaqMan probes were labeled with 5-FAM and 3-TAMRA, and all primers and probes were synthesized by GenScript (Nanjing, China).

[0056] Example 9: MTT assay for cell proliferation Using H1299 cells containing an inducible form of the wild-type p53 gene, MTT assays were performed on a Synergy H1 multiplate reader (Biotek, USA). The p53 protein was fused with a GFP protein marker. Overexpression of Mdm2 and MdmX was achieved by transfecting pCMV plasmids containing full-length genes encoding either Mdm2 or MdmX and RFP protein markers. Cells were cultured for 3 days in Dulbecco modified medium supplemented with 10% serum, penicillin, and streptomycin (37°C and 5% CO2), followed by replacement with RPMI 1640 supplemented with 3% FBS. Cells were grown at a growth rate of 1×10⁶ cells / year. 4 / cm 2Cells were seeded at a density of 100 μL. After 2 hours, Tn3a, nutlin-3a, and nutlin-3b were added. After 24 hours, 10 µL of MTT (5 mg / ml) solution was added to each well. Cells were then cultured in a CO2 incubator at 37°C for another 4 hours. The supernatant was discarded, and cells were washed with PBS. 100 µL of dimethyl sulfoxide (DMSO) was added to each well. The plates were shaken on a plate shaker for 10 minutes, and the OD values ​​were read at 520 nm. Data were processed using MicroCal Origin software (v2017, MicroCal, USA).

[0057] Example 10: Western blot analysis Cells were collected and extracted using RPMI lysis buffer containing a mixture of 50 mM Tris (pH 7.4), 150 mM NaCl, 1% Triton-X100, 0.1% sodium deoxycholate, 0.1% SDS, and 0.1% protein inhibitor. The concentration of the protein extract was first measured at OD 280 nm using a Nanodrop-2000c, and then the cell-actin content was calibrated using a mouse anti-β-actin monoclonal antibody from Proteintech (Wuhan, China; Cat#: HRP-60008; Gene ID60, 100 g / ml) at a dilution ratio of 1:10000. Protein samples were separated and calibrated using SDS-PAGE with standard molecular weight comparison. Each SDS-PAGE gel was electrotransferred to a polyvinyl fluoride (PVDF) membrane (Millipore, USA). Each PVDF membrane was blocked for 1 hour at room temperature with TBST buffer containing 5% skim milk, then incubated with the corresponding primary antibody for 2 hours. After washing three times with TBST buffer, the membrane was incubated with HRP-labeled secondary antibody for 1 hour. Protein bands on the PVDF membrane were detected using BioSharp's (Beijing, China) enhanced chemiluminescence (ECL) reagent on a Tanon 5200 Chemiliminescent Imager (Shanghai, China). Each blot was repeated until a high-quality image was obtained.

[0058] Rabbit anti-TP53 polyclonal antibody IgG from CUSABIO (Texas, USA; Catalog No.: CSB-PA15509AORB, Lot No.: F0912A) was used to blot p53 protein in cells at a dilution of 1:4000; mouse anti-RFP monoclonal antibody from Solarbio (Beijing, China; Catalog No.: K20016M) was used to determine RFP-labeled Mdm2 and MdmX proteins in cells at a dilution of 1:10000; and Elabscience (Wuhan, China; Catalog No.: E-AB-4009) was used to blot p53 protein in cells. 7) Rabbit anti-p21 polyclonal antibody was used to detect cell p21 protein at a dilution of 1:500; rabbit anti-human PUMA monoclonal antibody from Beyotime (Shanghai, China; Catalog No.: AF1204; Gene ID27113) was used to detect cell PUMA protein at a dilution of 1:1000; secondary antibody was HRP-conjugated goat anti-rabbit IgG (H+L) or HRP-conjugated goat anti-mouse IgG (H+L) (Biosharp, Guangzhou, China; Catalog No.: BL001A, 0.8 mg / ml) at a dilution of 1:10000.

[0059] Those skilled in the art should understand that the above embodiments are merely exemplary embodiments, and various changes, substitutions, and modifications can be made without departing from the spirit and scope of the present invention.

Claims

1. A dual inhibitor of Mdm2 / MdmX peptide analogues, wherein the structure of the dual inhibitor of Mdm2 / MdmX peptide analogues is as follows: .

2. The use of the Mdm2 / MdmX polypeptide analog dual inhibitor according to claim 1 in the preparation of a drug for treating non-small cell lung cancer.

Images