EMBOW derived peptide for targeting WDR5-MLL1 protein interaction and application of EMBOW derived peptide

Abstract

The invention relates to the technical field of small molecule polypeptides and leukemia treatment drugs, in particular to an EMBOW derived peptide targeting WDR5-MLL1 protein interaction and application of the EMBOW derived peptide, the amino acid sequence of the EMBOW derived peptide is shown as SEQ ID NO: 1, the N end of the EMBOW derived peptide is subjected to acetylation modification, and the C end of the EMBOW derived peptide is subjected to protective modification. The EMBOW derived peptide can target WDR5, inhibit protein interaction between WDR5-MLL1 and inhibit the methylation level of histone H3K4, and can be used for preparing a medicine for treating MLL1 gene rearrangement type leukemia.

Description

Technical Field

[0001] This invention relates to the field of small molecule peptides and leukemia treatment drugs, specifically to an EMBOW-derived peptide that targets the interaction between WDR5-MLL1 protein and its application. Background Technology

[0002] The WDR5 protein recognizes an arginine-containing WIN binding sequence from the catalytic component of the SET1 / MLL family complex via its WIN site, thereby promoting the methylation of histone H3K4. Within the SET1 / MLL complex, WDR5 also binds to another core protein, RbBP5, via its WBM site. This site simultaneously interacts with MYC, participating in MYC target gene recognition and tumorigenesis. WDR5 plays a unique role in epigenetic regulation through its diverse protein-protein interaction network, participating in multiple important cellular processes such as gene expression regulation, cell cycle, signal transduction, and epigenetic modification. Notably, WDR5 is overexpressed in various malignant tumors, including leukemia, breast cancer, and lung cancer, and promotes cancer development, progression, and metastasis through interactions with tumor-associated proteins, thus being considered a potentially significant cancer therapeutic target.

[0003] WDR5, along with MLL1, RbBP5, Ash2L, and DPY-30, constitutes the MLL1 methyltransferase complex. This complex plays a crucial role in hematopoietic system development and blood cell turnover by regulating the methylation level of histone H3K4. MLL1 protein, named for its close association with the development of mixed-lineage leukemia, can fuse with multiple genes to form MLL1 fusion proteins when it undergoes chromosomal translocation. Studies have shown that the production of MLL1 fusion proteins is closely related to the development and progression of acute lymphoblastic leukemia and acute myeloid leukemia, and some MLL1 fusion proteins have been shown to directly induce leukemia in animal experiments. From a molecular mechanism perspective, MLL1 promotes H3K4 methylation, thereby facilitating the development of mixed-lineage leukemia. Hox , Meis-1 Expression of leukemia-related genes. Among them, Hox Gene families play an important regulatory role in embryonic development and blood cell differentiation, HoxA9 Specific Hox Sustained high expression of genes is MLL1 Gene rearrangement-related leukemia is a prominent feature and plays a crucial role in the development and progression of leukemia. Against this backdrop, WDR5, as a core component of the MLL1 methyltransferase complex, plays a vital role in… MLL1The important role of gene rearrangement in the development of leukemia is increasingly concerned. Studies have found that MLL1 specifically binds to the WIN site of WDR5, and then recruits other components such as RbBP5, Ash2L and DPY-30 to form a complete MLL1 methyltransferase complex. Notably, in the absence of WDR5, even if other MLL1 complex components are present, MLL1 can only slowly catalyze the monomethylation of histone H3K4, and cannot exert the complete methyltransferase activity, which fully confirms that WDR5 is a key component indispensable to maintain the complete function of MLL1 methyltransferase. Based on the above mechanism, by targeting and inhibiting the interaction between WDR5 and MLL1, reducing the level of abnormal H3K4 methylation, and then regulating the expression of Hox 、 Meis-1 and related genes, it has become a key strategy for the treatment of MLL1 fusion protein type leukemia.

[0004] However, the development of efficient and specific WDR5-MLL1 interaction inhibitor molecules is still an urgent need in the current leukemia treatment research field. In recent years, with the rapid development of ribosome sequencing and proteogenomics technology, a large number of unannotated small proteins or peptides (collectively referred to as microproteins) with a length of less than 150 amino acids have been confirmed to be translated on ribosomes. Although such microproteins were once thought to be difficult to form stable spatial structures and play biological functions, recent studies have revealed that some microproteins have important regulatory roles, of which EMBOW is a typical representative. EMBOW (endogenous microprotein binder of WDR5, EMBOW) has a significant interaction with WDR5. EMBOW mainly negatively regulates the interaction between WDR5 and MLL1 protein. Endogenous EMBOW exerts regulatory effects by transiently binding to WDR5, thereby limiting the binding of WDR5 to MLL1 protein in the nucleus. This finding suggests that EMBOW has the potential to develop into a WDR5-MLL1 interaction inhibitor and is worthy of further study. SUMMARY

[0005] To solve the problems in the background art, the present application provides an EMBOW-derived peptide targeting WDR5-MLL1 protein interaction and its application. The EMBOW-derived peptide can target WDR5 and inhibit the protein interaction between WDR5 and MLL1, and can be used for preparing a drug for treating MLL1 gene rearrangement type leukemia. The technical solution of the present application to solve the above technical problems is as follows: In a first aspect, the present application provides an EMBOW-derived peptide targeting WDR5-MLL1 protein interaction, the amino acid sequence of which is shown in SEQ ID NO: 1, the N-terminus of which is acetylated and the C-terminus of which is protected.

[0006] According to the above scheme, the C-terminus of the EMBOW-derived peptide is acetylated, esterified, thioesterified, biotinylated or polyethyleneglycolated, or the C-terminal carboxyl group is protected by reduction to aldehyde or alcohol, conversion to hydrazine or hydrazide, or cyclization.

[0007] The acetylation of the N-terminus protects the N-terminus, and the introduction of acetyl group forms two intramolecular hydrogen bonds in the derived peptide, increases the stability of the derived peptide, and causes the flip of the first residue, enabling it to be more tightly embedded in the binding pocket of WDR5, significantly improving its binding force with WDR5; the various modifications of the C-terminus can protect the C-terminal carboxyl group.

[0008] In a preferred embodiment of the present application, the N-terminus of the EMBOW-derived peptide is acetylated, and the C-terminus is connected with an amino group for aminoacylation modification. Similarly, other group modifications of the C-terminus can also protect the C-terminal carboxyl group.

[0009] In a second aspect, the present application provides the use of the above-mentioned EMBOW-derived peptide targeting WDR5-MLL1 protein interaction in the preparation of a WDR5-MLL1 protein interaction inhibitor.

[0010] In a third aspect, the present application provides the use of the above-mentioned EMBOW-derived peptide targeting WDR5-MLL1 protein interaction in the preparation of a MLL1 methyltransferase activity inhibitor.

[0011] In a fourth aspect, the present application provides the use of the above-mentioned EMBOW-derived peptide targeting WDR5-MLL1 protein interaction in the preparation of a drug for reducing the methylation level of histone H3K4.

[0012] In a fifth aspect, the present application provides the use of the above-mentioned EMBOW-derived peptide targeting WDR5-MLL1 protein interaction in the preparation of a drug for inhibiting the proliferation of gene rearrangement leukemia cells. MLL1

[0013] In a sixth aspect, the present application provides the use of the above-mentioned EMBOW-derived peptide targeting WDR5-MLL1 protein interaction in the preparation of a drug for inhibiting the expression of leukemia-related genes HoxA7 , HoxA9 , and co-factors Meis1 .

[0014] ​In a seventh aspect, the present invention provides the above-mentioned EMBOW-derived peptide targeting WDR5-MLL1 protein interaction in the preparation of therapeutic... MLL1 Application in drugs for gene rearrangement-type leukemia.

[0015] According to the above protocol, the drug is an intravenous injection.

[0016] The beneficial effects of this invention are: This invention provides an EMBOW-derived peptide that exhibits significantly enhanced affinity for WDR5. As a small peptide, it can penetrate the cell membrane and enter the cell, inhibiting the protein-protein interaction between WDR5 and MLL1, thereby effectively inhibiting the intracellular histone H3K4 methylation level and targeting key genes. HoxA7 , HoxA9 and Meis1 The expression of all of them showed excellent downregulation effects and could effectively inhibit [the expression of all of them]. MLL1 The proliferation of gene-rearranged leukemia cells, in MLL1 It exhibits potent antitumor activity in a mouse model of gene-rearranged leukemia xenograft with no significant toxicity, and can be used to prepare therapeutic drugs. MLL1 Drugs for gene rearrangement-type leukemia. Attached Figure Description

[0017] Figure 1 The results of the interaction between EMBOW and WDR5 in this invention are shown in Figure 1A, which is a schematic diagram of the structural domains of EMBOW-like mutants and WDR5; Figure 1B is a schematic diagram of the binding ability between the full-length EMBOW / EMBOW mutant and WDR5, where "+" indicates that they can bind and "-" indicates that they cannot bind; Figure 1C is an ITC binding curve showing the interaction between the full-length EMBOW / EMBOW mutant and WDR5; and Figure 1D is a quantitative statistical graph of the binding affinity of the full-length EMBOW / EMBOW mutant and WDR5 in the ITC experiment. Figure 2 This invention relates to the effects of EMBOW on the in vitro H3K4 methyltransferase activity, intracellular H3K4 trimethylation, leukemia cell proliferation, and leukemia-related gene expression of the MLL1 core complex. Figure 2 A is EMBOW 1-120 and EMBOW 1- 120 Fitting curves and IC50 values ​​of R2A on the in vitro activity of H3K4 methyltransferase, a core complex of MLL1. 50 2B is EMBOW 1-120 and EMBOW 1-120 The results show the effect of R2A on intracellular H3K4 trimethylation. The left image is a Western blot plot, and the right image is the quantification plot of the left image. 2C represents EMBOW. 1-120and EMBOW 1-120 Effect of R2A on cell viability, 2D is EMBOW 1-120 and EMBOW 1-120 Effect of R2A on leukemia genes HoxA9 and Meis1 Effect on gene expression Figure 3 Interaction of EMBOW derived peptides with WDR5 and their effects on MLL1 core complex H3K4 methyltransferase activity in vitro, where 3A-3E are the crystal structures of Ac3-WDR5, Ac5-WDR5, Ac7-WDR5, Ac10-WDR5 and WT10-WDR5, respectively, Ac3, Ac5, Ac7, Ac10 (cyan) and WT10 (yellow) are shown as stick models (red characters), and WDR5 is shown as stick and cartoon (light green, black characters), 3F is the activity curve and IC 50 of Ac3, Ac5, Ac6, Ac7 and Ac8 in inhibiting H3K4 methyltransferase activity in vitro Figure 4 Effect of EMBOW derived peptides on leukemia cell lines, where A is a schematic diagram of cell experiments for evaluating derived peptides, B is a result graph for evaluating H3K4 methylation level by Western blot analysis, B-D are quantitative analysis results of Western blot results for methylation, dimethylation and trimethylation of H3K4, E and F are the effects of derived peptides on cell viability of MV-4-11 and MOLM13 cell lines, G is the selective growth inhibition effect of Ac7 treatment on leukemia cell lines, H-J are the expression levels of HoxA7 , HoxA9 and Meis-1 after treatment of MV4-11 cells with derived peptides, the data were normalized by GAPDH expression, and the data are expressed as mean ± standard deviation p <0.05, p <0.01, p <0.001, indicating statistically significant difference from the control group, and "ns" represents no statistically significant difference Figure 5Results of the effect of EMBOW-derived peptide Ac7 in a MV-4-11 xenograft mouse model, wherein A is a schematic diagram of the experiment to evaluate the antitumor effect of the derived peptide Ac7 in MV-4-11 human leukemia xenograft tumors, B is the volume of the tumors, C is the weight of the tumors at the end of the experiment, D is the curve of the change in the weight of the mice, E is TUNEL staining of tumor paraffin sections, brown cells indicate apoptosis (scale bar: 100 µm), F is the quantitative analysis of TUNEL staining, G is the image of the excised tumors from each group (n = 6 mice per group), the data are expressed as mean ± standard deviation. p <0.05, p <0.01, p <0.001, statistically significant difference compared with the control group. “ns” indicates no statistically significant difference. Control group: injection of the same volume of 0.9% saline; Figure 6 Representative HE staining images of heart, liver, spleen, lung and kidney tissues, scale bar: 100 µm. DETAILED DESCRIPTION

[0018] The principles and characteristics of the present application are described below in conjunction with the accompanying drawings and specific examples, which are only used to explain the present application and are not intended to limit the scope of the present application.

[0019] I. Interaction of EMBOW protein with WDR5 and inhibition of the interaction of MLL1 and WDR5 proteins by EMBOW protein and applications 1. Expression and purification of human WDR5 protein, EMBOW protein and EMBOW protein mutants The amino acid sequences of human WDR5 protein (aa 22-334) and EMBOW (aa 1-120) protein are shown in SEQ ID NO: 2 and SEQ ID NO: 3, respectively: WDR5 protein (aa 22-334) SATQSKPTPVKPNYALKFTLAGHTKAVSSVKFSPNGEWLASSSADKLIKIWGAYDGKFEKTIS GHKLGISDVAWSSDSNLLVSASDDKTLKIWDVSSGKCLKTLKGHSNYVFCCNFNPQSNLIVS GSFDESVRIWDVKTGKCLKTLPAHSDPVSAVHFNRDGSLIVSSSYDGLCRIWDTASGQCLK TLIDDDNPPVSFVKFSPNGKYILAATLDNTLKLWDYSKGKCLKTYTGHKNEKYCIFANFSVT GGKWIVSGSEDNLVYIWNLQTKEIVQKLQGHTDVVISTACHPTENIIASAALENDKTIKLWK SDC (SEQ ID NO: 2) EMBOW (aa 1-120) protein MRTEPRPPAPSPPSAAAGARAAHPHHAQVHPAVALQPARGVGGQAALFAAGRAGGDLPLQP QPGGAAARRQPAARAAQAFFPAAELAQAGPERQRDPAVASRGGQLHAAGGAGRVPERYP (SEQ ID NO: 3) Human WDR5 protein (aa 22-334) and EMBOW protein (aa 1-120) were obtained by genetic engineering, and four mutants of EMBOW protein were constructed: EMBOW 1-30+61-120 , EMBOW 1-60+91-120 , EMBOW 1-90 , EMBOW 31-120The specific method was as follows: DNA fragments of human WDR5 protein (aa 22-334), EMBOW protein (aa 1-120), and EMBOW protein mutants (1-30+61-120, 1-60+91-120, 1-90, 31-120) were cloned into the pET28-MHL vector to construct fusion proteins with N-terminal His6 tags (WDR5) or C-terminal His6 tags (EMBOW) for overexpression. The recombinant plasmids were transformed into *E. coli* BL21(DE3) cells and induced overnight expression with 0.5 mM IPTG at 14°C. After harvesting the cells, they were resuspended in lysis buffer (20 mM Tris, pH 7.5, 500 mM NaCl, 5% glycerol) and then lysed by sonication at 4°C. After sonication, the lysis buffer was centrifuged at 16,000 g for 40 minutes, and the supernatant containing the target protein was purified using Ni-NTA resin. After removing the His6 tag using TEV protease, the protein was diluted with buffer containing 20 mM Tris (pH 7.5) and further purified by anion exchange chromatography (HiTrap SP HP column, GE Healthcare) with a 0-1000 mM NaCl gradient. Finally, the protein was purified using a Superdex 200 gel filtration column (GE Healthcare) with 20 mM Tris (pH 7.5) and 150 mM NaCl, yielding EMBOW protein. 1-30+61-120 EMBOW 1-60+91-120 EMBOW 1-90 and EMBOW 31 -120 The purified protein was concentrated to 10-20 mg / mL using a concentration tube (Merck KGaA, Darmstadt, Germany) and stored at -80 °C for later use.

[0020] 2. Interaction between EMBOW and WDR5 2.1 Dissociation constant ( K d ) Measurement To investigate the region in EMBOW that binds to WDR5, isothermal titration calorimetry (ITC) was used to determine the binding intensity of WDR5. 22-334 Compared with the full-length EMBOW protein and four mutant EMBOW 1-30+61-120 EMBOW 1-60+91-120 EMBOW 1-90 and EMBOW 31-120 The dissociation constant between them ( K dThe specific experimental method is as follows: Full-length EMBOW / EMBOW mutant and WDR5 protein were prepared into 500 µM and 50 µM solutions, respectively. Isothermal titration thermodynamic experiments were conducted using a MicroCal PEAQ-ITC microcalorimeter under constant temperature conditions of 25 ± 0.1 ℃. Specific experimental parameters were set as follows: a high-concentration ligand solution (500 µM) was loaded into the titration needle, and a low-concentration protein solution (50 µM) was added to the sample cell; the initial delay time was set to 120 s, the stirring speed to 750 rpm, the titration interval to 180 s, and the titration volume to be 2 µL for a total of 19 titrations. The change in heat power was monitored in real time during the experiment to obtain the original thermodynamic curves. The nonlinear fitting analysis of the binding curves was performed using Origin 7.0 software to calculate the dissociation constant (…). K d Thermodynamic parameters such as )

[0021] Figure 1 A is a schematic diagram of the structural domains of human EMBOW and WDR5. Figure 1 B is a schematic diagram illustrating the binding ability between the full-length EMBOW / EMBOW mutant and WDR5, where "+" indicates binding and "-" indicates non-binding. 1C is an ITC binding curve showing the interaction between the full-length EMBOW / EMBOW mutant and WDR5. 1D is a quantitative numerical statistical graph of the ITC binding affinity between the full-length EMBOW / EMBOW mutant and WDR5, obtained from the dissociation constant. K d The results show that the total length of the EMBOW 1-120 With WDR5 K d The concentration was 0.99 ± 0.06 µM, and three EMBOW mutants (EMBOW) retained the N-terminal fragment. 1-30+61-120 EMBOW 1-60+91-120 and EMBOW 1-90 It has a similar affinity to WDR5. K d The values ​​are 1.01 ± 0.05 µM, 1.01 ± 0.08 µM, and 0.91 ± 0.03 µM, respectively, which are close to the full-length EMBOW. 1-120 The binding ability of EMBOW, while the EMBOW mutant lacking the N-terminal fragment, is... 31-120 The detection showed that it did not bind to WDR5, indicating that the N-terminal region is essential for the interaction between EMBOW and WDR5.

[0022] EMBOW was prepared 1-10 With EMBOW 11-120 The same method was used to determine its dissociation constant ( Kd ), and the results are shown in Figure 1 C and 1D, EMBOW 1-10 binding to WDR5 K d was 0.76 ± 0.07 µM, while EMBOW 11-120 did not bind, further confirming that the interaction of EMBOW with WDR5 is mainly mediated by the first ten amino acids at the N-terminus of EMBOW.

[0023] 2.2, Interaction site of EMBOW with WDR5 resolved by crystal diffraction Screening of protein crystallization conditions: Mosquito crystal protein crystal spotting robot was used to respectively add EMBOW 1-120 -WDR5 protein samples and EMBOW 1-10 -WDR5 protein samples in 96-well sitting plates, and Crystal Screen buffer (Crystal, pEGRX, MD1-46, etc.) was used for protein complex crystallization condition screening. The concentration of the protein in the plate was optimized, the protein crystallization fragment was changed, the molar ratio of the protein to the ligand, the ratio of the crystallization solution, and the temperature and humidity of the crystallization were adjusted, and higher quality protein complex crystals were obtained. Before crystal diffraction, soak in crystal protection solution containing 20% glycerol, and store in liquid nitrogen.

[0024] Diffraction data and structure resolution: The diffraction data of all crystals were collected at 100K at Shanghai Synchrotron Radiation Facility (SSRF). The diffraction data of WDR5-EMBOW 1-120 were collected at BL02U1 line station, and the diffraction data of other WDR5-EMBOW 1-10 , etc. were collected at BL19U1. HKL2000 software package was used for indexing, integration and merging. The PDB2H14 structure of WDR5 was used as the search model for molecular replacement, and all structures were resolved by Phaser. REFMAC and Coot were used for structure refinement, manual modeling and analysis.

[0025] Crystal structure shows that EMBOW 1-120 and EMBOW 1-10 both mainly bind to the WIN site of WDR5 through the first six amino acids at their N-terminus, and the interaction of the two is almost the same, and R2 is a key structural determinant for the formation of EMBOW 1-120 and EMBOW 1-10 complex with WDR5.

[0026] 3, Inhibition of EMBOW on the interaction of MLL1 and WDR5 protein 3.1 EMBOW 1-120 Effect on H3K4 methyltransferase activity in vitro MLL1 binds to the WIN site of WDR5 through its WIN motif, which is essential for the histone H3K4 methyltransferase activity of MLL1 complex. Comparing the crystal structures of EMBOW-WDR5 and MLL1-WDR5 complex shows that both bind to the WIN site of WDR5 and bind to the key residues through similar interactions. Given that EMBOW has a strong binding affinity to WDR5 and the interaction of WDR5 with MLL1 is essential for the H3K4 methyltransferase activity of MLL1 complex, we further tested the effect of EMBOW mutants on the protein interaction of MLL1 with WDR5 by in vitro methyltransferase activity assay. 1-120 R2A, and tested the effect of EMBOW 1-120 R2A on the protein interaction of MLL1 with WDR5.

[0027] The specific experimental method of in vitro methyltransferase activity assay is as follows: AlphaLISA method was used to evaluate the inhibitory effect of full-length EMBOW 1-120 protein on the methyltransferase activity of MLL1 core complex (MLL1, Ash2L, RbBP5, WDR5). First, prepare 1x reaction buffer (50 mM Tris-HCl, pH 9.0), sample solution (containing full-length EMBOW 1-120 protein or its mutant EMBOW 1-120 R2A, with an initial concentration of 100 µM, followed by ten 5-fold dilutions to obtain a concentration gradient solution), 3x enzyme solution (5 nM MLL1 complex), and 3x substrate solution (0.1 µM histone H3 (1-21) peptide and 2 µM S-adenosyl methionine SAM). At the beginning of the experiment, different concentrations of sample solution were added to each well of a 384-well plate, followed by the addition of 3x enzyme solution (except for the enzyme-free control), and then centrifuged and incubated at room temperature for 15 minutes. Then add 3x substrate solution to start the reaction, and after centrifugation, incubate at room temperature for 60 minutes. Then add AlphaLISA amplification buffer and reagents, prepare 1x detection buffer, and add anti-methylated histone H3K4me1 / 2 capture beads and Strep-Tactin® emission beads at a concentration of 2x, and finally add 15 µL per well. Centrifuge again, incubate at room temperature in the dark for 60 minutes, and then use a multimode reader (Enspire or EnVision) to measure the RFU value in Alpha mode (Ex680 / Em615). Use GraphPad Prism 7.0 to curve fit the data and calculate the IC 50 value. The results are as follows:Figure 2 As shown in A, EMBOW 1-120 It can significantly inhibit the methyltransferase activity of the MLL1 complex (IC50). 50 =116.61 nM), while EMBOW 1-120 The R2A mutant exhibited a weaker inhibitory effect, further confirming that R2 is an inhibitor of EMBOW. 1 -120 and EMBOW 1-10 A key structural determinant in the formation of the complex with WDR5.

[0028] 3.2 EMBOW 1-120 Effects of H3K4 trimethylation on MV-4-11 leukemia cells Western blot was used to further determine EMBOW 1-120 and EMBOW 1-120 The effect of R2A on H3K4 trimethylation in MV-4-11 leukemia cells. The results are shown in 2B, indicating that EMBOW... 1-120 It can also significantly inhibit H3K4 trimethylation in MV-4-11 leukemia cells, while EMBOW 1-120 The R2A mutant does not have an inhibitory effect.

[0029] 3.3 EMBOW 1-120 right MLL1 Effects of gene rearrangement on the proliferation of patient-derived leukemia cell line MV-4-11 EMBOW was further evaluated. 1-120 and EMBOW 1-120 Effect of R2A on the proliferation of MLL1 rearranged patient-derived leukemia cell line MV-4-11, human leukemia cell line MV-4-11 ( MLL1-AF4 Cells were cultured in Iscove's Modified Dulbecco's Medium (IMDM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. STR identification was performed, and PCR detection was used to ensure the absence of mycoplasma contamination. Cell proliferation was detected using the CCK-8 reagent according to the kit instructions. Cells were seeded in 96-well plates (5000 cells per well) and cultured overnight for 12 hours. Then, they were transfected with Lipofectamine 2000 into the empty pcDNA 3.1 vector or the vector containing pcDNA 3.1-EMBOW. 1-120 plasmid or pcDNA 3.1-EMBOW 1-120Plasmid R2A, and incubated for another 48 hours. After adding 10 μL CCK-8 reagent, the cells were incubated at 37 °C for 2 hours. The absorbance was detected at 450 nm wavelength using a microplate reader, and the data were analyzed using GraphPad Prism 7.0. The results are shown in Figure 2 C. The results of the CCK8 cell viability experiment showed that overexpression of EMBOW 1-120 significantly inhibited the proliferation of MV-4-11 cells, while EMBOW 1-120 R2A mutants had no effect on the proliferation of MV-4-11 cells.

[0030] 3.4 EMBOW 1-120 R2A mutants had no effect on the proliferation of MV-4-11 cells. HoxA9 and Meis1 expression of EMBOW R2A significantly inhibited the proliferation of MV-4-11 cells. 1-120 and EMBOW 1-120 R2A significantly inhibited the proliferation of MV-4-11 cells. HoxA9 and Meis1 R2A mutants had no effect on the proliferation of MV-4-11 cells. 1-120 and EMBOW 1-120 R2A mutants had no effect on the proliferation of MV-4-11 cells. Figure 2 D. The results showed that EMBOW 1-120 significantly reduced the mRNA levels of HoxA9 and Meis1 EMBOW 1-120 R2A mutants had no effect on the proliferation of MV-4-11 cells. p <0.05 and p <0.001).

[0031] The above results showed that EMBOW 1-120 by blocking the interaction between MLL1 and WDR5, leading to a decrease in H3K4me3 levels, further down-regulating the expression of two key target genes of MLL1 HoxA9 and Meis1 and thereby inhibiting the proliferation of MV-4-11 cells.

[0032] II. EMBOW-derived peptides 1. Binding affinity of EMBOW-derived peptides to WDR5 Based on the above research, EMBOW 1-120 was designed as a peptide inhibitor of WDR5. The first 10 amino acids of EMBOW 1-120 EMBOW 1-10MRTEPRPPAP, and the first 3, 5, 6, 7, 8, 10 amino acids of the amino acid sequence are designed into peptide segments, and the peptide segments are modified at both ends, wherein an acetyl group is introduced at the N-terminus, which can protect the N-terminus on one hand, and the introduction of the acetyl group can increase the stability of the peptide segment by forming intramolecular hydrogen bonds, and at the same time, the binding force of the derivative peptide with WDR5 is improved by conformational change. The C-terminus of the peptide end is protected by modification, and the C-terminus of the peptide end can be protected by any one of amidation, esterification, thioesterification, biotinylation or polyethylene glycol modification, or the C-terminal carboxyl group is reduced to aldehyde or alcohol, converted to hydrazine or hydrazide, or cyclized by any one of the following reactions to obtain a protective modification. The above-mentioned various modifications of the C-terminus can protect the C-terminal carboxyl group. In this embodiment, the N-terminus is modified by an acetyl group, and the C-terminus is modified by an amino group, and the EMBOW derivative peptides are prepared, and the prepared derivative peptides are named as Ac3, Ac5, Ac6, Ac7, Ac8 and Ac10, respectively. The structures of the derivative peptides are as follows: Ac3: Ac-MRT-NH2 Ac5: Ac-MRTEP-NH2 (SEQ ID NO: 4) Ac6: Ac-MRTEPR-NH2 (SEQ ID NO: 5) Ac7: Ac-MRTEPRP-NH2 (SEQ ID NO: 1) Ac8: Ac-MRTEPRPP-NH2 (SEQ ID NO: 6) Ac10: Ac-MRTEPRPP-NH2 (SEQ ID NO: 7), wherein Ac- is an acetyl group modified at the N-terminus, and -NH2 is an amino group modified at the C-terminus.

[0033] The first 3, 5, 7, 10 amino acid peptide segments are used as controls, and the peptide segments are named as WT3, WT5, WT7 and WT10, respectively, and the structures of the peptide segments are as follows: WT3: MRT WT5: MRTEP WT7: MRTEPRP WT10: MRTEPRPPAP The dissociation constants (Kd) of the derivative peptides Ac3, Ac5, Ac6, Ac7, Ac8 and Ac10, and the controls WT3, WT5, WT7, WT10 are determined by isothermal titration calorimetry (ITC) experiments. K d The results are shown in Table 1.

[0034] Table 1

[0035] From the above table, it can be seen that the dissociation constant of the derivative peptide with N-terminal acetyl group modification compared with the control without N-terminal acetyl group modification is significantly lower, showing stronger binding affinity. The N-terminal acetyl group modification in the derivative peptide significantly enhances its binding affinity, and Ac6, Ac7 and Ac8 have higher affinity than other derivative peptides. K d )is significantly lower, showing stronger binding affinity. The N-terminal acetyl group modification in the derivative peptide significantly enhances its binding affinity, and Ac6, Ac7 and Ac8 have higher affinity than other derivative peptides.

[0036] Co-crystallization and crystal structure analysis of derivative peptides Ac3, Ac5, Ac7, Ac10, control WT10 and WDR5 were performed, and the specific method was the same as that in “1”. The crystal structures of Ac3-WDR5, Ac5-WDR5, Ac7-WDR5, Ac10-WDR5 and WT10-WDR5 are shown in Figs. Figure 3 A-E, respectively. With the increase of peptide length, they interact more with the WIN site of WDR5. However, only the first six residues continuously interact with WDR5 in the structure, explaining why the binding affinity of Ac6 peptide reaches a plateau. By comparing the structures of EMBOW 1-10 (or WT10) and EMBOW 1-120 -WDR5 complexes, it is found that the introduction of N-terminal acetyl group in Ac3, Ac5, Ac7, Ac10 peptides forms two intramolecular hydrogen bonds in the peptide, increasing the stability of the derivative peptide, and the introduction of acetyl group causes the flip of the first residue. The acetyl group occupies the binding site of M1 residue in EMBOW 1-10 and EMBOW 1-120 complexes, thereby displacing the M1 residue. The results show that the M1 residue is more tightly embedded in the binding pocket formed by D107, Y131, F133 and F149 in Ac3, Ac5, Ac7 and Ac10 peptides, thus significantly improving the binding of the derivative peptide to WDR5.

[0037] 2. Inhibition effect of derivative peptides on H3K4 methyltransferase in vitro The effects of derivative peptides Ac3, Ac5, Ac6, Ac7 and Ac8 on the inhibition of H3K4 methyltransferase activity of MLL1 core complex on H3K4 in vitro were tested, using EMBOW 1-10 R2A mutant and SAH (S-adenosylhomocysteine, a potent product inhibitor of most methyltransferases, which is used as a methyltransferase inhibitor of MLL1 complex here) as negative and positive controls, respectively. The test method is the same as that in “1.3.1”. The activity curves and IC 50 as shown in Fig. Figure 3 F, EMBOW 1-10R2A mutants failed to inhibit H3K4 methyltransferase activity, SAH showed moderate inhibition, IC 50 = 1181.15 nM, among all the derived peptides, Ac6 (IC 50 = 123.11 nM), Ac7 (IC 50 = 135.21 nM) and Ac8 (IC 50 = 67.00 nM) showed significantly superior inhibitory activity.

[0038] Next, the effects of the above derived peptides on intracellular H3K4 methylation levels, leukemia cell proliferation and related gene expression were further tested, and the schematic diagram of the experiment is shown in Figure 4 A.

[0039] 3, Effects of derived peptides on intracellular H3K4 methylation (monomethyl, dimethyl and trimethyl) levels In view of the strong binding affinity of EMBOW derived peptides to WDR5 and the significant inhibitory effect on H3K4 methyltransferase activity in vitro, the effects of Ac3, Ac5, Ac6, Ac7, Ac8 and Ac10 on intracellular H3K4 methylation (monomethyl, dimethyl and trimethyl) levels were systematically evaluated, and no addition was added as a control. β-actin as an internal reference.

[0040] Western blot was used to detect the levels of H3K4 methylation (monomethyl, dimethyl and trimethyl). The specific steps of Western blot are as follows: protein was extracted with RIPA lysis buffer, separated by SDS-PAGE and transferred to PVDF membrane, blocked with 5% skim milk, then incubated with primary antibody according to the recommended ratio of the manufacturer. After washing the membrane, incubate with HRP labeled secondary antibody (anti-rabbit or anti-mouse) for 2 hours, the antibodies used include: anti-H3K4me1, H3K4me2, H3K4me3 and anti-β-actin, color development with ultra-sensitive ECL reagent, image using chemiluminescence imaging system, quantitative analysis using ImageJ software, statistics completed by GraphPad Prism 7.0.

[0041] The results are shown in Figure 4 B-D, among all the tested peptides, Ac7 inhibited histone H3K4 methylation (me1, me2 and me3) most significantly (P p <0.01, p <0.001 and p <0.001).

[0042] 4, Effects of derived peptides on MLL1 proliferation of leukemia cell lines derived from patients with gene rearrangement Further evaluation was conducted on the derived peptides Ac3, Ac5, Ac6, Ac7, Ac8, and Ac10 in two groups with... MLL1 The patient-derived leukemia cell line MV-4-11 (carrying gene rearrangements) MLL1 - AF4 ) and MOLM-13 (carrying MLL1 - AF9 The effect of the peptide on cell proliferation was investigated, and cell viability was assessed using the CCK-8 assay. The specific experimental method was as follows: 5000 cells / well were seeded into 96-well plates. Peptides were added to the culture medium at final concentrations of 500 µM, 100 µM, 20 µM, 4 µM, 0.5 µM, and 0.1 µM, respectively, and the plates were cultured for 48 hours. Then, 10 µL of CCK-8 reagent was added to each well, and the plates were incubated at 37°C for 2 hours. The absorbance was measured at 450 nm using a microplate reader. Results are as follows: Figure 4 As shown in E and 4F, all derived peptides Ac3, Ac5, Ac6, Ac7, Ac8, and Ac10 inhibited the proliferation of both MV-4-11 and MOLM-13 cell lines. Among Ac7, Ac7 showed the most significant inhibitory effect, with an IC50 of [missing value] against both MV-4-11 and MOLM-13. 50 The values ​​were 58.74 ± 3.12 µM and 79.49 ± 5.07 µM, respectively. These results indicate that the derived peptide Ac7 exhibited the strongest antiproliferative activity in the aforementioned leukemia cells MV-4-11 and MOLM-13.

[0043] To evaluate the antiproliferative specificity of the derived peptide Ac7 in different cell types, in the leukemia cell line K562 (non-leukemia) MLL1 Cytotoxicity tests were performed on gene-rearranged cell lines, and the results were as follows: Figure 4 As shown in G, Ac7 did not show significant inhibitory effects in K562 cells, indicating that the anti-leukemic activity of Ac7 depends on the presence of the MLL1 fusion protein, highlighting the specificity of its inhibition.

[0044] The above results indicate that the derived peptide Ac can be used as a therapeutic peptide to inhibit... MLL1 Proliferation of gene-rearranged leukemia cells.

[0045] 5. Derived peptide pairs HoxA9 and Meis1 Suppression of expression Further analysis of the effects of derived peptides Ac3, Ac5, Ac6, Ac7, Ac8, and Ac10 on... HoxA Family genes ( HoxA7 , HoxA9 ) and its cofactors Meis1 The expression of the result is shown in the following experimental steps: 1×106 Cells were seeded in 96-well plates at 1,000 cells / well, and after the addition of polypeptides to each group of medium at a final concentration of 100 µM, the cells were further incubated for 24 hours. According to the manufacturer's instructions, total RNA was isolated using the FastPure® Cell / Tissue Total RNA Isolation Kit V2 kit (RC112, Norgen) and reverse transcribed into cDNA using the HiScript® III 1st Strand cDNA Synthesis Kit (+gDNA wiper) kit (R312, Norgen). Then, according to the manufacturer's instructions, RT-qPCR was performed on the cDNA template using the Taq Pro Universal SYBR qPCR Master Mix (Q712, Norgen). The specific primers used for RT-qPCR detection were designed as shown in Table 2 below, and were synthesized and purchased from Shengong Bioengineering (Shanghai) Co., Ltd. Quantitative RT-PCR and analysis were performed using a Bio-Rad CFX96 PCR system. GADPH protein was used as an internal reference. The relative expression level of the target gene was determined using the 2−ΔΔCt method.

[0046] Table 2

[0047] Results as shown in Figure 4 H-J, Ac7 had the strongest inhibitory effect on the expression levels of HoxA7 , HoxA9 and Meis1 ( p <0.01, p <0.01 and p <0.001), with inhibition rates of 51.20%, 60.23%, and 41.15%, respectively. Ac7 had excellent down-regulation effects on the expression of key target genes HoxA7 , HoxA9 and Meis1 .

[0048] In summary, the derived peptide Ac7 consistently showed the strongest effect in inhibiting H3K4 methylation, leukemia-related gene expression, and MLL1 gene rearrangement type leukemia cell proliferation. The application prospect of Ac7 as a therapeutic peptide is worthy of further development for the treatment of MLL1 gene rearrangement type leukemia.

[0049] 6. Anti-leukemia efficacy and safety of the derived peptide Ac7 in a MV-4-11 xenograft mouse model The derived peptide Ac7 showed good anti-leukemia activity in vitro, and was therefore preliminarily evaluated in a MV-4-11 xenograft mouse model to explore its anti-leukemia effect in vivo, as shown in Figure 5 A, to explore the tumor volume growth inhibition percentage (GI) and tumor growth inhibition percentage (TGI) of the derived peptide Ac7 in mice, and to evaluate its anti-tumor effect and drug safety by TUNEL staining and HE staining, respectively.

[0050] The specific experimental methods are as follows: 6-week-old male BALB / c nude mice were provided by Jiangsu Huacheng Co., Ltd. and were raised under specific pathogen-free (SPF) conditions with a 12-hour light cycle. After one week of adaptation, they were randomly divided into 4 groups (6 per group) by weight. Logarithmic growth phase MV-4-11 cells were collected, washed with PBS, mixed with Matrigel™ matrix (1:1) to prepare a cell suspension of 5 × 10 7 6 / mL. Each mouse was injected subcutaneously with 200 μL in the right flank. After the tumor volume reached 100 ± 10 mm³, 20 mg / kg, 40 mg / kg and 80 mg / kg of Ac7 dissolved in normal saline were injected through the tail vein. The control group received the same volume of solvent. Tumor volume and animal body weight changes were monitored every two days, and the animals were sacrificed after 14 days, the tumors were removed, measured and weighed. The tumor volume was calculated using the formula V = L × W² × 0.5. The evaluation indicators included tumor volume growth inhibition percentage (GI, %) and tumor growth inhibition percentage (TGI, %). All animal experiments were approved by the Animal Protection Committee of Central China Normal University and were performed in accordance with the American NIH Guide for the Care and Use of Laboratory Animals. Statistical analysis was performed using GraphPad Prism 7.0.

[0051] Tumor volume, weight and mouse body weight are shown in Figure 5 B-D, compared with the control group, the tumor volume growth inhibition rate (GI) of the 20, 40 and 80 mg / kg dose groups was 17.91%, 60.43% and 66.97% (P < 0.01, p <0.01, p <0.001 and p <0.001), and the corresponding tumor weight growth inhibition rate (TGI) was 16.40%, 51.37% and 58.33% (P < 0.01, p <0.01, p <0.001 and p <0.001). No significant changes in animal behavior or body weight fluctuations were observed during the overall treatment, indicating good tolerance.

[0052] To further explore the anti-tumor mechanism of the derivative peptide Ac7, tumor tissues were collected after treatment, and the anti-tumor effect was evaluated by TUNEL staining. The specific operation of TUNEL staining is as follows: the tumor tissues from the sacrificed mice were fixed in 4% paraformaldehyde for 24 hours, dehydrated by gradient alcohol (70%, 80%, 90%, and 100% alcohol, 1 hour each step), and embedded in paraffin. Cut into 3 µm thick sections, deparaffinized with xylene for 10-15 minutes, and rehydrated with decreasing concentrations of alcohol (100%, 90%, and 70%). Pretreated with proteinase K (20 µg / mL) at 37°C for 15-30 minutes. Block endogenous peroxidase with 3% hydrogen peroxide, then incubate with TUNEL reagent from the TUNEL kit at 37°C for 60 minutes. Wash with PBS to stop the reaction, add streptavidin-horseradish peroxidase and incubate for 30 minutes, then develop with DAB. Nuclei were stained with hematoxylin, followed by dehydration, transparency, and mounting. Photographed with a microscope, and the proportion of TUNEL-positive cells was analyzed using ImageJ. The results are shown in Figure 5 As shown in Figures 5E and 5F, the number of apoptotic cells in the Ac7 dose groups (20, 40, and 80 mg / kg) increased in a dose-dependent manner p <0.01, p <0.001, and p <0.001.

[0053] To systematically evaluate the safety of the derivative peptide Ac7 in vivo, histopathological analysis of major organs (heart, liver, spleen, lung, and kidney) was performed after treatment using HE staining. The specific experimental operation is as follows: after Ac7 treatment, the animals were sacrificed, and the heart, liver, spleen, lung, and kidney were collected and fixed in 10% formalin for 24 hours. After fixation, the tissues were gradually dehydrated by gradient alcohol solutions (70%, 80%, 90%, and 100% ethanol), 1 hour each step, and then embedded in paraffin wax. Thin sections (3 µM) were cut and placed on glass slides. The sections were deparaffinized in xylene for 10-15 minutes, and then rehydrated by gradually reducing the concentration of ethanol (100%, 90%, and 70% ethanol) for 5 minutes each time. After rehydration, the sections were stained with hematoxylin for 3-6 minutes, washed, and then stained with eosin for 2-3 minutes. After staining, the sections were dehydrated again with gradually increasing concentrations of ethanol (70%, 90%, and 100%) for 5 minutes each, then washed with xylene for 10 minutes, and finally mounted with a cover glass. The histological changes of the tissues were observed under a microscope, and representative images were taken for analysis. The results are shown in Figure 6 As shown in Figure 5G, the histological structure of all organs was intact, and no pathological changes caused by the drug were observed, including necrosis, inflammatory infiltration, or degenerative changes.

[0054] The above results show that the derivative peptide Ac7 exhibits strong anti-leukemia activity in vivo, and has good safety and very low systemic toxicity in the used dose range. It has the value of preclinical development as a potential drug for treating leukemia.

[0055] The above merely describes the preferred embodiments of the present application and is not intended to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. An EMBOW-derived peptide targeting the WDR5-MLL1 protein interaction, characterized in that, The amino acid sequence of the EMBOW-derived peptide is shown in SEQ ID NO: 1, which has an acetylation modification at the N-terminus and a protective modification at the C-terminus.

2. The EMBOW-derived peptide targeting WDR5-MLL1 protein interaction according to claim 1, characterized in that, The C-terminus of the EMBOW-derived peptide has any one of acetylation, esterification, thioesterification, biotinylation or pegylation modification, or the C-terminal carboxyl is protected by any one of the following reactions: reduction of the C-terminal carboxyl to aldehyde or alcohol, conversion to hydrazine or hydrazide, or cyclization.

3. The EMBOW-derived peptide targeting WDR5-MLL1 protein interaction according to claim 2, characterized in that, The C-terminus of the EMBOW-derived peptide is modified with an amino group.

4. Use of the EMBOW-derived peptide targeting the WDR5-MLL1 protein interaction in the preparation of a WDR5-MLL1 protein interaction inhibitor according to any one of claims 1-3.

5. Use of the EMBOW-derived peptide targeting the WDR5-MLL1 protein interaction in the preparation of a MLL1 methyltransferase activity inhibitor according to any one of claims 1-3.

6. Use of the EMBOW-derived peptide targeting the WDR5-MLL1 protein interaction in the preparation of a drug for reducing the level of histone H3K4 methylation according to any one of claims 1-3.

7. Use of an EMBOW-derived peptide targeting the WDR5-MLLl protein interaction for the preparation of a medicament for inhibiting the proliferation of a leukemia cell of the gene rearrangement type. MLL1 leukemia.

8. Use of an EMBOW-derived peptide targeting the WDR5-MLL1 protein interaction for the preparation of a medicament for inhibiting the expression of leukemia-associated genes HoxA7 、 HoxA9 and co-factors Meis1 .

9. Use of an EMBOW-derived peptide targeting the WDR5-MLLl protein interaction for the manufacture of a medicament for the treatment of a gene rearrangement type leukemia according to any one of claims 1 to 3. MLL1 9. Use of an EMBOW-derived peptide targeting the WDR5-MLLl protein interaction for the manufacture of a medicament for the treatment of a gene rearrangement type leukemia according to any one of claims 1 to 3.

10. Use according to claim 9, characterized in that, The drug is an intravenous injection.

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