A polypeptide inhibitor targeting WDR5 and its use

Abstract

The present invention provides a polypeptide inhibitor targeting WDR5, whose amino acid sequence is Ala-Arg-Ala-Gln. The inhibitor is synthesized based on the four amino acids at the N-terminus of WDR5. The inhibitor can competitively inhibit the binding between the N-terminus of WDR5 itself and the WD40 domain, disrupt the phase separation of WDR5, and destroy the interaction between WDR5 and other proteins, thereby inhibiting the growth of tumor cells and ultimately achieving the purpose of cancer suppression. The inhibitor has potential value for biopharmaceutical development.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a polypeptide inhibitor targeting WDR5 and uses thereof. Background Art

[0002] WDR5 is a highly conserved WD40 family protein that functions as a core scaffold component in a variety of protein complexes, controlling transcriptional regulation and histone code modification as part of the epigenetic machinery. WDR5 consists of 334 amino acids, with a disordered structure of approximately 30 residues at its N-terminus and seven WD40 repeats at its C-terminus, forming a characteristic seven-bladed β-propeller structure resembling a doughnut. This structure provides two major binding sites for WDR5 as the interface for complex formation: a shallow cleft on one surface, termed the "WDR5 binding motif" (WBM), and an arginine-binding cavity on the other, termed the "WDR5 interaction" (WIN) site (Guarnaccia and Tansey, 2018).

[0003] The most well-studied interaction is between WDR5 and MLL1 (mixed lineage leukemia 1). By recognizing the WIN sequence, WDR5 forms an H3K4 methyltransferase complex with MLL1, RBBP5 (retinoblastoma binding protein 5), ASH2L (absent, small, orhomeotic 2-like), and DPY-30 (Dumpy-30). MLL1 is often dysregulated in acute myeloid leukemia and lymphocytic leukemia. The common defect is a gene translocation on chromosome 11, which leads to the formation of leukemic MLL1 fusion proteins. MLL1 fusion proteins lack the SET domain (SET domains are named after the first letters of the three genes where such domains were first discovered: Suppressor of variegation 3-9, Enhancer of zeste, and Trithorax. SET domains are contained in most transferases and are responsible for the enzymatic activity of methyltransferases) and therefore lack H3K4 methyltransferase activity. They synergize with wild-type MLL1 complexes to activate MLL1 target genes and promote leukemia (Karatas et al., 2013). MLL1 methyltransferase activity alone is weak, but disrupting the WDR5-MLL1 interaction significantly inhibits H3K4 methyltransferase activity, thereby suppressing the aberrant expression of downstream genes. Therefore, targeting the H3K4 methyltransferase activity of MLL1 may be a promising strategy for treating leukemias harboring MLL1 fusion proteins (Thiel et al., 2010).

[0004] WDR5 also associates with MYC through the WBM locus, where it is a key cofactor required for recruiting MYC to a subset of its genomic targets. WDR5 colocalizes with MYC on the chromatin of key target genes, and MYC requires WDR5 for efficient chromatin recognition of target genes (Thomas et al., 2015). The WDR5-MYC axis has been shown to be critically involved in the development and progression of pancreatic cancer (Carugo et al., 2016). Disrupting the interaction between MYC and WDR5 reduces MYC binding to approximately 80% of its chromosomal locations and prevents it from promoting induced pluripotent stem cell formation and driving tumorigenesis (Thomas et al., 2015).

[0005] In addition to the cancers mentioned above, overexpression of WDR5 is associated with adverse clinical outcomes in many human cancers, including neuroblastoma (Sun et al., 2015), breast cancer (Dai et al., 2015), bladder cancer (Chen et al., 2015), lung cancer (Xie et al., 2017), and colorectal cancer (Tan et al., 2017). Given the multifaceted roles of WDR5 in human malignancies, WDR5 has become an attractive drug target for the development of peptide or small molecule inhibitors.

[0006] In addition, in recent years, more and more research evidence has shown that phase separation is related to the pathological processes of various diseases. Research by Gang Greg Wang's team at the University of North Carolina at Chapel Hill and others has demonstrated that phase separation is a driving factor in the formation of cancer (Ahn et al., 2021). Phase separation is ubiquitous in cells, similar to the phenomenon of oil-water separation. Once molecules reach a certain concentration, they can phase separate, bringing similar components together to form signal bodies to accelerate reactions and achieve various biological functions. Research on the mechanism of phase separation will provide a theoretical basis for effective target molecules, and targeted phase separation has also become a direction for drug development with great potential. Therefore, it is possible to consider designing peptides or small molecule inhibitors that target phase separation to regulate protein function and thus develop new drugs. Summary of the Invention

[0007] The purpose of the present invention is to provide a polypeptide inhibitor targeting WDR5, which disrupts the phase separation of WDR5 and the interaction between WDR5 and other proteins, thereby playing a tumor suppressor role.

[0008] Another object of the present invention is to design a small molecular weight polypeptide inhibitor based on the structural characteristics of WDR5.

[0009] The technical solution of the present invention is to provide a polypeptide inhibitor targeting WDR5 (SEQ ID NO. 5), and its application in inhibiting the methyltransferase activity of the MLL1 protein complex and inhibiting tumor growth.

[0010] Specifically, the present invention provides the following technical solutions:

[0011] In one aspect, the present invention provides a polypeptide inhibitor targeting WDR5, characterized in that its amino acid sequence is Ala-Arg-Ala-Gln.

[0012] In some embodiments, the polypeptide inhibitor is characterized in that it competitively inhibits the binding of the N-terminus of WDR5 to its own WD40 domain.

[0013] In some embodiments, it is characterized in that the polypeptide inhibitor is synthesized based on the N-terminal four amino acids that bind to the WD40 domain.

[0014] In another aspect, the present invention provides an isolated nucleotide sequence, characterized in that the nucleotide sequence encodes the above-mentioned polypeptide inhibitor.

[0015] On the other hand, the present invention provides an expression vector, characterized in that the expression vector comprises the above-mentioned nucleotide sequence.

[0016] In another aspect, the present invention provides a host cell, characterized in that the host cell comprises the above-mentioned expression vector.

[0017] In another aspect, the present invention provides a pharmaceutical composition, characterized in that it comprises the above-mentioned polypeptide inhibitor and a pharmaceutically acceptable carrier.

[0018] In some embodiments, the pharmaceutically acceptable carrier includes, but is not limited to, excipients, adjuvants, fillers, sweeteners, disintegrants, wetting agents, and lubricants.

[0019] In some embodiments, the pharmaceutically acceptable carrier is selected from the group consisting of excipients, adjuvants, fillers, sweeteners, disintegrants, wetting agents, and lubricants.

[0020] In another aspect, the present invention provides use of the above polypeptide inhibitor in the preparation of a drug for treating tumors.

[0021] In some embodiments, the tumor is selected from leukemia, pancreatic cancer, neuroblastoma, breast cancer, bladder cancer, lung cancer, and colorectal cancer.

[0022] In some embodiments, the leukemia is acute myeloid leukemia or lymphocytic leukemia.

[0023] In some embodiments, the pancreatic cancer is adenocarcinoma.

[0024] In some embodiments, the pancreatic cancer is metastatic pancreatic cancer and / or in situ pancreatic cancer.

[0025] In some embodiments, the pancreatic cancer is metastatic pancreatic adenocarcinoma and / or in situ pancreatic adenocarcinoma.

[0026] definition

[0027] Polypeptide: A polypeptide is a compound formed by multiple amino acids linked by peptide bonds.

[0028] Peptide inhibitors are relatively short peptides, typically composed of a few to twenty amino acids, that can target proteins, reducing their activity or inhibiting biochemical reactions. Peptide inhibitors have a well-defined structure and are easy to synthesize. Compared to small molecule inhibitors, peptide inhibitors are primarily cleared through hydrolysis and renal filtration. The hydrolysis products are amino acids, so the toxicity of their metabolites is very low. Furthermore, peptide inhibitors are often specifically designed using endogenous peptides as templates and generally have high target affinity.

[0029] WDR5 (WD40 repeat protein 5): WDR5 is a highly conserved WD40 family protein and a key member of the human MLL and SET1 histone H3K4 methylase complex. It contains seven WD40 repeats arranged in a β-propeller pattern, which normally acts as a scaffold. WDR5 also plays a key role in the development and progression of various cancers.

[0030] Phase separation: Phase separation is one of the key mechanisms regulating the compartmentalized distribution of biomacromolecules (such as proteins and nucleic acids) in cells. Phase-separating proteins can be divided into two categories based on the different mechanisms involved in the phase separation process: one that spontaneously assembles to form phase-separated aggregates, and the other that relies on interactions with other biomacromolecules to undergo phase separation.

[0031] WD40: The typical WD40 domain is a propeller-shaped structure composed of seven repeating blades, each containing 40-60 residues and folded into four antiparallel β-strands. Repeat sequences and domains are conventionally named using the conserved tryptophan-aspartate (WD) motif and a length of 40. Often, a β-strand shift occurs between the repeating blades in the structure and the repeat units in the sequence, creating a "Velcro" that closes the β-propeller.

[0032] MLL1, also known as histone lysine methyltransferase 2 (KMT2A), is named for its role in inducing mixed-lineage leukemia. MLL1 is ubiquitous in human tissues, and the gene encoding it is located on chromosome 11q23. Following translation, the full-length MLL1 protein is cleaved by taspase 1 at residues 2666 / 2667 and 2718 / 2719, producing two peptides (the N-terminal and C-terminal peptides, MLL-C and MLL-N). These peptides are then linked by FYRN and FYRC, respectively, to form a fully active MLL1 protein.

[0033] Phase separation and cancer: Cancer development is closely linked to genetic abnormalities in proteins containing intrinsically disordered regions (IDRs). Research by Gang Greg Wang's team at the University of North Carolina at Chapel Hill and others has demonstrated that phase separation is a driver of cancer development (Ahn et al., 2021). Cancers acquire mutations through phase separation to establish oncogenic transcription factor condensates, which simultaneously enhance their genomic targeting and induce the formation of abnormal three-dimensional chromatin structure during neoplastic transformation. When normal liquid-liquid phase separation is disrupted by genetic or epigenetic mutations, abnormal biomolecular condensates may participate in tumorigenesis because they play a role in chromosome organization, signal transduction, and transcriptional dysregulation, thereby promoting cancer development. For example, abnormal expression and / or activity of stress granule components can lead to drug resistance and tumorigenesis in various cancers, including colorectal cancer (Bouchard et al., 2018), pancreatic cancer (Grabocka and Bar-Sagi, 2016), and leukemia (Podszywalow-Bartnicka et al., 2014). Cancer-associated SPOP (speckled POZ protein) mutations disrupt protein-substrate colocalization, affecting the protein's phase separation and tumor suppressor abilities (Bouchard et al., 2018). Because phase separation / phase transition is a highly complex process, many types of drugs, such as small molecules, antibodies targeting disordered regions, and designed peptides, can control the phase separation process and ultimately effectively treat cancer.

[0034] The relationship between WDR5 phase separation and cancer: WDR5 is a typical scaffold protein that plays an important role in regulating the biological function of phase separation (Alberti and Dormann, 2019). The phase separation protein WDR5 is often abnormally overexpressed in various cancers, affecting their ability to undergo normal and orderly phase separation, thereby affecting the corresponding biological processes that lead to tumorigenesis.

[0035] Beneficial results:

[0036] Research in the present invention shows that the polypeptide inhibitor (SEQ ID NO.5) can target the "donut" structure formed by WD40, competitively inhibit the binding between the N-terminus and C-terminus of WDR5 itself, disrupt the phase separation of WDR5 and the interaction between WDR5 and other proteins, inhibit the methyltransferase activity of the MLL1 protein complex, inhibit the proliferation of pancreatic cancer cells and leukemia cells, and play a tumor suppressor role.

[0037] Many drug targets, particularly kinases, have provided remarkable clinical results. However, many targets, such as RAS, MYC, and fusion proteins, are considered undruggable in cancer due to the lack of protein pockets amenable to small molecules (Dang et al., 2017). Interestingly, some anti-tumor drugs can selectively partition into aggregates, and cells can develop drug resistance by altering aggregate mechanisms (Klein et al., 2020), providing strong evidence that compounds can interfere with the misregulation of phase separation. Therefore, interfering with phase separation complexes may be a potential avenue for targeting undruggable proteins and making these undruggable targets druggable for treating disease.

[0038] The present invention proposes for the first time to inhibit the interaction between WDR5 and other proteins by destroying phase separation, thereby inhibiting the growth of related tumor cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1A The N-terminal amino acid of WDR5 protein was mutated in different protein buffers, and the effect on the formation of phase separation droplets was observed under a microscope.

[0040] Figure 1B The WDR5 protein N-terminal polypeptide (SEQ ID NO.1) and the mutated polypeptide (SEQ ID NO.2) were added to the WDR5 protein, incubated together in a protein buffer, and then their effects on the formation of WDR5 phase separation droplets were observed under a microscope.

[0041] Figure 2A and 2B After adding DMSO or polypeptide fragments of different sizes at the same concentration to the WDR5 protein, the areas of the formed phase separation droplets were observed under a microscope and counted.

[0042] Figure 3 Western-blot was used to detect the effect of polypeptide (SEQ ID NO.5) treatment on H3K4 trimethylation (H3K4me3) levels in MV4-11 cells for 96 hours.

[0043] Figure 4Pancreatic cancer cells AsPC-1 and BxPC-3 and leukemia cells MV4-11 were treated with different concentrations of the polypeptide (SEQ ID NO. 5), and cell viability was detected by CCK8.

[0044] The scale bars in the above experiments are all 5 μm. DETAILED DESCRIPTION

[0045] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments and with reference to the accompanying drawings.

[0046] The N-terminus of WDR5 is a disordered structure of approximately 30 residues, followed by seven WD40 repeats, which acts as a scaffold protein and can interact with various proteins, such as MLL1 through the WIN site. In addition to interacting with other proteins, interestingly, the N-terminal residues of WDR5 (aa11-17) can also bind to the top surface of the β-propeller structure of its own WD40 region (Guarnaccia and Tansey, 2018; Schuetz et al., 2006).

[0047] Previous reports suggest that WDR5 is a phase-separating protein (Zhou et al., 2019), and our in vitro experiments also confirmed that WDR5 is a phase-separating protein. While phase separation typically requires weak interactions, we hypothesize that WDR5 phase separation is driven by intermolecular interactions between the disordered N-terminal structure and the "donut" structure formed by WD40, and our research further supports this mechanism. The inventors synthesized an N-terminal sequence peptide (SEQ ID NO. 1) that binds to the WD40 region at GenScript, truncated it, and screened it, ultimately identifying the most suitable motif as the peptide inhibitor of the present invention and verifying its effectiveness in tumor cells.

[0048] The present invention studies the following aspects: 1. truncation and screening of polypeptide inhibitors; 2. protein immunoblotting detection of the effect of polypeptide fragments on H3K4 methyltransferase activity; 3. intracellular detection of the effect of polypeptide fragments on cancer cell proliferation.

[0049] The result:

[0050] We designed and synthesized a WDR5 N-terminal sequence peptide (SEQ ID NO.1) and determined the optimal peptide sequence (SEQ ID NO.5) through truncation screening. This disrupted the phase separation of WDR5 and the interaction between WDR5 and other proteins, thereby inhibiting the growth of tumor cells and playing a tumor suppressor role.

[0051] The present invention is further described in detail below with reference to the accompanying drawings:

[0052] Example 1 Truncation and screening of polypeptide inhibitors

[0053] The amino acid sequences of the linear peptide inhibitors of WDR5 (WDR5 is highly conserved, and the amino acid sequences at positions 11-17 of human WDR5 and mouse WDR5 are both EAARAQP) protein in this example are shown in Table 1. The sequences in the table were synthesized by Nanjing GenScript Biotechnology Co., Ltd.

[0054] The sequence of human WDR5 (SEQ ID NO.6) is as follows:

[0055] MATEEKKPETEAARAQPTPSSSATQSKPTPVKPNYALKFTLAGHTKAVSSVKFSPNGEWLASSSADKLIKIWGAYDGKFEKTISGHKLGISDVAWSSDSNLLVSASDDKTLKIWDVSSGKCLKTLKGHSNYVFCCNFNPQSNLIVSGSFDESVRIWDVKTGKCLKTL PAHSDPVSAVHFNRDGSLIVSSSYDGLCRIWDTASGQCLKTLIDDDNPPVSFVKFSPNGKYILAATLDNTLKLWDYSKGKCLKTYTGHKNEKYCIFANFSVTGGKWIVSGSEDNLVYIWNLQTKEIVQKLQGHTDVVISTACHPTENIIASAALENDKTIKLWKSDC

[0056] Table 1 Amino acid sequences of linear peptide inhibitors of WDR5 protein

[0057]

[0058]

[0059] This invention designs more effective peptide inhibitors targeting WDR5 based on the N-terminal domain of the WDR5 protein. The crystal structure of WDR5 reveals that its N-terminal residues (aa11-17) can bind to the top surface of the "donut" structure formed by the C-terminal WD40 domain (Guarnaccia and Tansey, 2018; Schuetz et al., 2006). Related reports (Zhou et al., 2019) and our experiments have confirmed that WDR5 is a phase-separating protein. Given the increasing evidence that phase separation is closely related to the occurrence and development of diseases and the important role of WDR5 in tumor development, we speculate that disrupting WDR5's weak self-interactions can inhibit its interactions with other proteins by inhibiting its phase separation, thereby suppressing related tumor growth.

[0060] The present invention synthesized a polypeptide based on the protein sequence of the N-terminal residues 11 to 17 of WDR5, and its sequence is shown in Table 1. Figure 1A As shown, we purified WDR5-GFP protein in vitro and incubated it with different protein buffers. For observation, a circle of Great Wall 7501 high vacuum silicone grease was first applied to a glass slide. The sample was then dropped onto the center of the slide, covered with a coverslip, and placed upside down on a 100x oil immersion lens. The sample was then observed using a laboratory confocal microscopy system.

[0061] The inventors first constructed a His-tagged WDR5-GFP plasmid and then purified the WDR5-GFP protein. The specific protein purification method is as follows:

[0062] 1) Prepare LB medium: Weigh 25 g of LB powder (Sangon, Catalog No. A507002-0250) into a culture flask, add 1 L of water, seal the flask, and sterilize in an autoclave.

[0063] 2) Transformation: Remove the Rosetta (DE3) competent medium and WDR5-GFP plasmid from the refrigerator. Add 1 μL of the plasmid to the Rosetta (DE3) competent medium. Place the culture in a 42°C water bath. Heat shock the culture for 90 seconds and immediately place on ice. Ice-bath the culture for 5 minutes. Spread the plate and incubate at 37°C for 16 hours.

[0064] 3) Primary culture: Add 5 mL of LB medium and the appropriate antibiotics to a 15 mL centrifuge tube. Use a pipette to pick a single large colony and place it in a centrifuge tube. Incubate the tube in a constant temperature shaker at 250 rpm and 37°C overnight.

[0065] 4) Expansion culture: Transfer the bacterial solution after the initial culture to 1 L of LB medium and continue shaking culture on a constant temperature shaker. After 4 to 5 hours, stop the culture when the OD value reaches 0.5 to 0.8.

[0066] 5) Cooling the bacterial solution: Stop the shaking culture and place the expanded bacterial solution in a 4°C refrigerator to cool for 2 hours.

[0067] 6) Induction: Remove the cooled bacterial suspension and add IPTG (isopropyl β-D-thiogalactopyranoside, Sigma, Catalog No. I6758) to a working concentration of 0.1 mM. Induce the bacterial suspension on a thermostatic shaker at 16°C, 180 rpm for at least 16 hours.

[0068] 7) Collecting bacteria: Pour the induced bacterial solution into a centrifuge tube, centrifuge at 5000 rpm for 10 minutes, discard the supernatant, and collect the bacteria.

[0069] 8) Resuspend the bacterial pellet in Binding Buffer. Centrifuge at 5000 rpm for 10 minutes at 4°C. Add 30 mL of pre-chilled Binding Buffer to fully resuspend the pellet. Resuspend the pellet completely until a sterile mass is obtained. Incubate on ice for 10 minutes.

[0070] The Binding Buffer formula (500 mL) is as follows:

[0071]

[0072] 9) Ultrasonic fragmentation in an ice-water bath: 25% power, ultrasonic for 2 seconds and then 4 seconds on, for 30 minutes, then adjust the probe position and continue fragmentation for 10 minutes.

[0073] 10) Collect the supernatant: After the cells are disrupted, centrifuge at 12,000 rpm for 30 minutes at 4°C and collect the protein supernatant into a new 50 mL centrifuge tube.

[0074] 11) Resin equilibration: Take out an appropriate amount of Ni-NTA (Qiagen, Catalog No.: 30210) and add it to the protein purification column. After the solution naturally drains, equilibrate the column with pre-cooled Binding Buffer.

[0075] 12) Column hanging: After the resin is equilibrated, add the protein supernatant after crushing and centrifugation to the purification column and allow the protein flow-through to slowly flow down under the action of gravity.

[0076] 13) Washing: Fill the protein purification column with pre-cooled Wash Buffer (Binding Buffer supplemented with 70 mM imidazole and adjusted to pH 8.0).

[0077] 14) Elution: Add pre-cooled elution buffer (based on Binding Buffer supplemented with 500 mM imidazole) to the protein purification column to elute the target protein.

[0078] 15) Concentration: Add the eluate to the pre-treated concentrator tube, place it in a 4°C pre-cooled centrifuge, and centrifuge at 3000 rpm until the volume of the protein solution is less than 1 mL. Concentration is complete.

[0079] 16) SDS-PAGE verification: SDS-PAGE was used to verify the expression and purification results of the target protein.

[0080] 17) Protein aliquoting: After successful protein purification, determine the protein concentration, aliquot it into small portions, and store in a -80°C freezer.

[0081] After obtaining the WDR5-GFP protein, it was diluted into a protein buffer to a final concentration of 10 μM. The corresponding protein buffer was prepared, controlling different NaCl concentrations and the crowding agent PEG 3350 (seebio, Cat. No.: 167228) or PEG 8000 (BioFroxx, Cat. No.: 1363GR500). WDR5-GFP was incubated with different protein buffers and observed under a microscope after 10 minutes of incubation. During observation, a circle of high vacuum silicone grease (Great Wall 7501) was first applied to the slide, and the protein sample was then dropped into the center. The slide was covered with a coverslip and placed upside down on a 100x oil immersion lens. The sample was then observed using a laboratory confocal microscopy system. The observation results are shown in Figure 1. The inventors found that the WDR5 protein could form regular circular droplets regardless of whether it was in low salt without a crowding agent or in high salt with a crowding agent.

[0082] Generally speaking, in vitro reconstituted phase-separated proteins can form regular, round droplets in vitro at appropriate protein and salt concentrations. These phase-separated droplets also exhibit fluidity. The inventors have also demonstrated fluidity in droplets formed by the WDR5 protein through FRAP and fusion experiments, but this is not demonstrated in this application.

[0083] However, after mutating the N-terminal amino acid of WDR5, that is, mutating the arginine at position 15, which is most important for binding, to alanine, it was found that the number and size of the phase-separated droplets formed after the mutation were reduced accordingly. In particular, in the group with the crowding agent PEG 8000, the radius of most of the phase-separated droplets formed after the mutation was reduced to 1 / 5 of the original radius, and the number was reduced by at least 1 / 2. Similarly, when the unmutated peptide (SEQ ID NO.1) or the mutated peptide (SEQ ID NO.2) was added to the normal WDR5 protein and incubated together, as shown in FIG. Figure 1BAs shown, the unmutated peptide competitively binds to WDR5, thereby disrupting WDR5 phase separation; whereas the mutated peptide cannot competitively bind to it and therefore has little effect on WDR5 phase separation. This suggests that the unmutated peptide fragment can competitively bind to the WD40 domain of WDR5, thereby disrupting the weak interaction that promotes phase separation and, in turn, disrupting WDR5 phase separation.

[0084] The polypeptide of SEQ ID NO.1 is easily dissolved in DMSO, but DMSO can produce toxic effects. The solubility of a polypeptide is closely related to the amino acid sequence of the entire peptide chain. Under normal circumstances, peptides with less than 5 amino acids are generally soluble in aqueous solutions. Water as a solvent is safer for cells and has higher targeting. Therefore, considering the water solubility and safety of the polypeptide, the present invention truncated the sequence of the above-mentioned polypeptide and synthesized gradually shortened polypeptides, whose sequences are SEQ ID NO.3, SEQ ID NO.4, and SEQ ID NO.5, respectively. The most suitable polypeptide is selected by comparing the effects of polypeptides of different lengths on the phase separation ability of WDR5. Figure 2A and 2B As shown in the figure, after incubating 10μM peptides of different lengths with the same concentration of WDR5 protein, the results after different peptide treatments were observed and photographed under a microscope. Given that the size and number of phase separations vary, we counted the area of ​​the phase separation droplets formed to show the different effects. The results showed that compared with the control group, the number of phase separation droplets formed after different peptide treatments decreased. The average droplet area in the control group was 2096μm 2 The average droplet area after treatment with peptide SEQ ID NO.1 was 265.5 μm 2 The average droplet area after SEQ ID NO.3 treatment was 249.65 μm 2 The average droplet area after SEQ ID NO.4 treatment was 459.25 μm 2 The average droplet area after SEQ ID NO.5 treatment was 213.05 μm 2 From the comprehensive results, SEQ ID NO.5 showed the best results after treatment, which may be due to its relatively small molecular weight and better targeting. At the same time, compared with SEQ ID NO.4, although the length of SEQ ID NO.5 is the same, SEQ ID NO.5 has a significantly better effect in inhibiting phase separation than SEQ ID NO.4. Therefore, we selected SEQ ID NO.5 as our peptide inhibitor.

[0085] Example 2 Effect of polypeptide inhibitors on methyltransferase activity

[0086] Based on the above experimental results, the inventors selected SEQ ID NO. 5 with the best effect as a WDR5 polypeptide inhibitor.

[0087] The interaction between WDR5 and MLL1 is crucial for the H3K4 methyltransferase activity of the MLL1 complex. The effect of WDR5 on the methyltransferase activity of the MLL1 protein complex can be tested by detecting the H3K4 trimethylation level by Western-blot.

[0088] The present invention selected human myelomonocytic leukemia cells MV4-11 (Shanghai Cell Bank, Chinese Academy of Sciences, SCSP-5031), which are closely related to MLL1 protein activity. In the experiment, MV4-11 cells were centrifuged, resuspended, and plated in six-well plates. The next day, when the cell density reached 70%-80%, the cells were treated with 0μM, 20μM, 50μM, and 100μM of the peptide. After further incubation for 96 hours, the cells were harvested and the expression of H3K4 trimethylation in the leukemia cells was detected by Western blot.

[0089] The specific experimental steps of Western-blot are as follows:

[0090] 1) Cell Sample Preparation and Processing: After harvesting cells, lyse them using RIPA lysis buffer (Biosharp, Catalog No. P0013B) for 10 minutes. After lysis, protein was quantified using a BCA protein concentration assay kit (Biosharp, Catalog No. BL521A). Protein concentration was determined and calculated. Based on the sample load, an appropriate amount of Protein Loading Buffer (Yeasen, Catalog No. 20315ES05) was added and the samples were boiled at 100°C for 10 minutes.

[0091] 2) Protein electrophoresis: Clean the glass plate and dry it in a 60°C oven. After preparing the separation gel, fill the gel, seal it with water, flatten the separation gel, wait for 30 minutes, pour out the liquid seal water, add the prepared concentrated gel to the gel plate, and insert a 10-hole or 15-hole comb. After waiting for 30 minutes, unplug the comb and add electrophoresis buffer. Add protein samples and protein markers (Tianneng, catalog number: 180-6003) to the loading wells. Run electrophoresis at 80V for about 50 minutes. When the protein sample enters the separation gel, switch to 120V. When the Loading Buffer reaches the appropriate position at the bottom of the separation gel, stop electrophoresis and prepare for membrane transfer.

[0092] 3) Transfer: Prepare transfer buffer and pre-chill in the refrigerator. Cut a piece of PVDF membrane about the size of a business card. Gently pry open the electrophoresis gel, remove the stacking gel, and cut the separating gel. Stack the sponge, filter paper, and PVDF membrane (Millipore, Cat. No. ISEQ00010) like a sandwich. Assemble the wet transfer system and fill it with 4°C pre-chilled transfer buffer to cover the sandwich. Transfer at 350 mA for 90 minutes.

[0093] 4) Immunoreaction: Prepare a blocking buffer of 5% skim milk powder and 1 L of TBST (containing 500 μL of Tween) (TBS: Biosharp, Catalog No. BL602A; Tween 20: BioFroxx, Catalog No. 1247ML500). Remove the PVDF membrane and rinse it with TBST. Add an appropriate amount of skim milk powder and block the membrane on a shaker at room temperature for 1-2 hours. Rinse the membrane with TBST 2-3 times to remove any residual skim milk powder. Prepare 5% BSA (Shanghai Sangon Biotechnology Co., Ltd., Catalog No. A500023) in TBST and dilute the primary antibodies (WDR5 antibody: Abcam, Catalog No. ab22512; H3K4me3 antibody: CST, Catalog No. 9751; H3 antibody: Santa Cruz, Catalog No. sc-517576; GAPDH antibody: Proteintech, Catalog No. 60004-1-Ig) according to the antibody manufacturer's instructions. Incubate overnight on a shaker at 4°C. Recover the primary antibody and store in a -20°C refrigerator until ready for use. Wash three times with TBST for 5 minutes each. Dilute the secondary antibody of the same species as the primary antibody in skim milk powder (Yili) and add the diluted secondary antibody. Incubate at room temperature for 1 hour.

[0094] 5) Development: After rinsing with TBST, prepare the developer solution in proportion and gently add it dropwise onto the PVDF membrane using a pipette tip. Develop using a UVP gel imaging system.

[0095] like Figure 3 As shown, the experimental results showed that compared with the control group, H3K4 trimethylation levels were significantly reduced after MV4-11 cells were treated with different concentrations of peptide. Compared with the group treated with 0μM, grayscale analysis showed that H3K4 trimethylation levels decreased by 29%, 34%, and 51%, respectively, after cells were treated with 20μM, 50μM, and 100μM peptide. The decrease in H3K4 trimethylation levels indicates that the peptide inhibitor of the present invention can effectively inhibit the activity of MLL1 histone methyltransferase. While MLL fusion protein alone cannot cause leukemia, the methyltransferase catalytic activity of wild-type MLL1 is essential for MLL1 fusion protein-induced leukemia. Therefore, the present inhibitor can treat leukemia by inhibiting MLL1 methyltransferase activity.

[0096] Example 3 Tumor cell activity experiment

[0097] Pancreatic cancer is a common malignant tumor of the digestive tract, prone to recurrence after surgery and carrying a high mortality rate. Early studies have shown that platinum-based chemotherapy (such as cisplatin and oxaliplatin) is effective, but long-term use of cytotoxic drugs can lead to cumulative toxicity and organ dysfunction (Chevalier et al., 2020; Von Hoff et al., 2013). Furthermore, 90% of pancreatic cancer patients harbor KRAS mutations, but KRAS lacks a clear binding site, making it difficult to synthesize targeted drugs. Consequently, there remains a lack of effective targeted therapies for pancreatic cancer. Numerous studies have reported on the role of WDR5 in pancreatic cancer, with some suggesting that WDR5 recruitment of c-Myc to chromatin is crucial for pancreatic cancer tumorigenesis (Carugo et al., 2016). Other studies have shown that inhibiting the WDR5-H3K4me3 epigenetic axis can effectively suppress pancreatic tumor immune evasion (Lu et al., 2021). Therefore, WDR5 may represent a potential target for the treatment of pancreatic cancer.

[0098] Multiple studies have shown that WDR5 plays a key role in the development and progression of pancreatic cancer and leukemia (Carugo et al., 2016, Lu et al., 2021, Thiel et al., 2010). Therefore, this study used human metastatic pancreatic adenocarcinoma cells AsPC-1 (TCHu 8, Shanghai Cell Bank, Chinese Academy of Sciences), human orthotopic pancreatic adenocarcinoma cells BxPC-3 (TCHu 12, Shanghai Cell Bank, Chinese Academy of Sciences), and human myelomonocytic leukemia cells MV4-11 as experimental subjects. The cells were digested and resuspended or centrifuged and resuspended, then counted. 4,000 cells were seeded in 100 μL of culture medium (AsPC-1 and BxPC-3 cells were cultured in RPMI 1640 medium, and MV4-11 cells were cultured in Iscove's Modified Dulbecco's Medium) in a 96-well plate. After the cells adhered, they were treated with 0μM, 20μM, 50μM, and 100μM of the peptide SEQ ID NO. 5. A blank control group was set up and the cells were cultured at 37°C and 5% CO2 for 96 hours. 10μL of CCK8 (Biosharp, Cat. No. BS350B) solution was added to each well and the cells were cultured in a cell incubator for another hour. The absorbance at 450nm was measured using a microplate reader, and cell viability was calculated based on the OD value. The CellCounting Kit-8 (abbreviated as CCK-8) is a rapid and highly sensitive detection reagent based on WST-8 that is widely used for cell proliferation and cytotoxicity. WST-8 is a compound similar to MTT. In the presence of an electron carrier (1-Methoxy PMS), it is reduced by mitochondrial dehydrogenases to a highly water-soluble orange-yellow formazan product (formazan). The more and faster the cells proliferate, the darker the color; the greater the cytotoxicity, the lighter the color. For the same cells, the depth of the color is proportional to the number of living cells. Therefore, this characteristic can be used to directly analyze cell viability.

[0099] like Figure 4 The results showed that the polypeptide inhibitor of the present invention had an inhibitory effect on the growth of all three tumor cells. At a 20 μM polypeptide concentration, the cell viability of AsPC-1, BxPC-3, and MV4-11 decreased by 21%, 17%, and 40%, respectively; at a 50 μM polypeptide concentration, the cell viability of AsPC-1, BxPC-3, and MV4-11 decreased by 44%, 69%, and 48%, respectively; and at a 100 μM polypeptide concentration, the cell viability of AsPC-1, BxPC-3, and MV4-11 decreased by 73%, 82%, and 65%, respectively, demonstrating that the polypeptide inhibitor of the present invention is effective. Furthermore, the ability to inhibit tumor cell activity at a concentration of 20 μM suggests that the inhibitor polypeptide has potential for drug development.

[0100] The minimum concentration of WDR5 inhibitors in the prior art for exerting their activity and their inhibitory effects on tumor cells are as follows:

[0101] WDR5-IN-1, IC50 = 2.2 nM, showed potent antiproliferative effects in CHP-134 (neuroblastoma) and Ramos (Burkitt lymphoma) cell lines;

[0102] WDR5-0103, IC50 = 450 nM, can reduce the activity of acute myeloid leukemia cells in vitro;

[0103] OICR-9429, IC50 = 5 μM, can reduce the activity of acute myeloid leukemia cells in vitro;

[0104] DDO-2093, IC50=8.6nM, selectively inhibits the catalytic activity of the MLL complex.

[0105] According to the current research status of WDR5 inhibitors, most inhibitors target the MLL1-WDR5 interaction to inhibit the activity of leukemia cells, and are categorized as peptidomimetic and non-peptidomimetic inhibitors. However, the peptide inhibitors of the present invention, by disrupting phase separation, can inhibit not only leukemia cells but also pancreatic cancer cells, thus showing great potential for pharmaceutical development. Furthermore, our discovery helps to elucidate the complex pathological mechanisms of pancreatic cancer and provides a promising new approach for pancreatic cancer treatment.

[0106] The above experiments demonstrate that SEQ ID NO. 5 can effectively inhibit the growth of tumor cells closely related to WDR5. Furthermore, the polypeptide of the present invention has a small molecular weight, is easy to synthesize, has good water solubility, and greatly improved safety. Therefore, the polypeptide inhibitor of the present invention has potential value for biopharmaceutical development.

[0107] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

[0108] References

[0109] Ahn,J.H.,Davis,E.S.,Daugird,T.A.,Zhao,S.,Quiroga,I.Y.,Uryu,H.,Li,J.,Storey,A.J.,Tsai,Y.H.,Keeley,D.P.,et al.(2021).Phase separation drivesaberrant chromatin looping and cancer development.Nature 595,591-595.

[0110] Alberti,S.,and Dormann,D.(2019).Liquid-Liquid Phase Separation inDisease.Annu Rev Genet 53,171-+.

[0111] Bouchard,J.J.,Otero,J.H.,Scott,D.C.,Szulc,E.,Martin,E.W.,Sabri,N.,Granata,D.,Marzahn,M.R.,Lindorff-Larsen,K.,Salvatella,X.,et al.(2018).CancerMutations of the Tumor Suppressor SPOP Disrupt the Formation of Active,Phase-Separated Compartments.Mol Cell 72,19-36e18.

[0112] Carugo,A.,Genovese,G.,Seth,S.,Nezi,L.,Rose,J.L.,Bossi,D.,Cicalese,A.,Shah,P.K.,Viale,A.,Pettazzoni,P.F.,et al.(2016).In Vivo Functional PlatformTargeting Patient-Derived Xenografts Identifies WDR5-Myc Association as aCritical Determinant of Pancreatic Cancer.Cell Rep 16,133-147.

[0113] Chen,X.,Xie,W.,Gu,P.,Cai,Q.,Wang,B.,Xie,Y.,Dong,W.,He,W.,Zhong,G.,Lin,T.,et al.(2015).Upregulated WDR5 promotes proliferation,self-renewal andchemoresistance in bladder cancer via mediating H3K4trimethylation.Sci Rep 5,8293.

[0114] Chevalier,H.,Vienot,A.,Lievre,A.,Edeline,J.,El Hajbi,F.,Peugniez,C.,Vernerey,D.,Meurisse,A.,Hammel,P.,Neuzillet,C.,et al.(2020).FOLFIRINOX De-Escalation in Advanced Pancreatic Cancer:A Multicenter Real-LifeStudy.Oncologist 25,e1701-e1710.

[0115] Dai,X.,Guo,W.,Zhan,C.,Liu,X.,Bai,Z.,and Yang,Y.(2015).WDR5ExpressionIs Prognostic of Breast Cancer Outcome.PLoS One 10,e0124964.

[0116] Dang,C.V.,Reddy,E.P.,Shokat,K.M.,and Soucek,L.(2017).Drugging the'undruggable'cancer targets.Nat Rev Cancer 17,502-508.

[0117] Grabocka,E.,and Bar-Sagi,D.(2016).Mutant KRAS Enhances Tumor CellFitness by Upregulating Stress Granules.Cell 167,1803-1813 e1812.

[0118] Guarnaccia,A.D.,and Tansey,W.P.(2018).Moonlighting with WDR5:ACellular Multitasker.J Clin Med 7.

[0119] Karatas,H.,Townsend,E.C.,Cao,F.,Chen,Y.,Bernard,D.,Liu,L.,Lei,M.,Dou,Y.,and Wang,S.(2013).High-affinity,small-molecule peptidomimetic inhibitorsof MLL1 / WDR5 protein-protein interaction.J Am Chem Soc 135,669-682.

[0120] Klein,I.A.,Boija,A.,Afeyan,L.K.,Hawken,S.W.,Fan,M.,Dall'Agnese,A.,Oksuz,O.,Henninger,J.E.,Shrinivas,K.,Sabari,B.R.,et al.(2020).Partitioning ofcancer therapeutics in nuclear condensates.Science 368,1386-1392.

[0121] Legrand,N.,Dixon,D.A.,and Sobolewski,C.(2020).Stress granules incolorectal cancer:Current knowledge and potential therapeuticapplications.World J Gastroenterol 26,5223-5247.

[0122] Lu,C.,Liu,Z.,Klement,J.D.,Yang,D.,Merting,A.D.,Poschel,D.,Albers,T.,Waller,J.L.,Shi,H.,and Liu,K.(2021).WDR5-H3K4me3epigenetic axis regulates OPNexpression to compensate PD-L1 function to promote pancreatic cancer immuneescape.J Immunother Cancer 9.

[0123] Podszywalow-Bartnicka,P.,Wolczyk,M.,Kusio-Kobialka,M.,Wolanin,K.,Skowronek,K.,Nieborowska-Skorska,M.,Dasgupta,Y.,Skorski,T.,and Piwocka,K.(2014).Downregulation of BRCA1 protein in BCR-ABL1leukemia cells depends onstress-triggered TIAR-mediated suppression of translation.Cell Cycle 13,3727-3741.

[0124] Schuetz,A.,Allali-Hassani,A.,Martin,F.,Loppnau,P.,Vedadi,M.,Bochkarev,A.,Plotnikov,A.N.,Arrowsmith,C.H.,and Min,J.(2006).Structural basisfor molecular recognition and presentation of histone H3 by WDR5.EMBO J 25,4245-4252.

[0125] Sun,Y.,Bell,J.L.,Carter,D.,Gherardi,S.,Poulos,R.C.,Milazzo,G.,Wong,J.W.,Al-Awar,R.,Tee,A.E.,Liu,P.Y.,et al.(2015).WDR5 Supports an N-MycTranscriptional Complex That Drives a Protumorigenic Gene ExpressionSignature in Neuroblastoma.Cancer Res 75,5143-5154.

[0126] Tan,X.,Chen,S.,Wu,J.,Lin,J.,Pan,C.,Ying,X.,Pan,Z.,Qiu,L.,Liu,R.,Geng,R.,et al.(2017).PI3K / AKT-mediated upregulation of WDR5promotes colorectalcancer metastasis by directly targeting ZNF407.Cell Death Dis 8,e2686.

[0127] Thiel,A.T.,Blessington,P.,Zou,T.,Feather,D.,Wu,X.,Yan,J.,Zhang,H.,Liu,Z.,Ernst,P.,Koretzky,G.A.,et al.(2010).MLL-AF9-induced leukemogenesisrequires coexpression of the wild-type Mll allele.Cancer Cell17,148-159.

[0128] Thomas,L.R.,Wang,Q.,Grieb,B.C.,Phan,J.,Foshage,A.M.,Sun,Q.,Olejniczak,E.T.,Clark,T.,Dey,S.,Lorey,S.,et al.(2015).Interaction with WDR5promotes target gene recognition and tumorigenesis by MYC.Mol Cell58,440-452.

[0129] Von Hoff,DD,Ervin,T.,Arena,FP,Chiorean,EG,Infante,J.,Moore,M.,Seay,T.,Tjulandin,SA,Ma,WW,Saleh,MN,et al.(2013).Increased survival inpancreatic cancer with nab-paclitaxel plus gemcitabine.N Engl J Med 369,1691-1703.

[0130] Xie,Q.,Li,Z.,and Chen,J.(2017).WDR5 positively regulates p53stabilityby inhibiting p53 ubiquitination.Biochem Biophys Res Commun 487,333-338.

[0131] Zhou,Y.,Su,JM,Samuel,CE,and Ma,D.(2019).Measles Virus FormsInclusion Bodies with Properties of Liquid Organelles.J Virol 93.

[0132] The specific embodiments described above further illustrate the objectives, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above are only specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A polypeptide inhibitor targeting WDR5, characterized in that The amino acid sequence of the polypeptide inhibitor is Ala-Arg-Ala-Gln.

2. A nucleic acid encoding a polypeptide inhibitor targeting WDR5, characterized in that The nucleic acid encodes the polypeptide inhibitor according to claim 1.

3. An expression vector, characterized in that The expression vector comprises the nucleic acid according to claim 2 or expresses the polypeptide inhibitor according to claim 1.

4. A host cell, characterized in that The host cell comprises the expression vector according to claim 3.

5. A pharmaceutical composition, characterized in that The invention comprises the polypeptide inhibitor according to claim 1 and a pharmaceutically acceptable carrier.

6. The pharmaceutical composition according to claim 5, characterized in that The pharmaceutically acceptable carrier is selected from fillers, sweeteners, disintegrants, wetting agents and lubricants.

7. Use of the polypeptide inhibitor according to claim 1 in preparing a drug for treating tumors, characterized in that: The tumor is selected from the group consisting of leukemia and pancreatic cancer.

8. The use according to claim 7, characterized in that The leukemia is acute myeloid leukemia or lymphocytic leukemia.

9. The use according to claim 7, characterized in that The pancreatic cancer is adenocarcinoma.

10. The use according to claim 7, characterized in that The pancreatic cancer is metastatic pancreatic cancer and / or in situ pancreatic cancer.

11. The use according to claim 7, characterized in that The pancreatic cancer is metastatic pancreatic adenocarcinoma and / or in situ pancreatic adenocarcinoma.

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