CN116859058A - A molecular screening method and application for anti-colorectal cancer activity based on NDR1-Myc interaction - Google Patents

Abstract

The invention belongs to the field of biomedical research, and specifically relates to a screening method and application of anti-colorectal cancer active molecules based on NDR1-Myc interaction. After extensive and in-depth research, the present invention discovered for the first time that the interaction between NDR1 and Myc can be used as a drug screening target to screen tumor therapeutic drugs. On this basis, a specific tumor drug screening method was developed. The present invention discovers active molecules for treating colorectal cancer by targeting the interaction between NDR1 and Myc, and is expected to provide new ideas for clinical treatment of colorectal cancer.

Description

A molecular screening method and application for anti-colorectal cancer activity based on NDR1-Myc interaction

Technical field

The invention belongs to the field of biomedicine research, and specifically relates to a screening method and application of anti-colorectal cancer active molecules based on NDR1-Myc interaction.

Background technique

Colorectal cancer (CRC) is the third most common malignant tumor in the world (10.2%) and ranks second in cancer-related mortality (9.2%). With the changes in people's lifestyles, the incidence and mortality of colorectal cancer are increasing year by year. There are 376,000 new cases in China every year, and more than half of the patients are in the mid-to-late stage when diagnosed. At present, radical resection is the preferred treatment for colorectal cancer, but the recurrence rate after surgery is high. Most advanced patients have limited efficacy of chemotherapy and combination/or targeted therapy and are prone to drug resistance. Therefore, there is an urgent need to find targeted drugs with new mechanisms of action to improve the overall survival and quality of life of patients with colorectal cancer.

Myc, also known as c-Myc (encoded by the MYC gene), is the first member of the proto-oncogene family to be discovered. The same family also includes n-Myc and l-Myc, which have different effects on tumor formation and prognosis. . Myc is overexpressed in more than 70%-80% of colorectal cancer patients. High Myc expression and distant tumor metastasis are late events in the progression of colorectal cancer, representing a more malignant and aggressive clinical phenotype, suggesting a poor prognosis for patients. Difference. Abnormal activation of signaling pathways such as WNT/APC/β-Catenin and RTK/RAS/MEK/ERK, which are closely related to the onset of colorectal cancer, will increase the expression and protein stability of Myc. Myc can also positively regulate integrins, such as ITGA6 (Integrin alpha-6), ITGB1 (Integrin beta-1) and ITGB4 (Integrin Beta 4), to accelerate the progression of colorectal cancer. Recent studies have reported that abnormal Myc expression can cause metabolic reprogramming in colorectal cancer, changing the expression of 121 metabolic genes and 39 transporter genes, and inducing at least 215 metabolic reactions. Knocking out Myc in colorectal cancer cells changes the metabolic pathways of cancer cells and inhibits cancer cell growth. Colorectal cancer can be inhibited by reducing the expression of Myc by inhibiting the translation initiation factor eIF4A, miR-487b, knocking out HOXB8, BET inhibitors, and the combination of Wnt or MAPK inhibitors and BET inhibitor JQ1. Therefore, the development of drugs targeting Myc has always been a hot area of anti-colorectal cancer research.

However, Myc, as a super transcription factor, also plays important functions under normal physiological conditions. In normal cells, Myc regulates approximately 15% of gene transcription in the entire genome, involving genes encoding or non-coding proteins, which are involved in different cellular functions, including DNA repair, transcription, translation, cell adhesion and skeleton, cell cycle, and signaling. Transduction, metabolism, protein biosynthesis and angiogenesis, etc. Therefore, direct inhibition of Myc expression will also have adverse effects on the normal physiological functions of Myc.

In fact, Myc is a naturally disordered protein that lacks obvious small molecule binding sites. Currently, there are no drugs that directly target Myc. Therefore, the search for small molecule inhibitors that directly act on the Myc protein has long been the subject of international drug development. Major problems. Researchers have tried to inhibit Myc overexpression by regulating Myc's transcription, translation, activation and stability and other methods. Inhibitors such as BRD4 (bromodomain protein 4), CDK7 (cyclin-dependent kinase 7) and CDK9 (cyclin-dependent kinase 9) can inhibit the expression of Myc at the transcriptional level; inhibit the PI3K/AKT/mTOR pathway Can block the translation of Myc; USP7 (ubiquitin-specific protease 7), AURKA (Aurora kinase A) and PLK1 (polo-like kinase 1) inhibitors can reduce the stability of Myc protein at the post-translational level; 10058-F4 and Omomyc blocks the Myc/Max dimer complex. Currently, drugs targeting BRD4 have shown good biological activity in various tumor models and have entered the clinical trial research stage. However, the structural types of inhibitors reported publicly in literature and patents are still relatively limited, and the druggability of small molecule compounds needs to be improved urgently. Therefore, exploring new mechanisms based on inhibiting Myc expression and activity is of great significance for the treatment of tumors with high Myc expression such as colorectal cancer.

NDR1 (Nuclear Dbf2-related kinase 1, nuclear Dbf2-related protein kinase 1, also called Serine/threonine-protein kinase 38, STK38) is a cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP)-dependent protein kinase and protein A member of the kinase C (PKC)-related protein kinase subfamily, the other three members are: NDR2 (STK38L), LATS1 and LATS2. Their sequences are highly conserved from yeast to humans. The N-terminal end of NDR1 is the MOB1 (MOBkinase activator 1B) coactivator binding domain (MBD), the middle is the protein kinase domain, and the C-terminal end is the hydrophobic motif (HM). In normal cells, NDR/LATS kinase activity is tightly regulated. When the accessory protein MOB1 binds to the MBD, it causes autophosphorylation within the kinase (Ser281/282 in NDR1/2 or Ser909/872 in LATS1/2), and then phosphorylates its upstream protein kinases MST1, MST2 and/or MST3 The sites in the HM region (Thr444/442 in NDR1/2 or Thr1079/1041 in LATS1/2) increase kinase activity and regulate many important physiological functions such as mitosis, cell morphology, cell proliferation and apoptosis. NDR1 plays an important role in the innate immune response. It can positively regulate the antiviral innate immune response, inhibit the production of inflammatory cytokines during host infection, and protect the host from inflammatory damage.

Regarding the relationship between NDR1 and colorectal cancer, Zhang et al. revealed that NDR1 and NDR2, as two highly similar isoforms in mammals, have functional compensatory effects. Knocking out NDR1/2 in small intestinal epithelial cells of C57BL/6 mice makes the mice susceptible to colorectal cancer. Further mechanistic studies confirmed that NDR1/2 functions as a key tumor suppressor upstream of YAP1 in human colorectal cancer. Although the current understanding of the function of NDR1 in the body is limited, existing studies have shown that NDR1 is essential for maintaining normal physiological functions of the human body and is also a key inhibitory factor for colorectal cancer.

Protein-protein interaction (PPI) has always been a hot topic in research on therapeutic drugs for tumors and other diseases. In 2017, the FDA approved Venetoclax, the first true Bcl-2/Bax PPI inhibitor, for the treatment of relapsed and refractory 17p deletion chronic lymphocytic leukemia (CLL) or small lymphocytic lymphoma (SLL). . Due to the challenges in developing drugs that directly target Myc, researchers have also explored drug research strategies targeting Myc PPI. However, most of these studies are in the discovery stage of PPI inhibitors, and it is still necessary to further explore the important role of these PPIs in tumorigenesis and whether blocking these PPI interfaces can play an anti-tumor role.

Contents of the invention

In order to overcome the problems existing in the prior art, the purpose of the present invention is to provide a method and application for screening anti-colorectal cancer active molecules based on NDR1-Myc interaction. Used to discover active molecules for the treatment of colorectal cancer by targeting NDR1/Myc interaction, the present invention is expected to provide new ideas for clinical treatment of colorectal cancer.

In order to achieve the above objects and other related objects, the present invention adopts the following technical solutions:

A first aspect of the present invention provides a screening method for tumor therapeutic drugs, using the interaction between NDR1 and Myc as a drug screening target.

Using the interaction between NDR1 and Myc as a drug screening target refers to examining drugs that have an impact on the interaction between NDR1 and Myc, thereby obtaining drugs for treating tumors.

Furthermore, the screening method selects inhibitors that can block the specific binding of NDR1 and Myc as active ingredients of tumor treatment drugs.

The candidate drug may be a single compound, a polypeptide, or a pharmaceutical composition.

More specifically, the screening method for tumor treatment drugs of the present invention includes the following steps:

(1) Establish the interaction system between NDR1 and Myc protein;

(2) Use the established protein interaction system to detect the ability of candidate substances to inhibit the specific binding of NDR1 and Myc.

Further, in step (2), the ability of the candidate substance to inhibit the specific binding of NDR1 to Myc can be tested at the cellular level.

Further, the screening method also includes a verification test of anti-tumor effect.

Furthermore, in the screening method, the substances obtained by screening are used as test subjects to conduct anti-tumor verification tests.

Furthermore, the anti-tumor verification test refers to the verification of cell proliferation experiments or targeting experiments.

Further, the tumor is selected from colorectal cancer. Correspondingly, the tumor cells are selected from colorectal cancer cells. The tumor cells are selected from HCT116, HT-29 or DLD-1 cells.

The active substances obtained by the drug screening method of the present invention can be used to prepare anti-tumor drugs. If the active substance is a compound, it generally has a clear structural formula.

The second aspect of the present invention provides a new use of the interaction between NDR1 and Myc as a drug screening target for screening tumor therapeutic drugs.

Furthermore, inhibitors that can block the specific binding of NDR1 to Myc are selected as active ingredients of tumor treatment drugs.

Further, the tumor is selected from colorectal cancer.

The third aspect of the present invention provides a new use of inhibitors of the interaction between NDR1 and Myc in the preparation of tumor treatment drugs.

Further, the inhibitor of the interaction between NDR1 and Myc refers to an inhibitor that can block the specific binding of NDR1 and Myc.

Further, the tumor is selected from colorectal cancer.

Furthermore, the inhibitor of the interaction between NDR1 and Myc can be a single compound, a polypeptide, or a pharmaceutical composition.

Further, the inhibitor of the interaction between NDR1 and Myc is selected from Manidipine, Benidipinehydrochloride or MN3.

Further, the inhibitor of the interaction between NDR1 and Myc is an active ingredient in tumor treatment drugs.

That is, the tumor treatment drug must include an inhibitor of the interaction between NDR1 and Myc, and the inhibitor of the interaction between NDR1 and Myc is an active ingredient of the tumor treatment drug.

In the tumor treatment drug, the active ingredient that exerts the therapeutic function may only be the inhibitor of the interaction between NDR1 and Myc, or may also include other molecules that can play similar functions.

That is, the inhibitor of the interaction between NDR1 and Myc is the only active ingredient or one of the active ingredients of the tumor treatment drug.

The tumor treatment drug may be a single-component substance or a multi-component substance.

The form of the tumor treatment drug is not particularly limited and can be in various forms such as solid, liquid, gel, semi-liquid, aerosol, etc.

The tumor treatment drugs are mainly targeted at mammals, such as rodents, primates, etc.

A fourth aspect of the present invention provides a method for treating tumors, which includes administering an inhibitor of the interaction between NDR1 and Myc to a subject.

The subject may be a mammal or a mammalian cell. The mammal is preferably a rodent, an artiodactyl, a perissodactyl, a lagomorph, a primate, etc. The primate is preferably a monkey, ape or human. The cells may be ex vivo cells.

The subject may be a patient suffering from a disease or an individual in need of treatment.

The inhibitor of the interaction between NDR1 and Myc can be administered to the subject before, during or after receiving treatment.

Further, the tumor is selected from colorectal cancer.

Furthermore, the inhibitor of the interaction between NDR1 and Myc can be a single compound, a polypeptide, or a pharmaceutical composition.

Further, the inhibitor of the interaction between NDR1 and Myc is selected from Manidipine, Benidipinehydrochloride or MN3.

The fifth aspect of the present invention provides a tumor therapeutic drug, including an effective dose of an inhibitor of the interaction between NDR1 and Myc.

Further, the tumor treatment drug includes an effective dose of an inhibitor of the interaction between NDR1 and Myc and a pharmaceutical carrier.

The tumor treatment drug must include an inhibitor of the interaction between NDR1 and Myc, and the inhibitor of the interaction between NDR1 and Myc is an active ingredient of the tumor treatment drug.

In the tumor treatment drug, the active ingredient that exerts the therapeutic function may only be the inhibitor of the interaction between NDR1 and Myc, or may also include other molecules that can play similar functions.

That is, the inhibitor of the interaction between NDR1 and Myc is the only active ingredient or one of the active ingredients of the tumor treatment drug.

The tumor treatment drug may be a single-component substance or a multi-component substance.

The form of the tumor treatment drug is not particularly limited and can be in various forms such as solid, liquid, gel, semi-liquid, aerosol, etc.

A drug is prepared using an inhibitor of the interaction between NDR1 and Myc as the main active ingredient or one of the main active ingredients. Usually, in addition to the active ingredients, the medicine also includes one or more pharmaceutically acceptable carriers or excipients according to the needs of different dosage forms. "Pharmaceutically acceptable" means that the molecular entities and compositions do not produce adverse, allergic or other adverse reactions when properly administered to animals or humans. "Pharmaceutically acceptable carrier or excipient" is compatible with NDR1 and the Myc interaction inhibitor, that is, can be blended with it without substantially reducing the effectiveness of the pharmaceutical composition under normal circumstances. Specific examples of substances that can be used as pharmaceutically acceptable carriers or excipients are sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium methylcellulose, Ethylcellulose and methylcellulose; tragacanth powder; malt; gelatin; talc; solid lubricants, such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils, such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil and cocoa butter; polyols such as propylene glycol, glycerin, sorbitol, mannitol and polyethylene glycol; alginic acid; emulsifiers such as Tween; wetting agents such as sodium lauryl sulfate; coloring agents; flavoring agents; tableting agents, stabilizers; antioxidants; preservatives; pyrogen-free water; isotonic saline solutions; and phosphate buffers, etc. These substances are used as needed to aid the stability of the formulation or to help enhance the activity or its bioavailability or to produce an acceptable mouthfeel or odor in the case of oral administration. In the present invention, unless otherwise specified, the pharmaceutical dosage form is not particularly limited and can be made into dosage forms such as injections, oral liquids, tablets, capsules, dropping pills, sprays, etc., and can be prepared by conventional methods. The choice of drug dosage form should match the mode of administration.

The tumor treatment drugs are mainly targeted at mammals, such as rodents, primates, etc.

Compared with the prior art, the present invention has the following beneficial effects:

After extensive and in-depth research, the present invention discovered for the first time that the interaction between NDR1 and Myc can be used as a drug screening target to screen tumor therapeutic drugs. On this basis, a specific tumor drug screening method was developed. By targeting active molecules that interact with NDR1/Myc to treat colorectal cancer, the present invention is expected to provide new ideas for clinical treatment of colorectal cancer.

Description of the drawings

Figure 1: The anti-tumor mechanism of NDR1 regulating Myc stability.

Figure 2: NanoLuc enzyme reaction principle.

Figure 3: NanoBiT technology detection principle.

Figure 4: Eight plasmid combinations for NanoBiT transfection.

Figure 5: Enzyme digestion identification results of eight plasmids.

Figure 6: Comparison of fluorescence values transfected with plasmid combinations. (A) Comparison of fluorescence values of 8 plasmid combinations; (B) Comparison of fluorescence values of positive plasmid combinations and negative plasmid combinations.

Figure 7: Screening of drugs that block NDR1/Myc interaction from FDA’s old drug library.

Figure 8: Myc1-113, Myc201-439 binds to NDR1.

Figure 9: Myc N-terminus blocks the Myc/NDR1 interaction. (A) Schematic diagram of Myc N-terminal truncation; (B) GST-PullDown detection of the effect of Myc N-terminal truncated fragment on NDR1/Myc interaction.

Figure 10: Myc N-terminal small peptides MN1, MN2 and MN3 block the Myc/NDR1 interaction.

Figure 11: Preliminary evaluation of the activity of MN1, MN2 and MN3 against colorectal cancer cells.

Detailed ways

Before further describing the specific embodiments of the present invention, it should be understood that the protection scope of the present invention is not limited to the following specific specific embodiments; it should also be understood that the terms used in the embodiments of the present invention are for describing specific specific embodiments, It is not intended to limit the scope of the present invention. Test methods without specifying specific conditions in the following examples usually follow conventional conditions or conditions recommended by each manufacturer.

When the examples give numerical ranges, it should be understood that, unless otherwise stated in the present invention, both endpoints of each numerical range and any value between the two endpoints can be selected. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, equipment, and materials used in the embodiments, those skilled in the art can also use methods, equipment, and materials described in the embodiments of the present invention based on their understanding of the prior art and the description of the present invention. Any methods, equipment and materials similar or equivalent to those in the prior art may be used to implement the present invention.

Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in the present invention all adopt conventional molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology and related fields in this technical field. conventional technology. These techniques have been well described in the existing literature. For details, see Sambrook et al. MOLECULAR CLONING: A LABORATORY MANUAL, Second edition, Cold Spring Harbor Laboratory Press, 1989 and Third edition, 2001; Ausubel et al., CURRENT PROTOCOLS INMOLECULAR BIOLOGY, John Wiley & Sons, New York, 1987 and periodic updates; theseries METHODS IN ENZYMOLOGY, Academic Press, San Diego; Wolffe, CHROMATINSTRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998; METHODS INENZYMOLOGY, Vol.304, Chromatin (P.M. Wassarman and A.P. Wolffe, eds .), Academic Press, San Diego, 1999; and METHODS IN MOLECULAR BIOLOGY, Vol. 119, Chromatin Protocols (P.B. Becker, ed.) Humana Press, Totowa, 1999, etc.

Example 1

This example uses NanoBiT technology to construct a high-throughput screening system for Myc/NDR1 protein interaction in living cells. NDR1 regulates 303 transcription factors including Myc. As a key upstream regulator of Myc, NDR1 can directly bind to Myc. Knocking down NDR1 can reduce the stability of Myc and inhibit B-cell lymphoma (Figure 1). Since Myc is highly expressed in colorectal cancer, it and NDR1 play an important role in maintaining normal physiological functions. Therefore, the present invention hopes to verify that blocking Myc/NDR1PPI can inhibit colorectal cancer with high Myc expression, while avoiding the impact on Myc and Effects of NDR1 on normal physiological functions.

1.1. Working principle of NanoBiT protein interaction system

Binary Technology (NanoBiT) is a protein fragment complementation analysis technology developed in recent years, which can detect protein interaction signals in real time under physiological conditions of living cells. The complete NanoLuc enzyme has a molecular weight of 61KDa. It is a genetically engineered small molecule enzyme (19.1kDa) that does not rely on ATP to cleave the substrate and emits fluorescence that is two orders of magnitude higher than that of firefly luciferase. /> The system consists of the large subunit Large BiT (LgBiT, 17.6kDa) and the small subunit Small BiT (SmBiT, 11 amino acids), which can be fused with target proteins A and B for expression.

When the fusion target protein with these two subunits interacts, an active NanoLuc enzyme will be formed, which will react with the substrate Furimazine that permeates the cell membrane to produce a luminescent signal. The reaction schematic is shown in Figure 2. This experiment takes the interaction between NDR1 and Myc as an example, and the model diagram is shown in Figure 3.

1.2. Construction of plasmid

The Myc (Uniprot ID: P01106) and NDR1 (Uniprot ID: Q15208) sequences were respectively fused to the LgBiT and SmBiT vectors provided by NanoBiT CMV MCS BiBit Ready Vectors (purchased from Promega, Cat. No. CS1603B32). If Myc and NDR1 proteins interact As a result, the structures of LgBiT and SmBiT are complementary to form a complete and active luciferase, which reacts with the substrate Furimazine to produce a luminescent signal. LgBiT and SmBiT were fused with Myc and NDR1 proteins respectively to form 8 plasmids, which were constructed by Nanjing Genscript Biotechnology Co., Ltd. The specific combination method is shown in Figure 4. The results of plasmid enzyme digestion identification are shown in Figure 5.

1.3. Confirm that NDR1 specifically binds to Myc

Spread HEK293 cells into a 6-well plate and incubate them in a 37°C, 5% CO ₂ incubator for 8-24 hours depending on the cell adhesion. Transfection can be started when the cells are completely attached. Change to pre-warmed serum-free DMEM medium before transfection. FuGENEHD transfection reagent and plasmid were transfected at a ratio of 1:1 (3μg/well). 6 hours after transfection, HEK293 cells were dispersed by blowing evenly, transferred to a 96-well plate, continued to culture for 18 hours, and added Live Cell Reagent determines the best combination of NDR1 and Myc by detecting fluorescence values. Replace the plasmid expressing SmBiT fusion protein with expression /> -SmBiT's NanoBiT negative control vector is the negative control.

After comparison, a combination with stable and higher fluorescence value was selected, that is, LgBiT was fused to the N-terminus of Myc and SmBiT was fused to the C-terminus of NDR1. The fluorescence value at this time is strong and has a large fold difference from the negative control. The experimental results are shown in Figure 6.

Example 2

In this example, the optimal plasmid combination of NDR1-Myc NanoBiT obtained in Example 1 of the present invention was screened for small molecule compounds.

2.1. Preparation of compounds

The compounds required for this experiment came from the FDA old drug library purchased by the research group. All compounds were dissolved in DMSO and stored in separate packages at -80°C.

2.2. Screening of small molecule compounds targeting NDR1/Myc interaction

According to the experimental results of Example 1, N-Large-Myc and NDR1-C-Small plasmids were co-transfected into HEK-293 cells. The transfected HEK-293 cells were transferred to a 96-well plate and cultured for 18 hours, then the monomeric compound to be tested was added with a final concentration of 20 μM and treated for 3 hours. Live Cell Reagent compares Luminescence readings to detect the ability of small molecule compounds to inhibit the specific binding of NDR1 to Myc. The results are shown in Figure 7. We found that the candidate compounds Cilnidipine, Tazarotene, Nitazoxanide, Isradipine, Nimodipine, Verteporfin, Diethylstilbestrol, Felodipine, Lacidipine, Benidipine hydrochloride, Manidipine, Nitrendipine, Etravirine, Nicardipine HCl, Tiratricol have good inhibitory effects. The specific inhibition rates are shown in Table 1.

Table 1 Names and inhibition rates of drugs obtained from FDA library screening

2.3. Re-screening and selection of candidate compounds

When the target protein pair interacts, LgBiT will complement the SmBiT structure to form a NanoLuc enzyme with complete catalytic function. The blocking activity of the compound on the target protein pair can be evaluated through the change in fluorescence value produced by the reaction between the substrate and the enzyme. In order to rule out that the compound itself has an inhibitory effect on NanoLuc enzyme and leads to false positive results, further NanoLuc enzyme inhibition experiments are required. The operation method is similar to the NanoBiT blocking experiment. When transfecting in a 6-well plate, the plasmid is replaced with 3 μg of the fusion expression vector pNLF1-N expressing NanoLuc enzyme. In addition, 100 μL was added to each well after compound treatment. Cytotoxicity detection reagent, incubate at room temperature for 10 minutes and perform chemiluminescence measurement to eliminate false positive results due to excessive cytotoxicity of the compound (see Table 2 for results). Among the candidate compounds, Isradipine, Benidipinehydrochloride, Manidipine and Etravirine not only have low cytotoxicity and NanoLuc enzyme inhibitory activity, but also have strong inhibitory effects on NDR1/Myc interaction.

Table 2 Compound re-screening and optimization results

Example 3

In this example, the anti-tumor activity of four small molecule compounds preferred in Example 2 of the present invention was measured.

3.1. Culture of tumor cells

Colorectal cancer cells HT-29 and HCT116 were grown in McCoy's 5A complete medium (containing 10% serum and 1% double antibody). Colorectal cancer cell DLD-1 was cultured in DMEM complete medium (containing 10% serum and 1% double antibody). All cells were cultured at 37°C in a cell culture incubator containing 5% _CO2 .

3.2. Determine the toxicity of compounds to colorectal cancer cells

Colorectal cancer cell lines HT-29, HCT116 and DLD-1 were spread in a 96-well plate with 10,000 cells per well, and cultured for 18 hours until the cells adhered. Carefully discard the culture medium with a pipette gun, add 100 μL of different concentrations of compounds prepared from the basic culture medium to each well, and continue to culture for 24 h. After discarding the original culture medium, add 100 μL of 10% CCK-8 solution to each well and incubate. 1-2h, the microplate reader detects the absorbance value of each well at 450nm, and calculates the cell viability according to the formula.

The results are shown in Table 3. Among the four compounds, only Manidipine has obvious killing effect on colorectal cancer cells HT-29, HCT116 and DLD-1, with IC ₅₀ values of 29.14, 12.76 and 18.02 μM respectively. In addition, the killing effect of Benidipinehydrochloride on HT-29 and HCT116 cells was stronger than that on DLD-1 cells, with IC ₅₀ values of 13.29, 22.10 and 45.36 μM respectively. However, Etravirine and Isradipine showed no anti-tumor activity against the three types of cells.

Table 3 Evaluation of anti-tumor activity of four compounds at cellular level

3.3. Basic properties of compounds

The structural formula of manidipine is as follows:

Manidipine is a calcium antagonist that has strong effects on relaxing arterial smooth muscle, dilating blood vessels, and reducing peripheral vascular resistance and arterial pressure. It is used to treat essential hypertension. It has a more obvious antihypertensive effect on low-renin hypertension and can improve uric acid metabolism.

The structural formula of Benidipine hydrochloride is as follows:

Benidipine hydrochloride is a cardiovascular drug that inhibits the influx of calcium ions, thereby dilating coronary arteries and peripheral blood vessels, resulting in antihypertensive effects.

Example 4

This embodiment expands the application scope of the platform constructed in Example 1 of the present invention to discover polypeptide inhibitors that block the NDR1-Myc protein interaction.

4.1. Identification of Myc fragments binding to NDR1 through NanoBiT technology

In order to obtain small peptides that block the NDR1-Myc protein interaction, we truncated the Myc protein into different regions according to the literature, namely Myc1-113, Myc113-185 and Myc201-439, and cloned them as N-terminal fusions Expressed in LgBiT vector. According to the experimental results of Example 1, the same method was used to co-transfect HEK293 cells with full-length or truncated Myc plasmid expressing N-terminal fusion LgBiT and NDR1-C-Small. 6 hours after transfection, HEK293 cells were dispersed and transferred to a 96-well plate and cultured for 18 hours. Live Cell Reagent determines the best Myc fragment that binds to NDR1 by detecting fluorescence values (results shown in Figure 8). The results show that NDR1 binds to Myc, Myc1-113, and Myc201-439. Among them, NDR1 binds to Myc1-113 slightly stronger than Myc201-439, but does not bind to Myc113-185.

4.2. Detect the blocking activity of N-terminal truncated fragment of Myc on NDR1/Myc interaction

HEK293T was spread into a 6-well plate and cultured for 18 hours until the cells adhered. Use PEI to transiently transfer NDR1 expression plasmid with Venus tag and Myc full-length expression plasmid with GST tag, or GST tag plasmid, or plasmid with Venus tag expressing truncated Myc N-terminal fragment. After 48 h, the culture medium was removed, and the cells were washed with cold PBS, and then 80 μL NP-40 lysis solution was added to each well and lysed at 4°C for 1 hour. At this time, process the GST beads, wash the GST beads 3 times with 3 times the volume of PBS, and centrifuge at 4°C and 12000 rpm for 10 s each time. Finally, add 2 times the volume of lysis buffer and mix well. Add 60 μL of a mixture of GST beads and lysis buffer to each tube. Centrifuge the cell lysate at 4°C and 12,000 rpm for 10 min, and collect the supernatant. Transfer the supernatant to a clean Ep tube, take 10 μL as input, add 6×SDS loading buffer, and freeze at -20°C. Then take 45 μL of the supernatant and mix it with the beads. After rotating and mixing at 4°C for 2 hours, centrifuge at 4°C and 12000 rpm for 2 seconds. Discard the supernatant. Wash the GSTbeads three times with 500 μL NP-40 lysis buffer, centrifuge at 4°C each time at 12,000 rpm for 10 s, remove the lysate, add 20 μL 2×SDS loading buffer, heat at 100°C for 5 min, centrifuge, and freeze at -20°C. Use Yazy PAGE gel kit to prepare the gel required for electrophoresis. Clamp the configured gel with an electrophoresis clamp and detect leaks. Place the electrophoresis clamp into the electrophoresis tank, and add sufficient electrophoresis solution to the electrophoresis tank and electrophoresis clamp. Inject the prepared protein sample and marker into the reserved lane of the gel. The electrophoresis conditions are 80V, 30min; 120V, 60min. Cover the activated PVDF membrane flatly on the gel, taking care to eliminate air bubbles. Set the transfer conditions according to the molecular weight of the target band. After the transfer was completed, the membrane was blocked with TBST containing 5% skimmed milk powder at room temperature for 1 h. Prepare the primary antibody according to the instructions and incubate it with the blocked PVDF membrane on a shaking table at 4°C overnight. Add TBST to wash the membrane, 10 min each time, repeat 3 times. Add secondary antibody and incubate at room temperature for 1 hour. Wash the membrane, 10 minutes each time, repeat 3 times. Add the developer dropwise to the PVDF membrane for development (results shown in Figure 9).

Myc 1-33, Myc 16-33, Myc 30-50 and Myc 114-130 showed the activity of blocking the binding of NDR1 to Myc, and Myc1-33 had the strongest blocking activity. Since Myc 1-33 contains Myc 16-33 and Myc 1-16 did not show a blocking effect, it was finally confirmed that Myc 16-33 (MN1), Myc 30-50 (MN2) and Myc 114-130 (MN3) can be effective Blocks the binding of NDR1 to Myc. The amino acid sequences of MN1, MN2 and MN3 are shown in Table 4.

Table 4 Amino acid sequences of MN1, MN2 and MN3

Example 5

In this example, the blocking activity verification and anti-tumor activity detection of the three Myc-derived small peptides discovered in Example 4 of the present invention were carried out.

5.1. NanoBiT technology detects the blocking activity of MN1, MN2 and MN3 on NDR1/Myc

The three peptides MN1, MN2 and MN3 were synthesized by Nanjing Genscript Biotechnology Co., Ltd. Their antitumor activity was first measured, and no cytotoxicity to tumor cells was detected. Considering that the peptides may not be able to enter tumor cells to exert their effects, the membrane-penetrating peptide sequence GGYGRKKRRQRRR was added to the C-termini of the three peptides respectively to obtain the peptides TAT-MN1, TAT-MN2 and TAT-MN3 (Table 5). According to the method in Example 2, the effects of TAT-MN1, TAT-MN2 and TAT-MN3 on NDR1/Myc interaction were detected. The results are shown in Figure 10. The results showed that the three small peptides had a concentration-dependent inhibitory effect on the interaction between the two proteins.

Table 5 Amino acid sequences of TAT-MN1, TAT-MN2 and TAT-MN3

5.2. CCK-8 detects the toxicity of TAT-MN1, TAT-MN2 and TAT-MN3 to colorectal cancer cells.

According to the method in Example 3, a preliminary evaluation of the cytotoxicity of TAT-MN1, TAT-MN2 and TAT-MN3 on colorectal cancer cell lines HCT116, DLD-1 and HT-29 was carried out (the results are shown in Figure 11). TAT-MN1 and TAT-MN2 had no inhibitory effect on the three types of colorectal cancer cells, while TAT-MN3 had a strong killing effect on all three types of cells.

Therefore, the small molecules Manidipine, Benidipine hydrochloride and polypeptide MN3 discovered in the present invention exert anti-colorectal cancer effects by blocking the NDR1/Myc interaction.

The above are only preferred embodiments of the present invention, and do not limit the present invention in any form or substance. It should be pointed out that for those of ordinary skill in the art, without departing from the methods of the present invention, they will also Several improvements and additions can be made, and these improvements and additions should also be considered as the protection scope of the present invention. Those skilled in the art who are familiar with the art can make slight changes, modifications and equivalent changes based on the technical content disclosed above without departing from the spirit and scope of the invention. Equivalent embodiments; at the same time, any equivalent changes, modifications and evolutions made to the above embodiments based on the essential technology of the present invention still fall within the scope of the technical solution of the present invention.

Claims (10)

1. A screening method of tumor therapeutic drugs uses the interaction of NDR1 and Myc as a screening target.

2. The method of claim 1, wherein an inhibitor capable of blocking NDR1 binding specifically to Myc is selected as the active ingredient of the tumor therapeutic agent.

3. The method according to claim 1 or 2, wherein the screening method comprises the steps of: (1) establishing an NDR1 and Myc protein interaction system; (2) The ability of the candidate substance to inhibit specific binding of NDR1 to Myc is detected using the established protein interaction system.

4. The method of claim 3, wherein the screening method further comprises a validation test of anti-tumor effects.

5. The method according to claim 4, wherein the substance obtained by screening is subjected to an antitumor test using tumor cells as a test subject.

6. The method of claim 5, wherein the anti-tumor validation test is a cell proliferation test or a targeting test.

7. The method of any one of claims 1-5, wherein the tumor is selected from colorectal cancer.

New use of NDR1 and Myc interaction as screening target for screening tumor therapeutic drugs.

New use of NDR1 and Myc interaction inhibitor in preparing tumor therapeutic medicine is provided.

10. The use according to claim 9, wherein the inhibitor of NDR1 and Myc interaction is selected from mantipine, benidipine hydrochloride or MN3.