CN113683664A - FOXM1 targeted degradation small molecule FOXM1-PROTAC and derivative and application thereof - Google Patents
FOXM1 targeted degradation small molecule FOXM1-PROTAC and derivative and application thereof Download PDFInfo
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- CN113683664A CN113683664A CN202110989421.1A CN202110989421A CN113683664A CN 113683664 A CN113683664 A CN 113683664A CN 202110989421 A CN202110989421 A CN 202110989421A CN 113683664 A CN113683664 A CN 113683664A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/001—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof by chemical synthesis
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
- A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
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Abstract
The invention discloses FOXM1 targeted degradation small molecule FOXM1-PROTAC and a derivative and application thereof, wherein the FOXM1 targeted degradation small molecule FOXM1-PROTAC comprises FOXM1 antagonist polypeptide and medicine with amino acid sequences shown in SEQ ID No. 1. FOXM1-PROTAC can be combined with FOXM1, the combination with FOXM1 leads to the degradation of FOXM1 protein and the blocking of downstream signal channels, thereby inhibiting the proliferation of liver cancer cells, promoting the apoptosis of liver cancer cells, providing effective treatment micromolecular medicines for liver cancer and the like, and being widely applied in the fields of medicine and biology.
Description
Technical Field
The invention belongs to the technical field of biotechnology and biomedicine, and relates to FOXM1 targeted degradation small molecule FOXM1-PROTAC, a derivative and an application thereof.
Background
Hepatocellular carcinoma (HCC) is one of the most common malignancies in the world. The International Agency for Research on Cancer (IARC) report of the world health organization indicates that the incidence of liver Cancer is 5 th after lung Cancer, breast Cancer and the like, the mortality is 3 rd after lung Cancer, colorectal Cancer and the like, and the liver Cancer is the only Cancer with the rising annual incidence of the five cancers with the highest mortality. The regional distribution of liver cancer is remarkably different in the world, and the incidence of liver diseases in developing countries is high. Risk factors include hepatitis b virus, hepatitis c virus, fatty liver, alcohol-related cirrhosis, smoking, obesity, diabetes, iron overload, and various dietary exposures.
Liver cancer has a poor prognosis. Only 5% to 15% of patients are eligible for surgical resection, which is only applicable to early stage patients and is usually free of cirrhosis due to reduced liver regeneration capacity; the risk of complications after right hepatectomy is higher compared to left hepatectomy. The treatment scheme for advanced liver cancer comprises: (a) through Arterial Chemoembolization (TACE), compared with conservative treatment of middle and late liver cancer, TACE can improve survival rate of 2 years by 23%; (b) oral sorafenib, a kinase inhibitor, is the most commonly accepted option for late-stage patients. However, less than one third of patients benefit from this treatment and within six months after the start of the regimen, resistance is evident. With long-term use, chemotherapeutic drugs, such as sorafenib, have problems such as toxicity or drug ineffectiveness. Thus, neither current ablative therapy nor chemotherapy is effective in improving the prognosis of this devastating disease. Further research is necessary to find better methods for treating liver cancer.
With the continuous development and research of medicine and molecular biology, it is gradually recognized that the occurrence and development of liver cancer is the evolution process of 'inflammation-hyperplasia-canceration' caused by the cooperation of multiple genes and the participation of multiple signal pathways. The research at home and abroad finds that the liver cancer is a solid tumor with a relatively complex molecular pathogenesis, and the occurrence and development of the liver cancer relate to the imbalance of multiple signal paths and are related to various genetic variations. According to the COSMIC Database data, the mutation frequency of 8 genes in the liver cancer gene mutation spectrum of western people is more than 5 percent, and the mutation frequency is mainly focused on signal paths such as genome instability, immortalization, Wnt and the like. According to GenomiCare data, more than 20 gene variation frequencies in Chinese population exceed 5%, mainly focusing on signal pathways such as genome instability, angiogenesis, cell cycle runaway, growth factors, PI3K-AKT-mTOR and the like.
Besides radiotherapy and chemotherapy, with the discovery and application of treatment means such as targeted therapy, gene therapy, neoadjuvant chemotherapy, immunotherapy and the like, more and better choices are provided for the treatment of liver cancer, and the liver cancer treatment is also an important component of the treatment of liver cancer. Molecular targeted drug therapy has significant efficacy in specific patients, and patients benefit from survival. However, the application of targeted drugs is limited mainly because the effective targets of the existing targeted drugs are few, and the corresponding molecular target detection needs to be carried out on patients before the appropriate targeted therapeutic drugs are selected. At present, VEGFR1-3, FGFR1-4, PDGFR alpha, RTK, KIT and RET are mainly used as targets related to liver cancer treatment and are all used clinically. Therefore, the key of the drug selection for the targeted therapy of liver cancer is the identification of the signal pathway driver and the liver cancer specific transcription factor related to pathogenesis.
FOXM1, an important transcription factor regulating cell cycle and cell progression, has been demonstrated that the FOXM1 regulatory network is a major predictor of poor prognosis in 18000 cancer patients in 39 human malignancies, which also reveals an important role of FOXM1 in cancer. However, it is not completely clear how FOXM1 exerts its oncogenic activity in human cells at all. One mechanism is associated with transcriptional activation of the FOXM1 target, which leads to a variety of pro-tumor effects, including enhanced cell proliferation. In addition, FOXM1 may act as an oncogene by interacting with other proteins, thereby supporting different oncogenic pathways. FOXM1 regulates key processes in tumorigenesis and progression. FOXM1, a typical proliferation-associated transcription factor, directly or indirectly activates expression of target genes at the transcriptional level and exhibits a spatiotemporal pattern whose dysregulation is involved in almost all characteristics of tumor cells. And a large amount of data and experiments prove that the expression level of FOXM1 is up-regulated in various hepatocellular carcinomas, and analysis of patient data shows that the expression level of FOXM1 is closely related to the prognosis of patients with liver cancer. FOXM1 is considered to be a fatal lesion of tumor, and becomes an important target for developing a new tumor marker and an anti-tumor drug for judging the occurrence and development of tumor. At present, the domestic related drug treatment targeting FOXM1 is still in the preclinical research stage, and no antitumor drug directly targeting FOXM1 is on the market or applied to the clinic.
The targeted degradation of proteins using proteolytic targeting chimeras (PROTACs) has become a new therapeutic approach in drug development. PROTACs mediate degradation of the desired target protein by hijacking the activity of E3 ubiquitin ligase for target protein ubiquitination and subsequent degradation by the 26S proteasome. Binding of proteins to ubiquitin, a small protein modifier, is essential for the 26S proteasome to regulate protein degradation. Although the ATP-dependent pathway of protein degradation was described at the end of the 20 th century 70 s, the use of this system for targeted protein degradation was first reported after 30 years. The proteolytic targeting chimera (PROTAC) is a heterobifunctional molecule consisting of three parts: (1) a ligand that binds to a target protein; (2) ligands for recruiting E3 ubiquitin ligase (E3 recruiting elements; E3RE) to facilitate ubiquitination of the target protein; (3) a linker connecting the ligands. To date, more than 100 articles have described PROTACs for targeted protein degradation and their use in chemical biology and drug development.
As a new and promising technology, PROTACs show great opportunity in the following respects. First, PROTACs have a particular sensitivity to drug-resistant targets. Chemotherapy has traditionally been the primary method of cancer treatment. Acquired resistance to chemotherapeutic drugs impedes clinical use and leads to disease recurrence. As research on new targets and new drug discovery technologies progresses, another powerful strategy is to directly and specifically inhibit the function of oncogenic proteins or receptors through small molecules. Notably, the discovery of kinase inhibitors does not address the resistance of cancer therapies, and PROTACs can address the potential resistance faced by current therapies by eliminating the entire target to affect protein function, thereby eliminating the full function of the target, both enzymatic and nonenzymatic. In addition, PROTACs are less sensitive to increased target expression and target protein mutations, and because they have catalytic effects, low doses of PROTACs are required to exert their beneficial effects.
Based on PROTACs, the protein-based protein target fusion protein can deeply degrade target proteins, has sustainability, can quickly and strongly inhibit downstream signals, and can permanently inhibit the occurrence and development of tumors in vitro, so that the protein degradation by using a proteolysis targeting chimera becomes a hotspot mode of current tumor treatment. Therefore, the development of chimera PROTAC targeted to the target FOXM1 in the liver cancer cell is necessary.
Disclosure of Invention
In order to solve the problems in the background art, the invention aims to provide FOXM1 targeted degradation small molecule FOXM1-PROTAC and derivatives and applications thereof. The FOXM1 targeted degradation small molecule FOXM1-PROTAC (derivative) comprises the FOXM1 antagonist polypeptide (derivative) and a drug, the FOXM1 antagonist polypeptide (derivative) and the target FOXM1 have specific high affinity, when the FOXM1 targeted degradation small molecule FOXM1-PROTAC (derivative) is combined with the FOXM1, the combination of the FOXM1 and other proteins or genes can be prevented, the degradation of the FOXM1 protein can be induced, and downstream related genes and signal channels can be influenced, the FOXM1 targeted degradation small molecule FOXM1-PROTAC (derivative) plays an important role in the aspects of targeted inhibition of liver cancer cell proliferation, promotion of liver cancer cell apoptosis and the like, and has great application value in the aspect of liver cancer targeted therapy.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows: in one aspect, the invention provides an FOXM1 antagonist polypeptide, wherein the amino acid sequence of the FOXM1 antagonist polypeptide is shown in SEQ ID No: 1.
In another aspect, the present invention provides a FOXM1 antagonist polypeptide derivative, wherein the FOXM1 antagonist polypeptide derivative is a product obtained by conventionally modifying an amino terminal or a carboxyl terminal of the FOXM1 antagonist polypeptide fragment;
or the FOXM1 antagonist polypeptide is connected with a label for polypeptide or protein detection or purification.
Further, the conventional modification comprises amination, amidation, hydroxylation, carboxylation, carbonylation, alkylation, acetylation, phosphorylation, esterification, glycosylation, cyclization, biotinylation, fluorescent group modification, polyethylene glycol (PEG) modification or immobilization modification;
preferably, the tag comprises His6GST, EGFP, MBP, Nus, HA, IgG, FLAG, c-Myc or ProfinityXact.
Further, the derivative of the FOXM1 antagonist polypeptide is a product obtained by amidation modification of the tail end of the FOXM1 antagonist polypeptide.
Further, the FOXM1 antagonist polypeptide and the derivatives thereof can be obtained by chemical synthesis using an automatic polypeptide synthesizer, according to methods known in the art; deducing a nucleotide sequence from the short peptide sequence, and cloning the nucleotide sequence into a vector for biosynthesis; it can also be extracted and purified in large quantities from existing organisms.
Further, the FOXM1 antagonist polypeptide and derivatives thereof can be derived from mammals or birds, such as primates (humans); rodents, including mice, rats, hamsters, rabbits, horses, cattle, canines, cats, and the like.
In another aspect, the invention provides FOXM1 targeted degradation of a small molecule FOXM1-PROTAC comprising the FOXM1 antagonist polypeptide and a drug as described above.
In still another aspect, the invention provides a FOXM1 targeted degradation derivative of a small molecule FOXM1-PROTAC, which comprises the above-described FOXM1 antagonist polypeptide derivative and a drug.
Further, the FOXM1 antagonist polypeptide can be linked with a drug or a derivative of the FOXM1 antagonist polypeptide can be linked with a drug.
Further, the medicament comprises pomalidomide.
Further, the FOXM1 targeted degraded small molecule FOXM1-PROTAC and the derivatives thereof are obtained by a method known in the prior art, and can be chemically synthesized by an automatic polypeptide synthesizer; deducing a nucleotide sequence from the short peptide sequence, cloning the nucleotide sequence into a vector for biosynthesis, and then linking the nucleotide sequence with a drug; it can also be extracted and purified in large quantities from an existing organism and then linked to a drug.
In another aspect, the present invention provides a polynucleotide encoding the FOXM1 antagonist polypeptide or a derivative of the FOXM1 antagonist polypeptide.
In another aspect, the invention provides an application of the FOXM1 targeted degradation small molecule FOXM1-PROTAC or the FOXM1 targeted degradation small molecule FOXM1-PROTAC derivative in preparation of a drug for preventing and/or treating a tumor highly expressing FOXM 1.
In another aspect, the invention provides an application of the FOXM1 targeted degradation small molecule FOXM1-PROTAC or the FOXM1 targeted degradation small molecule FOXM1-PROTAC derivative in preparation of a drug for inhibiting proliferation of tumor cells highly expressing FOXM1 and/or promoting apoptosis of tumor cells highly expressing FOXM 1.
Further, the tumor highly expressing FOXM1 comprises liver cancer, lung cancer, breast cancer and colorectal cancer, and is preferably liver cancer.
In still another aspect, the present invention provides a pharmaceutical composition or a detection reagent, comprising at least one of the above-mentioned FOXM 1-targeted degraded small molecule FOXM1-PROTAC, and the above-mentioned FOXM 1-targeted degraded small molecule FOXM1-PROTAC derivative.
Further, the pharmaceutical composition contains one or more pharmaceutically acceptable carriers.
Further, the pharmaceutically acceptable carrier includes diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption enhancers, adsorption carriers, surfactants, lubricants, or the like.
Furthermore, the pharmaceutical composition can be prepared into various forms such as tablets, granules, capsules, oral liquid or injection, and the like, and the medicines of various dosage forms can be prepared according to the conventional method in the pharmaceutical field.
Furthermore, the pharmaceutical composition can be prepared into various forms such as tablets, granules, capsules, oral liquid or injection, and the like, and the medicines of various dosage forms can be prepared according to the conventional method in the pharmaceutical field.
The invention has the beneficial effects that:
(1) the invention provides a FOXM1 targeted degradation small molecule drug FOXM1-PROTAC and derivatives thereof, wherein the FOXM1-PROTAC and the derivatives thereof can be specifically combined with FOXM1 and specifically combined with FOXM1 to induce FOXM1 degradation and inhibit a FOXM1 downstream signal channel.
(2) The FOXM1 targeted degradation small molecule drug FOXM1-PROTAC and the derivatives thereof provided by the invention can inhibit cancer cell proliferation by blocking a signal channel of FOXM1 and promote cancer cell apoptosis, can be used as a biodegradable small molecule drug of FOXM1 binding sites, and can be used for preparing a biological degradation type small molecule drug for preventing and/or treating tumors. Can be widely applied in the medical and biological fields and can generate huge social and economic benefits.
Drawings
FIG. 1 is a mass spectrum of synthetic pomalidomide-PEG2 in an example of the present invention;
FIG. 2 is a diagram showing the result of FOXM1 targeted degradation of small molecule FOXM1-PROTAC inhibiting the proliferation of hepatoma cell HepG 2;
FIG. 3 is a diagram showing the result of FOXM1 targeted degradation of small molecule FOXM1-PROTAC inhibiting FOXM1 high expression cancer cell proliferation;
FIG. 4 is a graph showing the results of FOXM1 targeted degradation of small molecules FOXM1-PROTAC of the present invention inducing FOXM1 degradation in HepG2 cells; wherein A: the expression level of FOXM1-PROTAC treated HepG 216 h FOXM1 was varied at different concentrations; b: at different times, the expression level of FOXM 1-PROTAC-treated HepG 220. mu.M FOXM1 was varied.
FIG. 5 is a diagram showing the result of FOXM1 targeted degradation of small molecule FOXM1-PROTAC of the present invention inhibiting the proliferation and migration of HepG2 cells; wherein, A: cloning to form; b: and (4) scratching cells.
Detailed Description
In order that the invention may be more clearly understood, it will now be further described with reference to the following examples and the accompanying drawings. The examples are for illustration only and do not limit the invention in any way.
In the examples, each raw reagent material is commercially available, and the experimental method not specifying the specific conditions is a conventional method and a conventional condition well known in the art, or a condition recommended by an instrument manufacturer.
FOXM1 targeted degradation of a small molecule FOXM1-PROTAC in the following examples of this application is pomalidomide-PEG2-SEQ ID No:1 (i.e., pomalidomide-PEG 2-GLSSMHSAPPLR-GRKKRRQRRRPPQQ).
Example 1 Synthesis of FOXM1-PROTAC
Firstly, synthesizing intermediate pomalidomide-PEG2 of FOXM1-PROTAC, connecting the intermediate with known polypeptide sequence, and finally synthesizing high-purity small molecule FOXM1-PROTAC, which is completed by Vast biological company in the Shanghai.
Wherein, the mass spectrogram of the intermediate pomalidomide-PEG2 is shown in figure 1, and the purity and the quality of the intermediate product can be seen from figure 1 to meet the requirements of subsequent synthesis.
Example 2 CCK-8 detection of the inhibitory Effect of FOXM1-PROTAC on HepG2 cells
Inoculating HepG2 cells into a 10cm culture dish, culturing the cells to reach the density of 95% by using 10% FBS DMEM medium, sucking the medium after the cells reach the required density, washing the cells for 2 times by using PBS, adding 1mL of pancreatin, incubating and digesting the cells for 1min at 37 ℃, adding 1mL of 10% FBS DMEM medium, beating the dispersed cells, sucking 10 mu L of cell suspension, adding the cell suspension into 1mL of DMEM medium to prepare cell suspension, counting the number of the cells in the prepared cell suspension by using a cell counting plate, and counting the number of the cells in each hole5×103One was inoculated in a 96-well plate and cultured for 24 hours. The following day, the cell culture medium was discarded, fresh medium and FOXM1-PROTAC of various concentrations were added to the plates, the plates were incubated in an incubator for 48 hours, 10. mu.L of CCK-8 solution (taking care not to generate bubbles in the wells, which would affect the OD reading) was added to each well, the plates were incubated in the incubator for 1-4 hours, the absorbance at 450nm was measured with a microplate reader, the cell viability was calculated according to the following formula, and an inhibition curve was plotted. Cell viability (%) ([ a (medicated) -a (blank)]/[ A (0 dosing) -A (blank)]X 100, a (dosed): OD values of wells with cells, CCK-8 solution and drug solution, a (0 dosing): OD of wells with cells, CCK-8 solution and no drug solution, A (blank): OD values for wells without cells. The results are shown in FIG. 2, and it can be seen from FIG. 2 that the cell viability decreases with increasing drug concentration and eventually levels off, and the IC50 value of the cells is about 20 μ M.
Example 3 CCK-8 detection of the inhibitory Effect of FOXM1-PROTAC on HepG2 and other FOXM1 high expressing cells
Firstly, respectively selecting four non-cancer cells of liver cancer HepG2, lung cancer A549, breast cancer MB-231 and colorectal cancer HCT116, respectively inoculating the four cells into a 10cm culture dish, culturing the cells to the density of 95% by using 10% FBS DMEM culture medium, sucking the culture medium after the cells reach the required density, washing the cells for 2 times by using PBS, adding 1mL of pancreatin, incubating and digesting the cells for 1min at 37 ℃, then adding 1mL of 10% FBS DMEM culture medium, beating the dispersed cells, sucking 10 mu L of cell suspension and adding the cell suspension into 1mL of DMEM culture medium to prepare cell suspension, counting the number of the cells in the prepared cell suspension by using a cell counting plate, and then counting the number of the cells in the prepared cell suspension by 5 multiplied by 10 per hole3One was inoculated in a 96-well plate and cultured for 24 hours. The following day, the cell culture medium was discarded, fresh medium and different concentrations of FOXM1-PROTAC were added to the plates, the plates were incubated in an incubator for 48 hours, 10. mu.L of CCK-8 solution (taking care not to generate bubbles in the wells, which would affect the OD reading) were added to each well, the plates were incubated in the incubator for 1-4 hours, and the absorbance at 450nm was measured with a microplate reader. The cell viability was then calculated according to the following formula and an inhibition curve was plotted. Cell viability (%) ([ a (medicated) -a: (b) ())Blank)]/[ A (0 dosing) -A (blank)]X 100, a (dosed): OD values of wells with cells, CCK-8 solution and drug solution, a (0 dosing): OD of wells with cells, CCK-8 solution and no drug solution, A (blank): OD values for wells without cells. The results are shown in fig. 3, and it can be seen from fig. 3 that the activity of all four cancer cells is reduced with the increasing concentration of the drug, and the sensitivity of different cells to the drug is different, wherein the liver cancer cell is most sensitive to the drug and has the lowest IC50 value.
Example 4 FOXM1-PROTAC can induce the degradation of FOXM1 in HepG2 cells
Firstly, the liver cancer cell HepG2 is mixed by 5 times 105Inoculating each cell/well in a 6-well cell culture plate, wherein the volume of a culture medium in each well is 2mL, and culturing for 24 hours;
adding FOXM1-PROTAC with different concentration gradients (0 μ M, 2 μ M, 5 μ M, 10 μ M, 20 μ M, 30 μ M, 50 μ M) for culturing for 16 hours, collecting cells, adding lysate to collect protein;
③ adding FOXM1-PROTAC with the same concentration gradient (20 mu M) to culture for different time (0h, 2h, 4h, 8h, 12h and 24h), collecting cells, adding lysate to collect protein;
preparing SDS-PAGE gel with the concentration of 10% according to the Yazyme gel preparation kit;
adding a proper amount of SDS-PAGE protein loading buffer 5X sample bf. into the collected protein sample, and mixing, wherein B-mercaptoethanol (500 mul bf +70 mul l B-mercaptoethanol) is added into the sample bf. The protein sample was mixed with sample bf and centrifuged for 10s at 12000 rpm. Denaturing the protein at 100 deg.C for 5-10 min. Centrifuge briefly at 12000rpm, and cool. After cooling to room temperature, the protein samples were loaded directly into SDS-PAGE gel loading wells. And aligning the sample loading groove according to the designed sequence, and slowly adding the sample and the pre-dyed protein Marker. Usually, the electrophoresis is stopped when bromophenol blue reaches near the bottom end of the gel during electrophoresis, or the electrophoresis is stopped when the target protein is expected to be properly separated according to the electrophoresis of the prestained protein molecular weight standard.
Sixthly, preparing a box and containing methanol, transfer bf, and removing the gel glass plate from the electrophoresis apparatus. The gel was removed and the concentrated gel and bromophenol blue at the lower end were excised with a razor blade. The length and width of the glue are measured with a ruler. Two pieces of filter paper of the same size and one piece of PVDF membrane were cut out. The membrane was shaken for 1min by treatment with methanol and then washed 5 times with RO water, 1 min/1 time. And then soaked with transfer bf for more than 10 minutes. Filter paper and glue were also soaked in transfer bf. The cotton blocks in the film converter can be cleaned by the idle time, then dried, and then wetted by a transfer bf, and the film converter blackboard is arranged in a sponge-filter paper-gel-PVDF film-filter paper-sponge sequence. The glue and the membrane are placed in sequence, and the contact surface of the glue and the membrane is noticed, so that air bubbles cannot exist. No air bubbles can be present between the membrane and the filter paper, and between the filter paper and the glue. The air bubbles can be removed by lightly rolling the glass tube back and forth with a smearing bar or a 15ml glass centrifuge tube. The double-layer effect of the filter paper is better. 260mA voltage for 100 min.
Seventhly, taking out the membrane with the protein surface facing upwards by using a pair of tweezers, and sealing in 5 percent of sealing liquid of skimmed milk powder (prepared by TBST, eg, 40mL of TBST solution and 2g of skimmed milk powder) for 1-1.5 h. The hybridization system was selected to be 5mL or 10mL, with reference to the primary antibody specification. If the amount of the primary antibody added is large, 5mL of hybridization system or even 3or 4mL is selected. The primary antibody was diluted with a diluent (1% blocking solution, eg, hybrid system 5 mL: 1mL blocking solution +4mL TBST or hybrid system 10 mL: 2mL blocking solution +8mL TBST) at the appropriate ratio, according to the instructions for the primary antibody. The primary antibody was recovered by incubation for 1h on a side-shaking table at room temperature or 4 ℃ with slow shaking. Washed 3 times with TBST, 5 min/time, and washed on a shaker with slow shaking. The secondary antibody was diluted in appropriate proportions with reference to the instructions for the secondary antibody. Incubate for one hour on a shaker with slow shaking. Washed 5 times with TBST, 5 min/time, and washed on a shaker with slow shaking. ECL Western fluorescent detection reagents were used to detect proteins. The results are shown in fig. 4, from which it can be seen that FOXM1-PROTAC degrades FOXM1 more over time, and FOXM1 degrades more at higher FOXM1-PROTAC concentrations over the same time.
Example 5 FOXM1-PROTAC can inhibit proliferation and migration of HepG2 cells and promote apoptosis
Inoculating 100 liver cancer cells HepG2 into a 6-hole cell culture plate in a volume of 2mL per hole of culture medium, and culturing for 24 hours; then 20 mu M FOXM1-PROTAC and a proper amount of fresh culture medium are added, the culture medium and the drug are replaced every two days by taking an unformed group as a control, the continuous culture is carried out until the cell mass contains more than 50 cells, after the cells of the experimental group and the control group are fixed by methanol, 0.1% of crystal violet staining is carried out, the number of formed clones in each group is counted, and the result is shown in figure 5A, and as can be seen from the figure, compared with the control group, the number of formed clones of the cells is obviously reduced after FOXM1-PROTAC is added, and the FOXM1-PROTAC can effectively inhibit the formation of the clones of the cells.
Secondly, enabling a 6-pore plate to overgrow the liver cancer cell HepG2 to be divided into 1: 2, inoculating the cells into a new 6-well cell culture plate, wherein the volume of a culture medium in each well is 2mL, culturing for 24 hours, scratching the cells by using a wall head after the cells grow, and washing away floating cells by using PBS (phosphate buffer solution); the scratch distance was recorded by microscopic photograph, 20 μ M FOXM1-PROTAC and appropriate amount of serum-free fresh medium were added, and the control group was not added with drug, floating cells were washed out after 48h, and the scratch distance was recorded by microscopic photograph, as shown in fig. 5B, it can be seen from the figure that FOXM1-PROTAC was added, the width of the scratch was wider, the migration of cells was slowed, and FOXM1-PROTAC was effective in inhibiting the migration of cells, compared with the control group.
SEQUENCE LISTING
<110> Shenzhen International institute for graduate of Qinghua university
<120> FOXM1 targeted degradation small molecule FOXM1-PROTAC, and derivatives and application thereof
<130> CP121010569C
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 26
<212> PRT
<213> Artificial sequence
<400> 1
Gly Leu Ser Ser Met His Ser Ala Pro Pro Leu Arg Gly Arg Lys Lys
1 5 10 15
Arg Arg Gln Arg Arg Arg Pro Pro Gln Gln
20 25
Claims (10)
1. An FOXM1 antagonist polypeptide, wherein the amino acid sequence of the FOXM1 antagonist polypeptide is shown in SEQ ID No. 1.
2. A derivative of the FOXM1 antagonist polypeptide, wherein the derivative of the FOXM1 antagonist polypeptide is the FOXM1 antagonist polypeptide of claim 1, and/or the FOXM1 antagonist polypeptide fragment of claim 1 is modified at the amino-or carboxy-terminus by conventional modifications;
or the FOXM1 antagonist polypeptide of claim 1 linked to a tag for polypeptide or protein detection or purification;
preferably, the conventional modification comprises amination, amidation, hydroxylation, carboxylation, carbonylation, alkylation, acetylation, phosphorylation, esterification, glycosylation, cyclization, biotinylation, fluorophore modification, polyethylene glycol (PEG) modification or immobilization modification;
preferably, the tag comprises His6GST, EGFP, MBP, Nus, HA, IgG, FLAG, c-Myc or ProfinityXact.
3. FOXM1 targeted degradation of a small molecule FOXM1-PROTAC comprising the FOXM1 antagonist polypeptide of claim 1 and a drug.
4. A FOXM1 targeted degradation derivative of a small molecule FOXM1-PROTAC comprising the FOXM1 antagonist polypeptide derivative of claim 2 and a drug.
5. The FOXM 1-targeted degraded small molecule FOXM1-PROTAC according to claim 3or the FOXM 1-targeted degraded small molecule FOXM1-PROTAC derivative according to claim 4, further comprising a linker linking the FOXM1 antagonist polypeptide of claim 1 and a drug, or the FOXM1 antagonist polypeptide derivative of claim 2 and a drug.
6. The FOXM 1-targeted degraded small molecule FOXM1-PROTAC according to claim 3or the FOXM 1-targeted degraded small molecule FOXM1-PROTAC derivative according to claim 4, wherein the drug comprises pomalidomide.
7. The use of the FOXM1 targeted degraded small molecule FOXM1-PROTAC of claim 3or the FOXM1 targeted degraded small molecule FOXM1-PROTAC derivative of claim 4 in the preparation of a medicament for preventing and/or treating a tumor highly expressing FOXM 1.
8. The use of the FOXM 1-targeted degraded small molecule FOXM1-PROTAC of claim 3or the FOXM 1-targeted degraded small molecule FOXM1-PROTAC derivative of claim 4 in the preparation of a medicament for inhibiting the proliferation of tumor cells highly expressing FOXM1 and/or promoting the apoptosis of tumor cells highly expressing FOXM 1.
9. The use of claim 7 or 8, wherein the tumor highly expressing FOXM1 comprises liver cancer, lung cancer, breast cancer, colorectal cancer, preferably liver cancer.
10. A pharmaceutical composition or a detection reagent comprising at least one of the FOXM 1-targeted degraded small molecule FOXM1-PROTAC of claim 3, the FOXM 1-targeted degraded small molecule FOXM1-PROTAC derivative of claim 4.
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