CN114917359A - PROTAC composition aiming at cell cycle multi-space-time distribution anti-cancer targets - Google Patents
PROTAC composition aiming at cell cycle multi-space-time distribution anti-cancer targets Download PDFInfo
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Abstract
The invention discloses a PROTAC composition aiming at multiple space-time distribution anti-cancer targets in a cell cycle. The inventors found that the combined use of 2 or more types of ProTACs for the same oncogenic protein by a large number of grops and screens, including the first resolution of the cell cycle expression correlation of the target protein with different E3 ubiquitin ligases using cell cycle arrest-release experiments, the first resolution of the cell response to PROTACs based on different E3 ubiquitin ligases using quantitative proteomics and transcriptomics experiments, the screening of the combined killing effect of PROTACs based on different E3 ubiquitin ligases at different ratios and concentrations using cell proliferation experiments, and the combined use of 2 or more types of PROTACs for the same oncogenic protein using in vivo administration experiments in animals and administration experiments to cancer primary patients, can obviously improve the curative effects of proliferation inhibition, cycle retardation, apoptosis induction and dryness inhibition on cancer cells, meanwhile, the method can effectively prevent cancer drug resistance and relieve one of the biggest challenges of single PROTAC, namely the hook effect.
Description
Technical Field
The invention belongs to the field of medicines, and particularly relates to a PROTAC composition for multiple spatial-temporal distribution anti-cancer targets in a cell cycle.
Background
The Ubiquitin-Proteasome Pathway (UPP) mediates degradation of more than 80% of proteins in cells and is one of the major modes of protein degradation. Based on the mechanism of regulating protein degradation by ubiquitination modification, technologies of proteolytic targeting chimeras (PROTAC) were first proposed in 2001 by Raymond J. Desheies and Craig M. Crews et al, Calif. university of science. The PROTAC is a bifunctional molecule consisting of three key parts, namely a Protein of interest (POI) binding ligand, a recruitment ligand of E3 ubiquitin ligase and a Linker connecting the two, promotes ubiquitination of target proteins by recruiting E3 ubiquitin ligase, and then degrades the target proteins through a ubiquitin-proteasome pathway. E3 ubiquitin ligases in cells are over 600, and MDM2 (2008), cIAP (2010), CRBN (2015), VHL (2015) and the like are currently used in PROTAC in many cases.
Theoretically, the PROTAC technology can degrade target proteins from the source, realize comprehensive inhibition on functions of pathogenic proteins, and has great advantages in the aspects of intervening pathogenic proteins and overcoming drug resistance. In recent years, people successfully realize targeted degradation of different proteins by using the PROTAC technology. However, from all the PROTAC data published at present, most of the PROTACs have a serious Hook Effect (Hook Effect), and moreover, the target protein is not completely degraded, so that the clinical application potential of the PROTAC is limited.
The hook effect is one of the most serious challenges of the current PROTAC, belongs to the congenital defect and is difficult to overcome. The principle of "hook effect" formation is as follows: the PROTAC must form an effective ternary complex with the target protein and the E3 enzyme, i.e., [ target protein-PROTAC-E3 ubiquitin ligase ], so that the target protein, RPOTAC and E3 ubiquitin ligase have proper concentrations and affinity. If the concentration of the PROTAC is too low, the target protein cannot be modified by E3 ubiquitin ligase and cannot play a role; if the concentration of the PROTAC is too high, a large number of binary complexes can be formed with the target protein and the E3 ubiquitin ligase respectively, so that a ternary complex cannot be formed, and the degradation effect of the PROTAC on the target protein is weakened. The hook effect causes the selection of the drug dosage to be more rigorous, and particularly when the drug is administered to a human body, the standardized administration mode is difficult to realize by considering that the absorption, the metabolism and the like of the drug are different for individuals. If the hook effect can be overcome or better relieved, the method has great significance for clinical application of the PROTAC. The presence of a "hook effect" may also lead to tumor resistance.
The "hook effect" results in a certain lack of the ability of PROTAC to degrade target proteins. However, from the results published at present, most of the procac cannot completely degrade the target protein even if the optimal concentration of the procac is selected in the in vitro cell line experiment. The possible reason for the insufficient degradation capability of procac is that the molecular weight of procac molecules is relatively large compared with that of conventional small-molecule kinase inhibitors, and the procac molecules are difficult to enter cells. More reasons remain to be explored.
Disclosure of Invention
The invention aims to overcome at least one defect of the prior art and provides a proteolysis targeting chimera composition and application thereof.
The technical scheme adopted by the invention is as follows:
in a first aspect of the present invention, there is provided:
a proteolytic targeting chimeric composition (PROTAC cocktail) comprising at least 2 proteolytic targeting chimeric molecules directed against the same target protein, said proteolytic targeting chimeric molecules acting on said target protein in different cell cycles.
In some examples of the proteolytic targeting chimeric compositions, the proteolytic targeting chimeric molecules are proteolytic targeting chimeric molecules based on a different E3 ubiquitin ligase.
In some examples of the proteolytic targeting chimeric compositions, the target protein is a oncogenic protein.
In some examples of proteolytic targeting chimeric compositions, the cell cycle is interphase and M-phase.
In some examples of proteolytic targeting chimeric compositions, the proteolytic targeting chimeric molecule is a proteolytic targeting chimeric molecule that targets the ubiquitination-degrading Aurora-a kinase.
In some examples of the proteolytic targeting chimeric composition, the cancer is selected from breast cancer, leukemia, lung cancer, liver cancer, esophageal cancer, pancreatic cancer, colorectal cancer, gastric cancer, cervical cancer, brain cancer, nasopharyngeal cancer.
In some examples of the proteolytic targeting chimeric composition, the oncogenic protein is Aurora-A, Aurora-B, PLK1, CDK1, CDK2, RAF, MEK, ERK, ALK, MET, PI3K, ABL, ER, BRD4, BTK, AR, CDK4, or CDK 6.
In some examples of the proteolytic targeting chimeric compositions, the proteolytic targeting chimeric molecules are CRBN-based Aurora-a procac and cIAP-based Aurora-a procac.
In some examples of proteolytic targeting chimera compositions, the proteolytic targeting chimera molecules are CRBN-based Aurora-a procac dabra 383 and cIAP-based Aurora-a procac dabra 450, the formulae for dabra 383 and dabra 450 are as follows:
in some examples of proteolytic targeting chimeric compositions, the compositions are injectable, oral, mucosal.
In some examples of the proteolytic targeting chimeric compositions, the cancer overexpresses Aurora-A, Aurora-B, PLK1, CDK1, CDK2, RAF, MEK, ERK, ALK, MET, PI3K, ABL, ER, BRD4, BTK, AR, CDK4, or CDK 6.
In some examples of proteolytic targeted chimeric compositions, the high expression refers to a protein that is expressed in significantly higher than normal levels. Such as 1.5-fold, 2-fold or more above normal levels.
In a second aspect of the present invention, there is provided:
the invention also provides application of the proteolytic targeting chimera composition in preparation of antitumor drugs.
In some examples of use, the tumor is selected from breast cancer, leukemia, lung cancer, liver cancer, esophageal cancer, pancreatic cancer, colorectal cancer, stomach cancer, cervical cancer, brain cancer, nasopharyngeal cancer.
In some examples of use, the tumor overexpresses Aurora-A, Aurora-B, PLK1, CDK1, CDK2, RAF, MEK, ERK, ALK, MET, PI3K, ABL, ER, BRD4, BTK, AR, CDK4, or CDK 6.
In a third aspect of the present invention, there is provided:
a method of treating a tumor comprising the steps of:
detecting a tumor patient to determine the expression condition of the oncogenic protein of the tumor patient;
administering to a patient having a tumor a therapeutic amount of a proteolytic targeting chimeric composition comprising at least two proteolytic targeting chimeric molecules directed against the same oncogenic protein, said proteolytic targeting chimeric molecules acting on the oncogenic proteins of different cell cycles.
In some examples of therapeutic methods, the proteolytic targeting chimera molecule is a proteolytic targeting chimera molecule based on a different E3 ubiquitin ligase.
In some examples of methods of treatment, the cell cycle is interphase and M-phase.
In some examples of the method of treatment, the tumor is selected from breast cancer, leukemia, lung cancer, liver cancer, esophageal cancer, pancreatic cancer, colorectal cancer, gastric cancer, cervical cancer, brain cancer, nasopharyngeal cancer.
In some examples of the method of treatment, the oncogenic protein is Aurora-A, Aurora-B, PLK1, CDK1, CDK2, RAF, MEK, ERK, ALK, MET, PI3K, ABL, ER, BRD4, BTK, AR, CDK4, or CDK 6.
In some examples of therapeutic methods, the proteolytic targeting chimeric molecule is a proteolytic targeting chimeric molecule targeting ubiquitination-degrading Aurora-a kinase.
In some examples of therapeutic methods the proteolytic targeting chimeric molecules are CRBN-based Aurora-a procac and cIAP-based Aurora-a procac.
In some examples of therapeutic methods, the proteolytic targeting chimera molecules are CRBN-based Aurora-a procac dAurA383 and cIAP-based Aurora-a procac dAurA 450.
In some examples of methods of treatment, the method of administration includes injection, oral, mucosal administration.
The beneficial effects of the invention are:
the inventor researches and invents that the expression and the function of the same oncogenic protein in different cell cycles or subcellular structures may be different, and the functional mechanism exerted by the same oncogenic protein may be different. Through a large amount of exploration and screening, the inventor finds that the combined use of 2 or more PROTAC molecules aiming at the same oncogenic protein can obviously improve the curative Effect, simultaneously can effectively prevent tumor drug resistance and relieve one of the biggest challenges facing a targeting chimera for degrading a single protein, namely Hook Effect.
Taking Aurora kinase (Aurora kinase) as an example, Aurora kinase (a kind of serine/threonine kinase in cells, wherein Aurora-A (aurka) promotes centrosome maturation and chromosome separation in M phase through the kinase activity, Aurora-A presents high expression in various tumors, is related to poor prognosis and is one of important driving factors of tumorigenesis and tumor deterioration.
Through Western Blot experiments and differential proteomic and transcriptomic detection, the inventor internationally discovers that Aurora-A degrading agents dAurA383 and dAurA450 based on different E3 ubiquitin ligases CRBN and cIAP regulate the proliferation and the dryness of leukemia cells through preferentially degrading Aurora-A in an M period and an interphase respectively for the first time. dAurA383 and dAurA450 were determined to synergistically inhibit cell survival by CCK8 experiments and CompuSyn software analysis. Further, CFSE cell proliferation experiments, cell cycle analysis experiments, methyl cellulose colony formation experiments and Annexin V/PI flow experiments prove that the composition Aurora-A PROTAC cocktail of dAurA383 and dAurA450 can effectively inhibit cell proliferation and colony formation, induce cycle arrest and apoptosis, and remarkably improve the inhibition effect of the Aurora-A degrading agent based on single E3 ubiquitin ligase on cells. In addition, the inventor also proves that the Aurora-A PROTAC cocktail can effectively relieve the Hook Effect (Hook Effect) generated by the Aurora-A degradation agent based on single E3 ubiquitin ligase at higher concentration through Western Blot experiment, and the application concentration range of the single protein degradation agent is expanded. Finally, the inventors demonstrated that Aurora-a procac cocktail efficiently degrades target proteins and induces apoptosis in animal models and patient specimens.
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FIG. 1 is the periodic expression of E3 ubiquitin ligase in cells.
FIG. 2 is a TMT quantitative proteomics analysis of the degradation of intracellular proteins by Aurora-A PROTAC.
FIG. 3 is a graph showing the RNA-seq analysis of changes in gene expression under Aurora-A PROTAC treatment.
FIG. 4 shows the optimization of the concentration ratio of the composition of the present invention.
FIG. 5 is a graph of the synergistic inhibition of leukemia cell survival of the compositions of the present invention.
FIG. 6 is a graph of the synergistic inhibition of leukemia cell proliferation of a composition of the present invention.
FIG. 7 is a graph of the composition of the present invention inducing leukemia cell cycle arrest.
FIG. 8 shows that the composition of the present invention promotes apoptosis of leukemia cells.
FIG. 9 shows the ability of the composition of the present invention to inhibit the formation of methylcellulose colonies in leukemia cells.
Fig. 10 is a graph of the mitigating effect of the proteolytic targeting chimera composition of the invention on hook effect.
FIG. 11 is a graph of the inhibition of leukemia cell growth in an animal model by a composition of the invention.
FIG. 12 is a graph of the composition of the invention degrading a target protein and inducing apoptosis in primary cells in a patient sample.
Detailed Description
The embodiments of the invention will now be described in detail with reference to the drawings, but the following embodiments and drawings are only suitable for understanding the invention, and do not limit the invention, which can be embodied in many different forms as defined and covered by the claims.
Chinese and English contrast:
fetal Bovine Serum (FBS), Propidium Iodide (PI), Mean Fluorescence Intensity (MFI), Hook Effect (Hook Effect).
Primary reagent
Fetal bovine serum was purchased from Invitrogen; basal medium RPMI 1640 was purchased from Invitrogen.
Cell line and culture medium
Four cell cultures are mentioned in the examples. No mycoplasma pollution is generated in the experimental process. The media and formulation methods used are described below.
1) KG1a cells (acute myelogenous leukemia cell line)
Culture medium: RPMI 1640 supplemented with 20% Fetal Bovine Serum (FBS)
37℃，5% CO 2 Culturing and once passage for 2-3 days.
2) Kasumi-1 cell (acute myelogenous leukemia cell strain)
Culture medium: RPMI 1640 supplemented with 20% fetal bovine serum
37℃，5% CO 2 Culturing and once passage for 2-3 days.
3) HL60 cell (acute myelogenous leukemia cell strain)
Culture medium: RPMI 1640 supplemented with 10% fetal bovine serum
37℃，5% CO 2 Culturing and carrying out passage once in 2-3 days.
4) U937 cell (acute myelogenous leukemia cell strain)
Culture medium: RPMI 1640 supplemented with 10% fetal bovine serum
37℃，5% CO 2 Culturing and once passage for 2-3 days.
dAura383, dAura425 and dAura450 were designed based on E3 ubiquitin ligase CEBN, VHL and cIAP, respectively, and the structural formulas are as follows:
firstly, the method comprises the following steps: cyclic expression of E3 ubiquitin ligase in cells
Expression of the relevant proteins in each cell phase following Thymidine-blocking release was examined by Western Blot. The method comprises the following specific steps:
1) cells were synchronized at the G1/S boundary using standard double Thymidine blocking;
2) cells were first treated with Thymidine (HY-N1150, MCE, 2 mM) for 16 h;
3) the cells were collected, washed twice with PBS (G0002, Servicebio);
4) changing the culture solution and releasing the culture solution in a fresh culture medium for 9 hours;
5) cells were treated with Thymidine (2 mM) for 16 hours;
6) synchronized cells were released into fresh medium and cells were harvested at designated time points for Western Blot analysis.
The results are shown in FIG. 1. The experimental results showed that Aurora-a, CEBN, VHL and cIAP were distributed differently in the cell cycle, suggesting that Aurora-a PROTACs based on different E3 ubiquitin ligases might work by targeting Aurora-a at different stages.
II, secondly: TMT quantitative proteomics analysis of degradation condition of Aurora-A PROTAC on intracellular protein
The method comprises the following specific steps:
1) acute myelogenous leukemia cell KG1a at 1.2X 10 per well 6 Density of individual cells dispersed into 6-well plates;
2) KG1A cells treated with Aurora-A PROTACs (500 nM) and DMSO for 6 hours;
3) collecting cells, lysing and quantifying;
4) equal amounts of protein were taken for TMT labeling for each treatment, DMSO-TMT-126, dAurA383-TMT-127, dAurA425-TMT-128, and dAurA450-TMT-129, respectively; carrying out mass spectrum identification on the mixed marked sample;
5) data were analyzed using MaxQuant software to compare protein abundance of Aurora-a PROTACs-treated versus DMSO-treated cells, the relative normalized abundance of the relevant proteins after Aurora-a PROTAC-treated cells is shown as a heatmap;
6) GO analysis was performed on the proteins with decreased expression of the first 100 proteins using g: Profiler tool.
The results are shown in FIGS. 2A-B, in which A, the relative normalized abundance of the relevant proteins after Aurora-A PROTAC treatment of cells, is shown as a heatmap. And B, performing GO analysis on the proteins with the reduced expression quantity of the first 100 proteins by using a g-Profiler tool. . The experimental result shows that the Aurora-A PROTACs effectively degrade Aurora-A; degradation of target proteins by Aurora-A PROTACs results in altered expression of associated cellular pathway proteins. Wherein, dAurA383 causes the change of related proteins such as "mitotic cell cycle process", and dAurA450 causes the change of related proteins such as "oxidative phosphorylation".
Thirdly, the method comprises the following steps: RNA-seq analysis of Gene expression Change under Aurora-A PROTAC treatment
The method comprises the following specific steps:
1) acute myelogenous leukemia cell KG1a at 1.2X 10 per well 6 The density of individual cells was dispersed into 6-well plates;
2) treatment with Aurora-A PROTAC (1. mu.M) or DMSO for 48 hours;
3) cells were harvested and RNA-seq analysis was performed by the company;
4) RNA-seq data were subjected to a Gene Set Enrichment Analysis (Gene Set expression Analysis, GSEA).
The results are shown in FIG. 3. Experimental results show that the Aurora-A PROTACs degrade target proteins to cause the change of related signal paths. Among these, treatment with dAurA383 resulted in a change in the relevant pathway such as "mitotic spindle" in the cell, and treatment with dAurA450 resulted in a change in the relevant pathway such as "stem cell" in the cell.
Fourthly, the method comprises the following steps: optimization of concentration ratio of the composition of the invention
The inventors previously synthesized a variety of Aurora-a PROTACs based on different E3 ubiquitin ligases. Through a large number of experiments in the early stage, the inventor designs a protein hydrolysis targeting chimera composition. The method comprises the following specific steps:
1) acute myelogenous leukemia cells KG1a and Kasumi-1 were administered at 5X 10 per well 3 The density of individual cells was dispersed into 96-well plates;
2) cells were treated for 72 hours with the addition of 2 μ M total final concentration of dAura383, dAura425 or dAura450 in combinations of two at different molar ratios;
3) mu.L of CCK8 reagent (40203 ES80, Yeasen) was added to each well and incubated at 37 ℃ for 2 hours. Measuring absorbance at the test wavelength of 450nm, and calculating the relative growth condition of the cells;
4) the data were imported into GraphPad to analyze the composition of proteolytic targeting chimeric compositions with significant growth inhibitory effect and the ratio of dAurA383, dAurA425, or dAurA 450.
The results are shown in FIG. 4. Experimental results show that the composition of dAura383 and dAura450 has a good effect when the ratio is close to 1: 1.
Fifthly: the composition of the invention synergistically inhibits the survival of leukemia cells
The composition of dAura383 and dAura450 showed better growth inhibition, and the inventor further proves that the composition of dAura383 and dAura450 synergistically inhibits the survival of leukemia cells through CCK8 experiments and software analysis. The method comprises the following specific steps:
1) acute myelogenous leukemia cells KG1a and Kasumi-1 were added at 5X 10 per well 3 Density of individual cells dispersed into 96-well plates;
2) treating cells with dAura383, dAura450, or Aurora-A PROTAC cocktail at the indicated concentrations for 72 hours;
3) add 10. mu.L of CCK8 reagent (40203 ES80, Yeasen) per well and incubate for 2 hours at 37 ℃; measuring absorbance at the test wavelength of 450nm, and calculating the relative growth condition of the cells;
4) and importing the data into CompuSyn software to calculate the synergy index CI value under each processing condition.
The results are shown in FIGS. 5A & B. Compared with a single Aurora-A degrading agent, the Aurora-A degrading agent composition designed by the inventor has a better growth inhibition effect, and the growth of leukemia cells is inhibited by the cooperation of dAurA383 and dAurA 450.
Sixthly, the method comprises the following steps: the composition of the invention synergistically inhibits leukemia cell proliferation
The effect of the combination of dAurA383 and dAurA450 on leukemia cell proliferation was determined by CFSE assay. The method comprises the following specific steps:
1) acute myeloid leukemia cells KG1a and Kasumi-1 were harvested by centrifugation, and the cells were resuspended in serum-free medium containing CFSE (S8269, Selleck, 0.5. mu.M) and stained for 15 min;
2) the stained KG1a and Kasumi-1 cells were washed with PBS at 3X 10 per well 5 The density of individual cells was dispersed into 24-well plates;
3) treating cells with dAura383 and dAura450 or Aurora-A PROTAC cocktail (500 nm) for 72 hours;
4) the cells were collected, washed with PBS, and then resuspended in 250 μ L PBS;
5) detecting the CFSE intensity under the FITC channel by using flow cytometry;
6) the relative Mean Fluorescence Intensity (MFI) of CFSE was analyzed using CytExpert and FlowJo software.
The results are shown in FIG. 6. Compared with a single Aurora-A degradation agent, the Aurora-A PROTAC cocktail designed by the inventor has better proliferation inhibition effect.
Seventhly, the method comprises the following steps: the composition of the invention induces cell cycle arrest in leukemia
The effect of the combination of dAurA383 and dAurA450 on the cell cycle distribution of leukemia was determined by PI staining and flow experiments. The method comprises the following specific steps:
1) acute myelogenous leukemia cells KG1a and Kasumi-1 were injected at 3X 10/well 5 The density of individual cells was dispersed into 24-well plates;
2) treating cells with dAurA383 and dAurA450 or Aurora-A PROTAC cocktail (500 nm) for 48 hours;
3) the cells were collected, washed with PBS, resuspended in 70% ethanol and fixed at 4 ℃ overnight;
4) subsequently, the cells were washed, resuspended in PBS staining solution containing 50. mu.g/mL propidium iodide (81845, Sigma), 100. mu.g/mL RNase A (10109169001, Roche), 0.2% Triton X-100 (10789704001, Roche), and incubated at 37 ℃ for 30 minutes in the absence of light;
5) cells were collected and washed by centrifugation, resuspended in 200 μ L PBS and PI intensity under PI/PE channel was detected by flow cytometry;
6) cell cycle distribution was analyzed using FlowJo software.
The results are shown in FIG. 7. Compared with a single Aurora-A degradation agent, the Aurora-A PROTAC cocktail designed by the inventor has stronger cell cycle arrest effect.
Eighthly: the composition of the invention promotes the apoptosis of leukemia cells
The effect of the combination of dAurA383 and dAurA450 on leukemia cell apoptosis was determined by Annexin V staining and flow experiments. The method comprises the following specific steps:
1) acute myelogenous leukemia cells KG1a and Kasumi-1 were injected at 3X 10/well 5 Density of individual cells dispersed into 24-well plates;
2) treating cells with dAurA383 and dAurA450 or Aurora-A PROTAC cocktail (500 nm) for 48 hours;
3) the cells were harvested, washed with PBS, resuspended in 100. mu.L of 1 Annexin V binding buffer (AP006, Yishan Biotechnology) containing 5. mu.L of Annexin V-647 and 10. mu.L of Propidium Iodide (PI), and incubated for 15 min at room temperature in the dark;
4) 400 μ L of 1 Annexin V Binding buffer was added to each sample and Annexin V positive cells were detected by flow cytometry.
The results are shown in FIG. 8. Compared with a single Aurora-A degradation agent, the Aurora-A PROTAC cocktail designed by the inventor has stronger apoptosis promoting effect.
Nine: the composition of the invention can inhibit the methyl cellulose colony forming ability of leukemia cells
The effect of the combination of dAurA383 and dAurA450 on the colony forming ability of leukemia cells was determined by a methylcellulose colony forming assay. The method comprises the following specific steps:
1) pretreating acute myeloid leukemia cells KG1a and Kasumi-112 hours with dAurA383, dAurA450, Aurora-A PROTAC cocktail (500 nm) or DMSO;
2) resuspending the cells at 500 cells/well in 250. mu.L of cell culture medium containing 2 times of the drug concentration, mixing with 2% methylcellulose (M0512, Sigma) in the same volume, and inoculating to a 24-well cell culture dish for 14 days;
3) the cell colony formation under each treatment condition was photographed under a microscope; the scale bar is 50 μm;
4) primary colonies were collected, inoculated in the same experimental protocol, and subjected to secondary methylcellulose colony formation experiments.
The results are shown in FIG. 9. The Aurora-A PROTAC cocktail designed by the inventor has stronger colony formation inhibition effect compared with a single Aurora-A degradation agent.
Ten: the proteolytic targeting chimeric composition of the invention has a hook effect alleviating effect
The inventor further detects the degradation of the proteolytic targeting chimera composition to Aurora-A by Western Blot technique. The method comprises the following specific steps:
1) acute myelogenous leukemia cells KG1a, Kasumi-1, HL60 and U937 were administered at 3X 10/well 5 Density of individual cells dispersed into 24-well plates;
2) treating cells with a specified concentration of dAurA383 and dAurA450 or Aurora-a proctac cocktail for 12 hours;
3) the cells were collected, washed with PBS, lysed to obtain total protein and quantified, and the effect of a single degradant and Aurora-A PROTAC conjugate on Aurora-A protein was examined using Western Blot technique.
The results are shown in FIGS. 10A-C. The inventor designs Aurora-A PROTAC collektail which can effectively relieve the Hook Effect (Hook Effect) generated by a single Aurora-A degradation agent at higher concentration and expand the application concentration range of the single protein degradation agent.
Eleven: the compositions of the invention inhibit the growth of leukemia cells in animal models
The inventors further tested the effect of the proteolytic targeting chimeric compositions in animal models. The method comprises the following specific steps:
1) BALB/c nude mice (female, 6 weeks old) were used to evaluate the effect of Aurora-A PROTACs in vivo;
2) KG1A cells were cultured at 5X 10 6 Resuspend in PBS at a density of 100. mu.L, subcutaneously inoculate the right side of the mice;
3) after 6 days, dAurA383, dAurA450, proctac conjugate tail was injected intraperitoneally once daily with an equimolar dose of 30 μmol/kg/d (dAurA383=28.51 mg/kg/d, dAurA450=29.16 mg/kg/d), 100 μ l each of vehicle (DMSO 10%, PEG 30040%, Tween-805%, saline 45%);
4) measuring the long axis (A) and the short axis (B) of the tumor every 2 days, calculating the tumor volume V = A multiplied by B/2, and drawing a time-volume curve of the tumor growth;
5) after 2 weeks, mice were sacrificed, tumors dissected and weighed.
The results are shown in FIGS. 11A & B. The Aurora-A PROTAC cocktail designed by the inventor enhances the cancer inhibition effect of a single Aurora-A degradation agent.
Twelve: compositions of the invention degrade target proteins and induce apoptosis of primary cells in patient specimens
The inventors further tested the effect of the proteolytic targeting chimera composition in patient specimens. The method comprises the following specific steps:
1) carrying out density gradient centrifugation enrichment on primary cells by using Ficoll-Hypaque solution (LTS1077, TBD), washing and counting, and carrying out further treatment;
2) primary cells were plated at 3X 10 per well 5 The density of individual cells was dispersed into 24-well plates; cells were treated with dAura383 and dAura450 or Aurora-A PROTAC cocktail at the indicated concentrations for 12 hours; then collecting cells, and detecting the degradation condition of Aurora-A protein through a Western Blot experiment;
3) primary cells were plated at 3X 10 per well 5 The density of individual cells was dispersed into 24-well plates; cells were treated with dAurA383 and either dAurA450 or Aurora-A PROTAC cocktail (1 μm) for 48 hours. Apoptosis was then determined by Annexin V staining.
The results are shown in FIGS. 12A & B. The Aurora-A PROTAC cocktail designed by the inventor enhances the degradation effect and the cancer inhibition effect of a single Aurora-A degradation agent on Aurora-A.
Therefore, summarizing, the inventors have internationally first discovered, by Western Blot experiments and omics experimental analysis, that Aurora-a degradants dAurA383 and dAurA450 based on E3 ubiquitin ligase CRBN and cIAP regulate the proliferation and sternness of leukemia cells by preferentially degrading Aurora-a in M-phase and interphase, respectively. The proportion of dAurA383 and dAurA450 that synergistically inhibited cell survival was optimized and determined by CCK8 experiments and CompuSyn software analysis. Further, CFSE cell proliferation experiments, cell cycle analysis experiments, methyl cellulose colony formation experiments and Annexin V/PI flow experiments prove that the composition Aurora-A PROTAC cocktail of dAurA383 and dAurA450 can effectively inhibit cell proliferation and colony formation, induce cycle arrest and apoptosis, and remarkably improve the inhibition effect of the Aurora-A degrading agent based on single E3 ubiquitin ligase on cells. In addition, the inventor proves that the Aurora-A PROTAC cocktail can effectively relieve the Hook Effect (Hook Effect) generated by a single E3 ubiquitin ligase-based Aurora-A degrading agent at a higher concentration through a Western Blot experiment, and the application concentration range of the single protein degrading agent is expanded. Finally, the inventors demonstrated that Aurora-a procac cocktail efficiently degrades target proteins and induces apoptosis in animal models and patient specimens.
The foregoing is a further detailed description of the invention and is not to be taken in a limiting sense as the invention is defined by the appended claims. It will be apparent to those skilled in the art that various modifications, additions and substitutions can be made without departing from the spirit and scope of the invention.
Claims (10)
1. A proteolytic targeted chimera composition comprising at least 2 proteolytic targeted chimera molecules for the same target protein, characterized in that: the proteolytic targeting chimeric molecules act on the target proteins of different cell cycles.
2. The proteolytic targeting chimera composition of claim 1, wherein: the proteolytic targeting chimeric molecules are based on different E3 ubiquitin ligases; and/or
The target protein is an oncogenic protein; and/or
The cell cycle is interphase and M-phase.
3. The proteolytic targeting chimera composition of claim 2, wherein: the proteolytic targeting chimeric molecule is a proteolytic targeting chimeric molecule of a targeted ubiquitination degradation Aurora-A; and/or
The cancer is selected from breast cancer, leukemia, lung cancer, liver cancer, esophageal cancer, pancreatic cancer, colorectal cancer, gastric cancer, cervical cancer, brain cancer, and nasopharyngeal cancer; and/or
The oncogenic protein is Aurora-A, Aurora-B, PLK1, CDK1, CDK2, RAF, MEK, ERK, ALK, MET, PI3K, ABL, ER, BRD4, BTK, AR, CDK4 and CDK 6.
4. The proteolytic targeting chimera composition of claim 1, wherein: the proteolytic targeting chimeric molecules are CRBN-based Aurora-A PROTAC and cIAP-based Aurora-A PROTAC.
5. The proteolytic targeting chimera composition of claim 4, wherein: the proteolytic targeting chimeric molecules are Aurora-A PROTAC dAurA383 based on CRBN and Aurora-A PROTAC dAurA450 based on cIAP, and the molecular formulas of dAurA383 and dAurA450 are as follows:
6. the proteolytic targeted chimera composition of any one of claims 1-5, wherein: the composition can be made into injection, oral preparation, and mucosa administration preparation.
7. The proteolytic targeting chimera composition of claim 3, wherein: the cancer highly expresses Aurora-A, Aurora-B, PLK1, CDK1, CDK2, RAF, MEK, ERK, ALK, MET, PI3K, ABL, ER, BRD4, BTK, AR, CDK4, or CDK 6.
8. The use of a proteolytic targeted chimeric composition as claimed in any one of claims 1 to 6 in the preparation of an anti-tumor medicament.
9. Use according to claim 8, characterized in that: the tumor is selected from breast cancer, leukemia, lung cancer, hepatocarcinoma, esophageal cancer, pancreatic cancer, colorectal cancer, gastric cancer, cervical cancer, brain cancer, and nasopharyngeal carcinoma.
10. Use according to claim 9, characterized in that: the tumor highly expresses Aurora-A, Aurora-B, PLK1, CDK1, CDK2, RAF, MEK, ERK, ALK, MET, PI3K, ABL, ER, BRD4, BTK, AR, CDK4 or CDK 6.
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