Diketone compound with non-small cell lung cancer resisting activityTechnical Field
The invention relates to the field of pharmacy, in particular to a diketone compound with the activity of resisting non-small cell lung cancer.
Background
Lung cancer is largely divided into two major categories, small Cell Lung Cancer (SCLC) and non-small cell lung cancer (NSCLC), with non-small cell lung cancer accounting for up to 85%. The layered targeting treatment aiming at the cancerogenic factors has obviously improved the treatment effect of lung cancer patients, in particular to lung cancer patients carrying specific cancerogenic factors, and the survival rate of the lung cancer patients is greatly improved. Gene fusion-activated protein kinases are a major class of oncogenes closely related to hematopoietic malignancies and solid tumors, which are often produced by chromosomal translocation or other rearrangement events, and the encoded protein products of which are often ideal targets for the development of anticancer drugs.
Researchers rely on clinical sample resources of the Asian lung cancer genome screening platform (LC-SCRUM-Asia) to perform full transcriptome sequencing analysis on non-small cell lung cancer samples with unknown driver genes. Specific transcripts encoding CLIP1-LTK proteins were finally found in tumor samples of these patients by RT-PCR techniques, sanger sequencing, fluorescence In Situ Hybridization (FISH) and other experimental means. Further transformation experiments showed that the CLIP1-LTK fusion protein, depending on its own kinase activity, exhibited oncogenic transformation capacity in NIH3T3 fibroblasts and Ba/F3 pro B lymphocytes. The discovery proves that the CLIP1-LTK fusion protein is a brand-new oncogenic driving factor and provides a key new target for the development of targeted therapeutic drugs for non-small cell lung cancer.
CLIP1 is taken as a member of microtubule terminal tracer protein family, is combined with microtubules through CAP-Gly structural domain at the N end of the microtubule, and participates in various microtubule-related intracellular activities such as connection formation of centromeres and microtubules in the mitosis process, cell directional migration and the like. In addition, CLIP1 can also interact with other microtubule-binding proteins, such as EB1 proteins and CLASP family proteins, which are also located at the positive end of microtubules, to thereby locate more precisely at the positive end of microtubules.
LTK is a member of the receptor tyrosine kinase family, whose protein sequence includes an extracellular region, a transmembrane region, and an intracellular tyrosine kinase region. The intracellular region is composed of a membrane proximal region, a protein kinase domain and a carboxy terminus, wherein the protein kinase domain comprises a smaller N-terminal leaflet and a larger C-terminal leaflet with an ATP binding site located therebetween. LTK is highly homologous to ALK protein sequences, especially the kinase domain sequences can be 79%, but the extracellular domain structure of LTK is relatively small, comprising only two MAM domains. Currently, there are few studies on the biological function of wild-type LTKs, the only study suggesting a possible role in animal autoimmunity and neurodevelopment. Protein fusion of CLIP1 with LTK is critical for oncogenic transformation of cells. Importantly, in tumor samples positive for the CLIP1-LTK fusion gene, other known oncogene driving gene detection is negative, which indicates that the CLIP1-LTK fusion gene and other known driving genes are mutually exclusive, and therefore the CLIP1-LTK fusion gene is expected to become a potential target point for the treatment of a CLIP1-LTK fusion positive cancer patient.
At present, selective small molecule inhibitors for LTK have not been reported, and the protein structure of LTK has not been resolved. Those skilled in the art are continually striving to develop more structurally novel, better effective LTK inhibitors in an effort to expand the scope of clinical drug options.
Is a commercial compound of the compound library, IUPAC denominated 4-phenyl-7,8,9, 10-tetrahydroo-2H-isochromeno [3,4-d ] pyridazine-1,6-dione, which has not been studied to show that it has particular activity.
Disclosure of Invention
The invention aims to overcome at least one defect of the prior art and provide diketone compounds with the activity of resisting non-small cell lung cancer.
The technical scheme adopted by the invention is as follows:
in a first aspect, the invention provides an application of a diketone compound or an acceptable derivative thereof in preparing a non-small cell lung cancer resistant medicament, wherein the diketone compound has a structure shown in the following formula:
；
wherein, the R group is selected from H, C alkyl groups of 1 to 4.
In some examples, the R group is H.
In some examples, the derivative of the diketone compound is a pharmaceutically acceptable salt thereof.
In some examples, the non-small cell lung cancer disease includes adenocarcinoma, squamous cell carcinoma, and large cell carcinoma.
In some examples, the non-small cell lung cancer is a non-small cell lung cancer that is positive for a CLIP1-LTK fusion gene.
In some examples, the medicament further comprises pharmaceutically acceptable excipients including at least one of solvents, fillers, lubricants, disintegrants, buffers, co-solvents, antioxidants, bacteriostats, emulsifiers, binders, or suspending agents.
In some examples, the pharmaceutical dosage form is an intravenous injection, a suppository, a gel, a foam, an enteric, a decoction, a mixture, a syrup, a granule, a pill, a capsule, a gel, or a nano-preparation.
In a second aspect, the invention provides an application of a diketone compound or an acceptable derivative thereof in preparing an LTK inhibitor for experiments, wherein the diketone compound has a structure shown in the following formula:
；
wherein, R groups are selected from H, C alkyl groups with 1 to 4 carbon atoms;
the use does not include treatment of disease.
In some examples, the R group is H.
In some examples, the derivative of the diketone compound is a pharmaceutically acceptable salt thereof.
The beneficial effects of the invention are as follows:
When the diketone compounds with different concentrations provided by the invention are used for carrying out inhibition activity experiments on Ba/F3-CLIP1-LTK cells and LTK enzymes, the compounds show good inhibition activity, and the semi-inhibition concentration IC50 is 0.49 mu M/L and 4.23 mu M/L respectively.
Drawings
FIG. 1 shows inhibition curves of Ba/F3-CLIP1-LTK by diketones of different concentrations provided in the present invention.
FIG. 2 shows the inhibition curves of LTK by diketones at different concentrations provided in the present invention.
Detailed Description
The following disclosure provides many different embodiments, or examples, for implementing different aspects of the invention.
Anti-cell proliferation Activity assay
Experimental principle: the anti-cell proliferation effect of the compounds was determined using the MTT (thiazole blue) assay. MTT is reduced by succinate dehydrogenase in the cell mitochondria to water insoluble blue-violet crystalline formazan and deposited in living cells, whereas dead cells do not. Dimethyl sulfoxide can dissolve formazan in cells, and the light absorption value of the formazan can be measured at 570nm wavelength by an enzyme-linked immunosorbent assay, so that the number of living cells can be indirectly reflected.
The experimental method comprises the following steps: MTT colorimetric assay was used to evaluate cell proliferation activity in the constructed Ba/F3-CLIP1-LTK cell line.
During the experiment, a Ba/F3-CLIP1-LTK cell suspension was first prepared using RPMI-1640 medium without IL-3 and 90. Mu.L of the cell suspension was inoculated per well in a 96-well plate. Subsequently, the 96-well plate was placed in a cell incubator for 2 hours, after which 8 sets of concentration gradients (concentrations of 0.87nmol/l,4.35nmol/l,21.75nmol/l,32nmol/l,543.64nmol/l,2718.22nmol/l,13591.11nmol/l and 67955.56 nmol/l) of diketones or dimethyl sulfoxide were added to each well.
After 72 hours of incubation, 10 μl of MTT solution was added to each well and incubation was continued for 4 hours. Thereafter, 100. Mu.L of SDS-HCl-PBS triple buffer solution was added to dissolve the formazan crystals formed. Finally, absorbance values for each well at 570nm were monitored using a microplate reader, the data fitted to a concentration-response curve with variable slope, and IC50 values calculated therefrom.
Experimental results: the anti-proliferation experiment of Ba/F3-CLIP1-LTK cells by adopting diketone compounds with different concentrations shows that the compound has good inhibitory activity on Ba/F3-CLIP1-LTK cells, and the semi-inhibitory concentration IC50 is 0.49 mu M/L (figure 1).
Enzyme inhibition Activity assay of LTK
Experimental principle: measurement of LTK inhibitory Activity the kinase inhibitory activity of candidate compounds was determined using LANTHASCREENTM kinase assay based on TR-FRET technology.
The experimental method comprises the following steps: all experiments were performed on 384 well plates. Before the start of the experiment, 8 groups of solutions of diketones (0.87 nmol/l,4.35nmol/l,21.75nmol/l,32nmol/l,543.64nmol/l,2718.22nmol/l,13591.11nmol/l and 67955.56nmol/l, respectively) were prepared. Subsequently, 4 sets of recombinant human LTK proteins and 2 sets of substrate/ATP mixtures were prepared in buffer solutions containing 50mM HEPES (pH 7.5), 0.01% BRIJ-35, 10mM MgCl2,4 mM MnCl2, 1mM EGTA and 2mM DTT4, respectively.
The kinase reaction system finally contained 10. Mu.L of a mixture comprising 5. Mu.L of a1 Xsubstrate/ATP mixture (0.2. Mu.M substrate and 5. Mu.M ATP), 2.5. Mu.L of LTK protein (5 ng/ml) and 2.5. Mu.L of 1 Xtest compound containing the desired concentration. The assay plate was placed on a shaker and shaken for 30 seconds to ensure adequate mixing of the reactants. Next, the kinase reaction was allowed to proceed for 1 hour at room temperature (20-25 ℃).
Thereafter, 10. Mu.L of a pre-prepared stop solution (containing 20 mM EDTA and 4 nM Tb-labeled antibodies) was added to stop the kinase reaction and trigger antibody binding, and the assay plates were incubated at room temperature for 1 hour. Subsequently, using a fluorescence plate reader (BioTek SynergyTM) a delay time of 100 μs and an integration time of 200 μs were set and the emission signals of fluorescein and terbium were measured (excitation wavelength 340 nm; emission wavelengths 520 nm and 495 nm, respectively).
Finally, the inhibition activity was calculated from the TR-FRET emission ratio (i.e. the ratio of the intensity of fluorescein emission to the intensity of terbium emission) and plotted against the concentration of inhibitor. The data were fitted to a concentration-response curve with variable slope and IC50 values were calculated from this.
Experimental results: enzyme inhibition activity experiments of LTK are carried out by adopting diketone compounds with different concentrations, and the compound has good inhibition activity, and the half inhibition concentration IC50 is 4.23 mu M/L (figure 2).
The above description of the present invention is further illustrated in detail and should not be taken as limiting the practice of the present invention. It is within the scope of the present invention for those skilled in the art to make simple deductions or substitutions without departing from the concept of the present invention.