CN109207551B - Method for screening folic acid metabolism related drugs - Google Patents
Method for screening folic acid metabolism related drugs Download PDFInfo
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- CN109207551B CN109207551B CN201810745013.XA CN201810745013A CN109207551B CN 109207551 B CN109207551 B CN 109207551B CN 201810745013 A CN201810745013 A CN 201810745013A CN 109207551 B CN109207551 B CN 109207551B
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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Abstract
The invention discloses a method for screening folic acid metabolism related medicines. The method comprises the following steps: (1) Mixing a cell line with stable physiological state with a compound to be detected with different concentrations respectively, wherein the cell line has a serine hydroxymethyl transferase expression function; (2) Adding stable isotope labeled serine, and incubating until the reaction is complete; (3) Taking a cell culture solution supernatant, and detecting the glycine content marked by the stable isotope; (4) And judging the activity of the compound to be tested by utilizing the glycine content and the corresponding concentration of the compound to be tested. The detection method has high screening success rate, and the screened compound has stronger effect and smaller side effect.
Description
Technical Field
The invention belongs to the field of biological detection, and particularly relates to a method for screening folic acid metabolism related medicines.
Background
Dihydrofolate reductase (DHFR, ec 1.5.1.3) is an oxidoreductase that uses NADPH to reduce dihydrofolate in organisms to produce tetrahydrofolate. Tetrahydrofolate and its derivatives are intracellular one-carbon unit transmitters, which are essential coenzymes involved in DNA and amino acid synthesis. DHFR is an important target in drug development, and DHFR inhibitor research has long been an important direction in anticancer drug development: when the tumor grows rapidly, folic acid mediated one-carbon unit metabolism is needed to provide sufficient DNA and protein synthesis raw materials for the tumor; the DHFR inhibitor can block the function of DHFR by combining with the DHFR specifically, so that the dihydrofolate can not be converted into tetrahydrofolate, the folic acid metabolism is blocked, the synthesis of DNA and protein is interfered, and cancer cell death is finally led, so that the purpose of treating cancer is achieved. Once inhibited, DHFR will bring about a change in the synthesis rate of the corresponding biomarker.
The existing drug screening platform aiming at DHFR in the industry is an enzymatic reaction detection method, and cannot effectively evaluate the physiological reaction of the drug in cells. A representative DHFR enzymatic reaction assay was developed by Aiso et al at Tokyo university of agricultural technologies in 1999 (Kenji Aiso, tomoko Nozaki, minoru Shimoda, eiichi Kokue, assay of Dihydrofolate Reductase Activity by Monitoring Tetrahydrofolate Using High-Performance Liquid Chromatography with Electrochemical Detection, analytical Biochemistry, volume 272,Issue 2,1999,Pages 143-148, ISSN 0003-2697). Taking this method as an example: firstly, many compounds screened by the method have no good cell permeability and are difficult to enter cells to play a role; second, this method fails to detect side effects or toxicity of the compound; finally, certain inherently inactive compounds may become metabolically active after entry into the cell-this method does not detect these compounds. Therefore, many compounds screened by this method cannot be practically used in downstream animal experiments or clinical experiments.
Disclosure of Invention
The invention aims to overcome the defects that in the prior art, the screening of drugs aiming at DHFR by using an enzymatic reaction detection method cannot effectively evaluate physiological reactions caused by the drugs in cells, and the screened compounds cannot be applied to downstream in vivo experiments or clinical experiments, and the like, and provides a method for screening drugs related to folic acid metabolism. The method can effectively evaluate physiological response of the drug in cells, and has high screening success rate, stronger effect and smaller side effect of the screened compound.
Intracellular production of glycine and serine is dependent on the one-carbon unit pathway associated with folate metabolism: SHMT (serine hydroxymethyltransferase) catalyzes the conversion of serine and tetrahydrofolate to glycine and methylenetetrahydrofolate, which is the acceptor of one carbon unit in this reversible reaction. Once DHFR is inhibited, the physiological process of converting serine to glycine, lacking tetrahydrofolate in the cell, cannot be accomplished. The invention creatively combines the folic acid metabolism related approach with a biomarker detection method, and establishes a novel detection method for evaluating the bioactivity based on detecting the synthesis rate of the intracellular biomarker: the rate of formation of glycine, the biomarker detected, may reflect the effect of the drug on DHFR.
One of the technical schemes for solving the technical problems is as follows: a method of screening for a folate metabolism-related agent, the method comprising the steps of:
(1) Mixing or contacting a cell line with stable physiological state with a compound to be detected with different concentrations respectively, wherein the cell line has serine hydroxymethyl transferase expression function; preferably HCT-116, MV-4-11, HL-60 or HEK293;
(2) Adding stable isotope labeled serine to the cell line, and incubating until the reaction is complete;
(3) Taking a cell culture solution supernatant, and detecting the glycine content marked by the stable isotope;
(4) And judging the activity of the compound to be tested by utilizing the glycine content and the corresponding concentration of the compound to be tested.
Wherein, in the step (1), a cell line with serine hydroxymethyl transferase expression function is used, so that the cells are more sensitive to effective compounds, a stronger detection signal is obtained, and the experimental sensitivity is improved.
In the present invention, the cell line stable in physiological state in the step (1) is a cell which grows in an adherent manner after being cultured for 16 to 48 hours, preferably 18 to 24 hours; preferably, the culture medium for cell culture is McCoy's5a Medium Modified+10%FBS.
In the present invention, the concentration in step (1) is 2-fold diluted 8 concentrations starting from 1E-5M.
In the present invention, in order to better reflect the detection result, the step (1) further comprises a positive control, wherein the positive control is preferably an inhibitor of DHFR, and more preferably Pemetrexed.
In the present invention, the reaction in step (2) is sufficient to mean that most of the stable isotope-labeled serine and tetrahydrofolate are converted into glycine and methylene tetrahydrofolate in the cell; preferably, the incubation time is 3.5 to 4.5 hours, more preferably 4 hours.
In the present invention, the stable isotope of step (2) is one that has not been found to decay by itself as is conventional in the art, e.g 8 O、 2 H or 13 Conventional stable isotopes such as C, several labeling schemes are listed below:
the method comprises the steps of carrying out a first treatment on the surface of the Preferably 2 H。
In the present invention, the final concentration of the stable isotope-labeled serine in the step (2) is 15 to 25. Mu.M, preferably 20. Mu.M. The final concentration of the stable isotope labeled serine is set to enhance the detection signal and improve the experimental sensitivity, and excessive raw material consumption and excessive background of the experimental signal are avoided.
In the invention, the time interval between the step (2) and the step (1) is less than 30s.
In the present invention, the detection in step (3) is LC-MS detection.
In the present invention, the determining the activity of the compound in step (4) is calculating the IC50 value of the compound to be tested.
The second technical scheme for solving the technical problems is as follows: a method for assessing folate metabolism-related pharmaceutical activity comprising the above method for screening for folate metabolism-related drugs.
On the basis of conforming to the common knowledge in the field, the above preferred conditions can be arbitrarily combined to obtain the preferred examples of the invention.
The reagents and materials used in the present invention are commercially available.
The invention has the positive progress effects that: the detection method established by the invention evaluates the compound by researching the physiological reaction of the cell to the compound, can evaluate the permeability of the compound entering the cell and the toxicity or side effect of the compound to the cell, and can measure the conversion of the activity intensity of the compound entering the cell after metabolism; the detection method has high screening success rate, and the screened compound has stronger effect and smaller side effect.
Drawings
FIG. 1 is a comparison of the results of a prior art enzymatic reaction assay with the biomarker assay of the present invention.
FIG. 2 is a linear fit of the data obtained for the enzymatic method and the method of the present invention.
FIG. 3 is a graph comparing the effect of different concentrations of stable isotope labeled serine on the experiment: FIG. 3a is a 10. Mu.M versus 20. Mu.M; FIG. 3b is 20. Mu.M versus 30. Mu.M; the abscissa indicates the concentration of the compound to be measured, and the ordinate indicates the content of stable isotope-labeled glycine.
FIG. 4 is a comparison of results using HEK293 cells and SHMT1 knockout cells; the horizontal and vertical axes are indicated in fig. 3.
FIGS. 5 a-5 c are experimental results using MV-4-11, HL-60 and HEK293, respectively; the horizontal and vertical axes are indicated in fig. 3.
Detailed Description
The invention is further illustrated by means of the following examples, which are not intended to limit the scope of the invention. The experimental methods, in which specific conditions are not noted in the following examples, were selected according to conventional methods and conditions, or according to the commercial specifications.
HCT-116 cell line: purchased from ATCC (cat# CCL-247);
McCoy's5a Medium Modified cell culture medium: purchased from ATCC (cat No. 30-2007);
96-well cell culture plate: available from Corning (cat No. CLS 3595);
stable isotope [ ] 2 H) Marking serine: purchased from Cambridge Isotope Lab (cat No.:CDNLM-6813-PK)。
example 1 construction of the detection method
(1) HCT-116 cells were plated at 80,000 cells/well, 100. Mu.L/well in 96 well cell culture plates. At 37 ℃,5% CO 2 Incubate overnight in incubator.
(2) The test compounds were diluted 2-fold in gradient at appropriate concentrations using cell culture broth (McCoy's 5a Medium Modified+10%FBS) as diluent. Pemetrexed is a positive control, and the test compound and the positive compound are each diluted 2-fold for 8 concentrations starting from 1E-5M (final concentration). The specific concentration is as follows: 1E-5M, 5E-6M, 2.5E-6M, 1.25E-6M, 6.25E-7M, 3.13E-7M, 1.56E-7M, and 7.81E-8M.
(3) Diluted test compounds and Pemetrexed were added to the corresponding cell culture plate wells at 10 μl/well. Then 10. Mu.L/well 240. Mu.M stable isotope was added immediately 2 H-labeled serine to a final concentration of 20. Mu.M. At 37 ℃,5% CO 2 Incubate in incubator for 4 hours.
(4) Collecting cell culture supernatant, and detecting stable isotope by liquid chromatography-mass spectrometry (LC-MS) 2 H-tagged glycine content (molecular weight about 79.07).
(5) And (3) taking the glycine content marked by the stable isotope as a variable, taking the concentration of the compound to be tested as an independent variable, and plotting by using four-parameter fitting according to the conventional prior art. Compound IC50 values were obtained. The IC50 value will be used as a criterion for judging the activity of the compound.
Example 2 verification of the detection method
The activity of Pemetrexed [ available from Saikovia biosciences Inc. ] was tested according to the method in example 1 using MV-4-11 (available from American ATCC), HL-60 (available from American ATCC), or HEK293 (available from American ATCC), respectively. Specific results are shown in FIGS. 5a to 5c and Table 1 below.
TABLE 1
| Cells | MV-4-11 | HL-60 | HEK293 |
| IC50(M) | 1.99E-06 | 2.29E-06 | 3.21E-06 |
It can be seen that by carrying out experiments according to the method, the activity of Pemetrexed can be tested.
Effect examples
88 compounds were tested using the assay described above and the assay of the existing enzymatic reactions (see table below).
88 names of compounds (all compounds were purchased from Saikang, all inhibitors of folate metabolism related pathways)
The results are shown in FIG. 1: the correlation of the results obtained by the two detection methods is very low, and the correlation coefficient R2=0.05. We analyzed compounds that were greatly different in the two detection results, broadly divided into the following three categories:
(1) Strong activity in enzymatic reaction assays, but weak activity in biomarker assays: the compound cannot enter cells to play a role;
(2) Strong activity in biomarker assays, but weak activity in enzymatic reaction assays: the compounds are metabolically converted to strongly active compounds after entry into the cell.
(3) Compounds with cytotoxicity or strong side effects: cell death is caused in biomarker assays.
Comparative example 1
| IC50 for enzymology experiment (M) | HEK293 cell IC50 (M) | |
| Pemetrexed | 1.83E-08 | 2.03E-07 |
| Diaveridine | 1.41E-08 | 1.48E-06 |
| Methotrexate | 2.51E-09 | 2.42E-07 |
| Pyrimethamine | 2.00E-09 | 2.00E-07 |
| Dicoumarol | 1.01E-09 | 1.04E-07 |
| Pralatrexate | 4.07E-10 | 5.89E-08 |
| AEBSF HCl | 6.45E-10 | 8.31E-07 |
The results of the enzymatic method compared with the method of the present invention are shown in fig. 2, where the linear fit r2= 0.8690, indicating that the method is consistent with the enzymatic method results. Because the method is a cell experiment, the ability of the compound to enter cells and the effect of the compound in the intracellular environment can be reflected: as the compound AEBSF, the IC50 in the present method is significantly weaker, indicating that it cannot enter the cell or lacks stability in the cell. This was not found in enzymatic experiments. The method can reflect the activity of the compound under physiological conditions more truly compared with an enzymology experiment.
Comparative example 2
The serine concentrations were set to 10. Mu.M and 30. Mu.M, respectively, in the same manner as in example 1.
As shown in FIG. 3, a stable isotope labeled serine of 20. Mu.M is a better fit to a four parameter curve; neither 30 nor 10 μm fit a complete curve and experiments were not useful for evaluating compound activity.
Comparative example 3
Cells that do not express SHMT1 or that have too low an expression level are not suitable for this experiment. According to the method of example 1, HCT-116 cells were replaced with SHMT1 knockout HEK293 cells, and it was found that the concentration of isotopically labeled serine in the experimental culture medium was not changed by the action of the active compound, and the screening objective could not be achieved, as shown in FIG. 4.
Claims (15)
1. A method for screening for drugs associated with folate metabolism, said method comprising the steps of:
(1) Mixing or contacting a cell line with stable physiological state with compounds to be detected with different concentrations respectively, wherein the cell line has serine hydroxymethyl transferase expression function and dihydrofolate reductase expression function;
(2) Adding stable isotope labeled serine to the cell line, and incubating until the reaction is complete, wherein the final concentration of the stable isotope labeled serine is 20 mu M;
(3) Taking a cell culture solution supernatant, and detecting the glycine content marked by the stable isotope;
(4) And judging the activity of the compound to be tested by utilizing the glycine content and the corresponding concentration of the compound to be tested.
2. The method of claim 1, wherein the cell line in step (1) is HCT-116, MV-4-11, HL-60, or HEK293.
3. The method of claim 1, wherein the cell line stable in physiological state in step (1) is an adherently growing cell after 16-48 hours of culture.
4. The method of claim 3, wherein the cell line stable in physiological state in step (1) is an adherently growing cell after 18 to 24 hours of culture.
5. The method of claim 4, wherein the cultured broth is McCoy's a Medium Modified +10% FBS.
6. The method of claim 1, wherein the concentration of step (1) is 2-fold diluted 8 concentrations starting from 1E-5M;
and/or, step (1) further comprises a positive control.
7. The method of claim 6, wherein the positive control is an inhibitor of DHFR.
8. The method of claim 7, wherein the positive control is Pemetrexed.
9. The method of claim 1, wherein the incubation in step (2) is for a period of 3.5 to 4.5 hours.
10. The method of claim 9, wherein the incubation of step (2) is for a period of 4 hours.
11. The method of claim 1, wherein the stable isotope of step (2) is 2 H。
12. The method of claim 1, wherein the time interval between step (2) and step (1) is less than 30s.
13. The method of claim 1, wherein the detection of step (3) is an LC-MS detection.
14. The method of claim 1, wherein the determining the activity of the test compound in step (4) is calculating the IC50 value of the test compound.
15. A method of assessing folate metabolism related pharmaceutical activity comprising the method of any one of claims 1 to 14.
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| CN1291228A (en) * | 1998-01-23 | 2001-04-11 | 新生物生物公司 | Enzyme catalyzed therapeutic agents |
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| CN1291228A (en) * | 1998-01-23 | 2001-04-11 | 新生物生物公司 | Enzyme catalyzed therapeutic agents |
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| Title |
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| Marina Marani et al..A pyrazolopyran derivative preferentially inhibits the activity of human cytosolic serine hydroxymethyltransferase and induces cell death in lung cancer cells.Oncotarget.2015,第7卷(第4期),第4570-4583页. * |
| YASUKO HASHIMOTO et al..Simple Separation of Tritiated Water and [3H]Deoxyuridine from [5-3H]Deoxyuridine 5’-Monophosphate in the Thymidylate Synthase Assay.ANALYTICAL BIOCHEMISTRY.1987,第167卷第340-346页. * |
| 贾蕊等.二氢叶酸还原酶抑制剂的高效毛细管电泳法筛选模型的建立与应用.色谱.2007,第25卷(第2期),第189-192页. * |
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