CN112156095A

CN112156095A - M2 type pyruvate kinase small molecule activator and application thereof

Info

Publication number: CN112156095A
Application number: CN202011166333.3A
Authority: CN
Inventors: 刘焕香; 葛慧珍; 刘瑞娟; 袁苗苗; 郭婧筠; 彭立增; 姚小军
Original assignee: Lanzhou University
Current assignee: Lanzhou University
Priority date: 2020-10-27
Filing date: 2020-10-27
Publication date: 2021-01-01

Abstract

The invention provides a small molecule activator taking human M2 type pyruvate kinase (PKM2) as a target spot and application thereof, belonging to the technical field of pharmacy. The invention takes PKM2 as a target spot, and utilizes the combination of virtual screening based on docking and in-vitro biological activity evaluation to screen the small molecule activator. In vitro activity test results show that two of the screened compounds have better PKM2 activation activity and better cancer cell inhibition activity, and the found small molecules can provide a basis for developing new drugs for treating PKM 2-related diseases, such as cervical cancer, breast cancer, ovarian cancer, prostate cancer, liver cancer, lung cancer, pancreatic cancer, colorectal cancer, blood cancer, melanoma and multiple myeloma. The lead compound can be further structurally optimized, and has a good application prospect.

Description

M2 type pyruvate kinase small molecule activator and application thereof

Technical Field

The invention belongs to the technical field of medicinal chemistry, and particularly relates to application of a lead compound which takes M2 type pyruvate kinase (PKM2) protein as a target point to activate the kinase activity of human PKM2 in preparation of drugs for diseases related to PKM 2.

Background

In cells, the conversion of glucose metabolism into lactic acid is called glycolysis, and the reaction process can be divided into two stages; the first stage is called glycolytic pathway (glycolytic pathway), a process by which glucose is decomposed into pyruvate; the second stage is the conversion of pyruvate to lactate. The last reaction of the glycolytic pathway is catalyzed by Pyruvate Kinase (PK), which transfers phosphate from phosphoenolpyruvate (PEP) to Adenosine Diphosphate (ADP), producing Pyruvate and ATP.

The tumor cells take up and metabolize nutrients in a different way than normal cells, which provides a possible strategy for the research on the inhibition of the growth of the tumor cells. Under aerobic conditions, normal cells are mainly metabolized through oxidative phosphorylation, namely, glucose in the cells mainly enters mitochondria through pyruvic acid generated by glycolysis pathway, and enters tricarboxylic acid cycle through generating acetyl coenzyme A through oxidative decarboxylation, so that the acetyl coenzyme A is completely oxidized into water and carbon dioxide, and a large amount of energy is released for normal operation of organisms; only a small amount of pyruvate remains cytosolic and is converted to lactate. Under hypoxic conditions, normal cells are metabolized primarily by glycolysis, i.e., intracellular glucose is converted to a large amount of lactate via the glycolytic pathway producing pyruvate, which is excreted in vitro, and a small amount of energy is released. However, tumor cells are different, and as tumors grow, the biosynthetic demand for cancer cell proliferation is increasing. Even under oxygen-rich conditions, tumor cells take glycolysis as the main metabolic mode, and the produced pyruvate is converted into lactate and excreted outside the cell (aerobic glycolysis). This phenomenon, also known as the Warburg effect, was discovered by the german chemist Otto Warburg et al and in the 20's of the last century. Later, it was discovered that expression and post-translational modification of various key enzymes of the glycolytic pathway (such as pyruvate kinase, hexokinase, etc.) can cause deregulation of the glycolytic pathway, thereby affecting normal cellular metabolic patterns and leading to tumorigenesis.

Pyruvate kinases coexist in 4 subtypes, L, R, M1 and M2, respectively. Both PKL and PKR are encoded by the Pkrl gene and are expressed in the liver and red blood cells, respectively. Both PKM1 and PKM2 are encoded by the Pkm gene, and PKM1 is common in tissues that require rapid supply of large amounts of energy, such as brain and muscle tissues; PKM2 appears in some differentiated tissues such as adipose tissue, lung, retina and islet cells, as well as in all proliferating cells such as normal proliferating cells, embryonic cells, and adult stem cells, particularly tumor cells. In recent years, a great deal of research results show that the content of pyruvate kinase M2 (PKM2) is closely related to the stage of tumors, the over-expression of low-activity PKM2 (dimer) is confirmed to obviously enhance the Warburg effect of tumor cells, and the targeting activation of PKM2 enables the tetramer form to exist stably, so that the Warburg effect of the tumors can be reversed, and the occurrence and the development of the tumors are effectively inhibited.

The crystal structure of the PKM2 homotetramer is shown in FIG. 1, and taking peptide chain A as an example, we can see four different binding sites: (1) substrate binding Site (Active Site) the Active center to which PKM2 binds to the substrate PEP. During the reaction, the substrate PEP can transfer high-energy phosphate groups to ADP, and simultaneously synthesize ATP for supplying energy. (2) Allosteric regulatory site (Allosteric pocket) the ligand that binds to this binding site is called a modulator. The crystal structure of the PKM2 complex reflects that phenylalanine Phe and serine Ser can both act with the binding site, so that the conformation change of PKM2 is caused, and the binding of a substrate PEP is influenced, thereby regulating the activity of PKM2 pyruvate kinase. (3) An endogenous FBP binding site (Effect site) which is the action site of an endogenous activator FBP, wherein the binding of the FBP to the site can activate PKM2 and enhance the interaction force between C-C' interface amino acid residues, thereby leading PKM2 to form a stable tetramer activation state. (4) The PKM2 activator binding site (Activators binding site) is a symmetric pocket formed on the tetramer A-A' interface of PKM2, and the binding of allosteric Activators (Activators) can stabilize the activation state of the tetramer of PKM 2.

Research shows that PKM2 in tumors is mainly in a low-activity dimer form; the PKM2 activator converts its dimeric form to a highly active tetrameric form by activating PKM2, allowing more intermediates of glucose metabolism to be diverted to energy-producing pathways rather than to anabolism. This results in a decrease in lactate production and an increase in cellular oxygen consumption, thereby reversing the Warburg effect and effectively inhibiting the development and progression of tumors. Therefore, in recent years, people have gradually focused on the development of activators of PKM 2. However, the types and number of PKM2 agonists that have been reported to date are still quite limited, and there is a need to design and discover activators with novel backbones that specifically target PKM 2.

With the application of computer technology in the field of drug research and development, computer virtual screening provides a rapid and efficient screening technology for discovery of drug lead compounds. The technology aims at the three-dimensional structure of important disease specific target biomacromolecules, and searches compounds well combined with targets from the existing micromolecule database, so that the quantity of experimental screening compounds can be greatly reduced, and the research and development period is shortened.

Disclosure of Invention

The invention aims to provide application of a lead compound targeting human PKM2 protein in preparation of PKM2 kinase activators. Wherein the PKM2 mediated disease is selected from at least one of the following diseases: cervical cancer, breast cancer, ovarian cancer, prostate cancer, liver cancer, lung cancer, pancreatic cancer, colorectal cancer, blood cancer, melanoma, and multiple myeloma.

On the basis of research on an activator binding pocket of a target PKM2 protein, a small molecule activator compound I and a compound II of a target PKM2 protein with new structural entities are obtained by screening, and the small molecule inhibitor of the target PKM2 protein is a compound aiming at the small molecule structure of a binding site of PKM2 and Activators (shown in figure 1).

According to the invention, through virtual screening based on docking, and through multi-factor comprehensive analysis such as Lipinski's five-rule screening, scoring function scoring, free energy calculation, activation test on human PKM2 protein kinase activity and the like, a compound I and a compound II with activation activity on PKM2 are finally obtained.

Experiments prove that the compound I and the compound II have obvious activity of activating human PKM2 kinase in vitro and have better cancer cell inhibitory activity. The compound can be further structurally optimized to prepare a preparation or a medicine for treating diseases related to PKM2 protein (cervical cancer, breast cancer, ovarian cancer, prostate cancer, liver cancer, lung cancer, pancreatic cancer, colorectal cancer, blood cancer, melanoma and multiple myeloma).

According to the screening result, the compounds are selected to carry out PKM2 enzyme activation experiments and cytotoxicity MTT experiments, and the results show that two screened compounds have better PKM2 activation activity and better cancer cell inhibition activity; the compound has the following characteristics:

a compound I is prepared by reacting a compound I,

english name:

ethyl((2-methyl-5-(4-(m-tolylamino)phthalazin-1-yl)phenyl)sulfonyl)glycinate

the structure of the compound is as follows:

a compound II which is a compound of formula (I),

english name:

(R)-2-(3,4-dimethylphenyl)-3-(3-(trifluoromethyl)phenyl)thiazolidin-4-one

structure of compound

Table 1 shows the physicochemical properties and PKM2 enzyme activation data for the 2 compounds obtained from the screening.

TABLE 1

Table 2 shows the half Inhibitory Concentrations (IC) of 2 compounds against the human liver cancer cell line HepG2, the human cervical cancer cell line Hela and the human non-small cell lung cancer cell line A549 cancer cells, as determined by MTT assay₅₀)

TABLE 2

For the convenience of understanding, the small molecule activator targeting human PKM2 protein of the present invention will be described in detail below with reference to specific drawings and examples.

Drawings

FIG. 1 is a plot of the PKM2 tetramer and its interaction site with a substrate.

FIG. 2 is a diagram of the docking pattern of 3SZ and PKM2 target proteins.

FIG. 3 is a schematic diagram of the docking of compound-I with the PKM2 target protein.

FIG. 4 is a schematic diagram of the docking of compound-II with the PKM2 target protein.

Detailed Description

1. Virtual screening method of small molecule compounds I and II

(1) Protein preparation

A three-dimensional structure of PKM2 (PDB code:3ME3) was searched and obtained in a protein database (https:// www.rcsb.org /), and since PKM2 is in a tetrameric form (shown in FIG. 1), the structure is symmetrical along the C-C 'interface, and the binding site of a small molecule activator is on the A-A' interface, a dimer of the A chain and the B chain was selected as a study object. Preparation was performed using the Protein preparation wizard module in maestro contained in the schrodinger9.4.5 software package, first hydrogenation of the Protein followed by energy optimization and minimization of the Protein under the OPLS — 2005 force field.

(2) Ligand preparation

Docking small molecules were established from the Chembridge and SPECS databases, respectively. Wherein the Chembridge database contains about 40 or more ten thousand small molecules; the SPECS database contains over 30 ten thousand compounds; and (3) optimizing the ligands of the databases by using a Ligprep module in maestro contained in a Schrodinger9.4.5 software package, and screening the compounds before docking by respectively using the five rules of Lipinski's to determine whether the compounds have reactive groups.

(3) Lattice point generation

Grid point files were generated using Grid Generation in schrodinger9.4.5 with ligand 3SZ as the center of the Grid point box.

(4) Molecular docking-based virtual screening

And docking the prepared small molecule ligand with the target protein. Firstly, a Virtual Screening Workflow (VSW) in a Schrodinger9.4.5 software package is used, a 3ME3 crystal structure compound structure is used as a target, Screening methods with three precision (HTVS, SP and XP) of Glide are respectively used step by step for carrying out layered Screening on a Chembridge database and an SPECS database, and output results respectively keep appropriate proportions.

PKM2 enzyme activation assay

The activity of PKM2 was measured by an enzyme system coupled to lactate dehydrogenase, during which pyruvate produced by PKM2 was reduced to lactate, while NADH (reduced coenzyme) was oxidized to NAD + (oxidized coenzyme). After the reaction had proceeded, the oxidation state of the cofactor was spectrophotometrically determined at 340nm using a Cytation5 cell imaging multimode detector. Briefly, 48. mu.L of substrate mixture (final concentration, 50mM Tris-HCl, pH 8.0,200mM KCl,15mM MgCl)₂0.1mM PEP,4.0mM ADP,0.2mM NADH and 1 unit LDH) were dispensed into 96-well plates and 1 μ L of compound was delivered, FBP was used as positive control and DMSO was used as solvent control. Then 1. mu.L of PKM2 enzyme (final concentration, 4. mu.g) was added. The plates were immediately placed in a cytostation 5 cell imaging multimode detector, the conversion of NADH to NAD + was monitored at 340nm with 30s exposures every 3-6 min, and AC was calculated in SPSS19.0₅₀The value is obtained.

MTT assay to determine the median Inhibitory Concentration (IC) of an effective compound on cancer cells₅₀)。

The MTT colorimetric method is convenient and efficient, and is the most common method for cytotoxicity experiments. MTT is known as 3- (4,5) -dimethylhiahiahiazo (-z-yl) -3, 5-di-phenylyttrazolimide, a yellow fuel. Succinate dehydrogenase in mitochondria of living cells can metabolize and reduce MTT, and blue or blue-violet water-insoluble Formazan (Formazan) is generated under the action of cytochrome C and has ultraviolet absorption at 490 nm. Since the number of living cells is generally proportional to the amount of formazan produced, the number of living cells can be estimated from the optical density OD value. Whereas the dead cells did not contain succinate dehydrogenase, there was no response to the addition of MTT. The method has the advantages that the required cell amount is small during detection by the MTT method, the experimental steps are relatively simple, and the detection period is short, so that the MTT method is selected for cytotoxicity experiments, and the specific steps are as follows:

digesting and collecting cancer cells in logarithmic phase, using DMEM complete culture medium to resuspend the cells, and adjusting the cell concentration to be 5-10 multiplied by 10⁴And/ml. In a 96-well plate, 100. mu.L of cell suspension was added to each well at a cell concentration of 5000-10000/well, and cultured in an incubator containing 5% carbon dioxide at 37 ℃ for 6 hours to allow the cells to adhere to the wall. And sequentially adding a prepared candidate compound gradient concentration solution designed in advance into the experimental well group, adding DMSO with the same volume into a control group, setting a blank control group, and continuously culturing the cells for 48 hours. mu.L of MTT solution (5mg/ml) was added to each well and incubation was continued for 4 hours. After 4 hours, the supernatant was discarded, 150. mu.L of DMSO was added to each well, and the mixture was shaken on a shaker at a low speed for 10 minutes to dissolve the crystals sufficiently. The absorbance value at 490nm was measured for each well using a microplate reader.

We determined the toxicity of the two compounds on three cell lines of human hepatoma cell line HepG2, human cervical carcinoma cell HeLa and human non-small cell carcinoma cell A549 according to the above MTT experimental procedure. Final determination the final concentration tested of the compound in the experiment was: the concentration gradient of the compound I is 5 mu M, 10 mu M, 20 mu M, 40 mu M and 80 mu M; compound II was added at a concentration of 20. mu.M, 40. mu.M, 60. mu.M, 80. mu.M, 120. mu.M. Deducting the inhibitory effect of DMSO (dimethyl sulfoxide) as solvent on cancer cell proliferation, comparing with blank control group, calculating the inhibition rate of candidate compound on cancer cell proliferation at different concentrations according to formula (1) and obtaining respective IC₅₀The value is obtained.

Claims

1. The application of the compound I and the compound II and the medicinal salt thereof in preparing the medicine targeting human PKM2 protein.

2. The use of two compounds of claim 1 and their pharmaceutically acceptable salts for the manufacture of a medicament for the treatment of at least one of the diseases cervical, breast, ovarian and prostate cancer mediated by PKM2 kinase.

3. The use of two compounds of claim 1 and their pharmaceutically acceptable salts for the manufacture of a medicament for the PKM2 kinase mediated disease of at least one of liver cancer, lung cancer, pancreatic cancer, and colorectal cancer.

4. The use of two compounds of claim 1 and their pharmaceutically acceptable salts for the manufacture of a medicament for the treatment of at least one of the diseases leukemia, melanoma and multiple myeloma mediated by PKM2 kinase.

Compound I

Compound II