阅读说明：本技术 人源已糖激酶2小分子抑制剂及其应用 (Human hexokinase 2 small molecule inhibitor and application thereof ) 是由 刘焕香 谈硕彦 袁苗苗 刘瑞娟 郭婧筠 彭立增 姚小军 于 2020-03-11 设计创作，主要内容包括：本发明提供了一种以人源已糖激酶2为靶点的小分子抑制剂及其应用,属于制药技术领域。本发明将已糖激酶2作为受体,选取其抑制剂结合口袋,利用Glide程序进行分子对接,从160多万个化合物中虚拟筛选得到靶向人源已糖激酶2的小分子抑制剂LY2019-001,并进行酶活抑制实验和MTT细胞增值抑制实验,验证了化合物LY2019-001对已糖激酶2的抑制作用以及对宫颈癌细胞HeLa、肝癌细胞HepG2及肺癌细胞A549的生长抑制作用。所发现的小分子及其药用盐可以为治疗已糖激酶2有关的疾病,例如肝癌、肺癌、子宫内膜癌、乳腺癌、卵巢癌、胰腺癌或前列腺癌的新药研发提供基础。本发明的先导化合物可进行进一步的结构优化,具有较好的应用前景。(The invention provides a small molecule inhibitor taking human hexokinase 2 as a target point and application thereof, belonging to the technical field of pharmacy. The invention takes hexokinase 2 as a receptor, selects an inhibitor binding pocket thereof, utilizes a Glide program to carry out molecular docking, virtually screens more than 160 ten thousand compounds to obtain a small molecular inhibitor LY2019-001 targeting human hexokinase 2, carries out an enzyme activity inhibition experiment and an MTT cell proliferation inhibition experiment, and verifies the inhibition effect of the compound LY2019-001 on hexokinase 2 and the growth inhibition effect on cervical cancer cells HeLa, liver cancer cells HepG2 and lung cancer cells A549. The found small molecules and medicinal salts thereof can provide a basis for the development of new drugs for treating diseases related to hexokinase 2, such as liver cancer, lung cancer, endometrial cancer, breast cancer, ovarian cancer, pancreatic cancer or prostate cancer. The lead compound can be further structurally optimized, and has a good application prospect.)

1. Application of compound LY2019-001 and its medicinal salt in preparing medicine for targeting human hexokinase 2 is disclosed.

2. Use of compound LY2019-001 according to claim 1 and pharmaceutically acceptable salts thereof for the manufacture of a medicament for the treatment of liver cancer.

3. Use of compound LY2019-001 according to claim 1 and pharmaceutically acceptable salts thereof for the manufacture of a medicament for the treatment of lung cancer.

4. Use of compound LY2019-001 according to claim 1 and pharmaceutically acceptable salts thereof for the manufacture of a medicament for the treatment of endometrial cancer.

5. Use of compound LY2019-001 according to claim 1 and pharmaceutically acceptable salts thereof for the manufacture of a medicament for the treatment of breast cancer.

6. Use of compound LY2019-001 according to claim 1 and pharmaceutically acceptable salts thereof for the manufacture of a medicament for the treatment of ovarian cancer.

7. Use of compound LY2019-001 according to claim 1 and pharmaceutically acceptable salts thereof for the manufacture of a medicament for the treatment of pancreatic cancer.

8. Use of compound LY2019-001 according to claim 1 and pharmaceutically acceptable salts thereof for the manufacture of a medicament for the treatment of prostate cancer.

Technical Field

The invention belongs to the technical field of biochemical pharmacy, in particular relates to discovery of a small molecular inhibitor for inhibiting the activity of human hexokinase 2 by taking hexokinase 2(HK2) as a target spot, and discusses the growth inhibition effect of the discovered small molecular inhibitor on cervical cancer cells HeLa, liver cancer cells HepG2 and lung cancer cells A549.

Background

As early as the early twentieth century, Warburg et al found that unlike most cells, tumor cells have a greater choice of directly converting pyruvate to lactate (i.e., the glycolytic pathway) as the primary means of energy production, even under conditions where oxygen supply is sufficient, and glycolysis appears in higher rates especially in those malignant cells that grow more rapidly, a phenomenon known as the "Warburg effect". Hexokinase (HK) is a rate-limiting enzyme that catalyzes the first step of the glycolytic pathway, catalyzing the transfer of phosphate from ATP to glucose and converting it to glucose-6-phosphate, which is an irreversible reaction. Mammalian tissues contain four hexokinase subtypes, hexokinase 1-4. Expression of hexokinase in normal tissues is low and its distribution also differs from tissue to tissue. HK1 is distributed mainly in brain tissue; HK2 is found primarily in adipose and skeletal muscle tissue; HK3 is only present in small amounts in tissues such as liver, kidney and intestine; HK4 (i.e., glucokinase GK) is present only in tissues such as liver and pancreas, and phosphorylates hexoses other than glucose. However, hexokinase expression was significantly increased in malignant cells, and HK2 was predominant among the increased hexokinases (John E Wilson et al J Exp Biol,2003,206(Pt 12), 2049-57). HK2 was found to be highly expressed in a variety of human tumor tissues, tumor cells, tumor models and rapidly proliferating cells (e.g., embryonic cells). The higher the activity of HK bound to the outer mitochondrial membrane of tumors, the higher the rate of tumor glycolysis (Bustamante E et al J Biol Chem,1981,256(16): 8699-8704.). If the activity of the enzyme is inhibited, glycolysis of tumor cells is inhibited, so that the tumor cells cannot obtain enough glucose-6-phosphate, and the aim of treating malignant tumors is fulfilled.

Although HK2 is a potential cancer therapeutic target, few effective HK2 inhibitors have been found. Metformin (Met), 2-deoxyglucose (2-DG) and 3-bromopyruvate (3-BrPA) are the most common inhibitors of HK2 today. However, the traditional Chinese medicine composition has the defects of low therapeutic activity, instability, obvious adverse reaction and the like (Salani B et al Sci Rep-UK.2013,3:2070.), and an improved novel HK2 inhibitor still needs to be found.

Virtual screening (virtual screening) is a common method in the development of new drugs, and the method screens out chemical molecules with possible therapeutic effects by selecting a small molecule database and performing a calculation method, researches the binding effect of the chemical molecules and drug targets, and finally selects a compound with highest interaction binding force as a lead compound and uses the compound as a next step for research. Compared with the physical drug screening method, the virtual screening method has obvious advantages of using a computer, finding new candidate drugs more quickly and economically, reducing the cost of drug screening and shortening the research and development period of new drugs.

Disclosure of Invention

The invention aims to provide a lead compound targeting human hexokinase 2 and application of a medicinal salt thereof in preparing a hexokinase 2 inhibitor. Wherein said hexokinase 2 mediated disease comprises the following cancers: lung cancer, liver cancer, breast cancer, ovarian cancer, endometrial cancer, pancreatic cancer, and prostate cancer.

On the basis of the research on the target hexokinase 2 inhibitor binding pocket, the invention obtains the small molecule inhibitor LY2019-001 which has a new structural entity and targets human hexokinase 2 through screening.

The invention finally obtains the active compound LY2019-001 which has pharmacological activity and targets the human hexokinase 2 by virtual screening based on the protein structure of the hexokinase 2, and multi-factor comprehensive analysis such as Ribes-based rule screening, scoring of scoring function, inhibition experiment of the human hexokinase 2 activity, MTT method detection compound inhibition effect on cell proliferation and the like.

Enzyme level and cell level experiments prove that the compound LY2019-001 has the effect of inhibiting the activity of human hexokinase 2 in vitro (IC50 is 17.5 mu M), and has the effect of inhibiting the growth of cervical cancer cells HeLa, liver cancer cells HepG2 and lung cancer cells A549 (IC50 is 62.1 mu M, 62.5 mu M and 54.6 mu M respectively). The compound LY2019-001 can be further structurally optimized to prepare a preparation or a medicine for treating diseases related to the hexokinase 2 protein (including lung cancer, liver cancer, breast cancer, ovarian cancer, endometrial cancer, pancreatic cancer and prostate cancer).

The compound targeting human hexokinase 2 protein has the following advantages:

1. has the functions of targeting hexokinase 2 protein and inhibiting the activity of hexokinase 2;

2. has effects of inhibiting growth of cervical cancer cell HeLa, liver cancer cell HepG2 and lung cancer cell A549

3. The compound LY2019-001 and the pharmaceutically acceptable salt derivative thereof can be used for preparing preparations or medicaments for diseases (including lung cancer, liver cancer, breast cancer, ovarian cancer, endometrial cancer, pancreatic cancer and prostate cancer) with the hexokinase 2 protein as a target;

for ease of understanding, the compounds targeting human hexokinase 2 of the present invention will be described in detail below with specific figures and examples. It is specifically noted that the specific examples and figures are for illustrative purposes only and it will be apparent to those skilled in the art that, in light of the description herein, various modifications and changes can be made in the invention which are within the scope of the invention.

Drawings

FIG. 1 is the chemical structural formula of compound LY2019-001 of the present invention.

FIG. 2 shows the inhibition of hexokinase 2 activity at the protein level by the small molecule compound LY 2019-001.

Detailed Description

Example 1

1. High-throughput virtual screening method of small molecule compound LY2019-001

(1) Molecular docking-based virtual screening

Downloading a crystal structure (PDB code: 5HEX) of hexokinase 2 from a protein structure database PDB, optimizing the structure, adding charges and hydrogen atoms, selecting an inhibitor combination pocket, and performing high-flux virtual screening based on molecular docking on more than 130 ten thousand compounds provided by two compound databases of Chemdiv and specs by using a Glide module of Schrodinger software. Finally, the Chemdiv database kept 49 compounds, the Specs database kept 13, and a total of 62 compounds (see the following structural formula) were used in the next enzyme level activity inhibition experiment.

2. Inhibitory effect of virtual screening obtained 62 small-molecule compounds on hexokinase 2 activity

Enzyme level inhibition experiments of 62 small-molecule compounds are carried out by using a hexokinase-II inhibitor screening kit (Abcam), and the small-molecule compound LY2019-001 is found to have good kinase activity inhibition effect on hexokinase 2 (as shown in figure 2), and the half effective inhibition concentration of the small-molecule compound LY2019-001 for inhibiting hexokinase 2 is 17.5 mu M.

3. MTT (methyl thiazolyl tetrazolium) experiment determination of cell growth inhibition effect of small molecule compound

MTT (methyl thiazolyl tetrazolium) experiments are utilized to measure the growth inhibition effect of the small molecular compound LY2019-001 on various cancer cells, and the half effective inhibition concentrations of the small molecular compound LY2019-001 on the growth inhibition of cervical cancer cells HeLa, liver cancer cells HepG2 and lung cancer cells A549 are respectively 62.1 mu M, 62.5 mu M and 54.6 mu M.