Application of desloratadine in preparation of medicine taking TGF-beta type I receptor as target
阅读说明：本技术 地氯雷他定在制备以TGF-βI型受体为靶点的药物中的应用 (Application of desloratadine in preparation of medicine taking TGF-beta type I receptor as target ) 是由 芦秀丽 王浩臻 卢琛 高兵 于 2021-08-04 设计创作，主要内容包括：本发明提供了地氯雷他定在制备以TGF-βI型受体为靶点的药物中的应用。本发明首次发现地氯雷他定是转化生长因子β(TGF-β)I型受体的ATP竞争性抑制剂,通过抑制TGF-βI型受体的ATP酶活性进而抑制下游信号转导通路,达到治疗肺纤维化或者癌症的目的,具有重要应用前景。(The invention provides application of desloratadine in preparing a medicament taking a TGF-beta type I receptor as a target spot. The invention discovers for the first time that the desloratadine is an ATP competitive inhibitor of transforming growth factor beta (TGF-beta) type I receptor, and the downstream signal transduction pathway is inhibited by inhibiting the ATPase activity of the TGF-beta type I receptor, so that the purpose of treating pulmonary fibrosis or cancer is achieved, and the desloratadine has important application prospect.)
1. The desloratadine is used alone or in combination with other medicines in the preparation of medicines taking TGF-beta type I receptors as targets.
2. The use of claim 1, wherein desloratadine is used alone or in combination with other drugs in the preparation of anti-pulmonary fibrosis drugs targeting TGF- β type I receptors.
3. The use of claim 2, wherein desloratadine is used alone or in combination with other drugs for the preparation of an anti-pulmonary fibrosis drug targeting TGF- β type I receptors for the treatment of combined chronic idiopathic urticaria and perennial allergic rhinitis.
4. The use of claim 1, wherein desloratadine is used alone or in combination with other drugs in the preparation of an anti-cancer medicament targeting TGF- β type I receptors.
5. The use of claim 4, wherein desloratadine is used alone or in combination with other drugs for the preparation of an anticancer drug targeting TGF- β type I receptors for the treatment of combined chronic idiopathic urticaria and perennial allergic rhinitis.
Technical Field
The invention relates to application of Desloratadine (Desloratadine) in preparation of anti-pulmonary fibrosis drugs or anti-cancer drugs.
Background
Transforming growth factor-beta belongs to one of the TGF-beta superfamily, and plays a key role in maintaining and regenerating mature tissues from early embryonic development as well as in many developmental and degenerative diseases. TGF-beta is one of the most widely studied pro-fibrosis factors at present, is a key factor in pulmonary fibrosis, is also a key factor of a plurality of cancer molecular mechanisms, and is a common target of anti-pulmonary fibrosis or anti-cancer drugs. TGF-. beta.type I receptors have an ATP binding domain and appear to be more regulatable, with kinases being active after activation of the type I receptor. Pirfenidone (Pirfenidone) obtained SFDA new drug certificate in china in 2011 and 9 months, and obtained Food and Drug Administration (FDA) approval for marketing in 2014. In a bleomycin-induced idiopathic fibrosis mouse model, pirfenidone can reduce TGF-beta content in alveolar lavage fluid compared with a control group, and the expression level of TGF-beta is reduced by one third. Clinically, pirfenidone also shows a more significant effect of delaying the progression of pulmonary fibrosis. However, because the number of clinical cases participating in observation is limited, the anti-pulmonary fibrosis effect needs to be further evaluated, and the anti-pulmonary fibrosis effect has many side effects such as light allergy and the like, and the price is high, so that the clinical application of the anti-pulmonary fibrosis effect is greatly limited, and the research on new anti-pulmonary fibrosis drugs is always a hot spot for research and development of new drugs at present. At present, the TGF-beta receptor inhibitor drugs for inhibiting the enzymatic activity of the TGF-beta receptor so as to realize pulmonary fibrosis resistance do not appear clinically.
The marketing of a new drug takes a lot of time and research and development costs on average and is subject to strict safety tests by departments such as the national food and drug administration and other regulatory agencies. The development of many new drugs is eventually abandoned because of their safety issues. The new use of old drugs, as the name suggests, is the idea that some original drugs can be used to treat other diseases after having found a completely new medical value. Some "old drugs" are occasionally found to produce unusual therapeutic effects on other diseases in addition to the corresponding symptomatic diseases, and have very low side effects. Therefore, the innovative method for rapidly and efficiently finding a new place for the existing drugs is increasingly paid attention and adopted by many international pharmaceutical enterprises.
Virtual Screening (VS) is a computational method by which a database of compounds can be searched to identify novel small molecules with desired biological activities. Molecular docking is a core technique of virtual screening, aimed at predicting the structure of a complex formed between two or more constituent molecules. The basic steps of virtual screening include: establishing a receptor model; establishing a small molecule compound library; virtual screening of a server; post-treatment of the hit compound. The aim of virtual screening is to find out compounds with potential inhibitory activity from the existing drug database, thereby saving a great deal of time.
Desloratadine (English name: Desloratadine) has a structural formula shown in (I).
The molecular formula of the desloratadine compound is C19H19N2Cl, white or off-white crystalline powder, molecular weight 310.82, CAS number 100643-71-8. The compound is mainly used for relieving systemic and local symptoms of chronic idiopathic urticaria and perennial allergic rhinitis at present, and no report on desloratadine for treating pulmonary fibrosis or cancer is found so far.
Disclosure of Invention
Based on the above, the invention carries out computer virtual screening of ATP competitive inhibitor drugs of TGF-beta type I receptors and drug experiments at cell and enzyme activity level by a new method for old drugs, and can provide experimental basis for research and development of new drugs taking TGF-beta receptors as targets so as to meet the effect of pulmonary fibrosis resistance and treat or/and prevent related diseases caused by excessive TGF-beta.
The technical scheme adopted by the invention is as follows: the desloratadine is used alone or in combination with other medicines in the preparation of medicines taking TGF-beta type I receptors as targets.
Furthermore, the desloratadine is singly used or combined with other medicines to prepare the anti-pulmonary fibrosis medicine taking the TGF-beta type I receptor as a target.
Furthermore, the desloratadine is singly used or combined with other medicines to prepare the anti-pulmonary fibrosis medicine which treats combined chronic idiopathic urticaria and allergic rhinitis and takes a TGF-beta I type receptor as a target.
Furthermore, the desloratadine is singly used or combined with other medicines to prepare the anti-cancer medicine taking TGF-beta type I receptor as a target.
Furthermore, the desloratadine is singly used or combined with other medicines to prepare the anti-cancer medicine which treats combined chronic idiopathic urticaria and allergic rhinitis and takes a TGF-beta type I receptor as a target.
The invention has the beneficial effects that:
1. the invention provides a new application of desloratadine, namely the application of desloratadine in preparing a medicament taking a TGF-beta I type receptor as a target, belongs to the new application of old medicaments, and has important application value and significance in the aspect of pulmonary fibrosis resistance or cancer resistance.
2. Through early drug screening research, the desloratadine is found to be an enzyme inhibitor of TGF-beta I type receptor, so that the desloratadine can be supposed to play a role as a novel anti-pulmonary fibrosis drug or an anti-cancer drug. In the invention, HEK293 cell experiments are utilized to prove that desloratadine can inhibit cell proliferation induced by TGF-beta at a cell level. At the enzyme activity level, the activity of TGF-beta type I receptor mediated downstream cell signal transduction pathway can be inhibited. Desloratadine may therefore be effective inhibitors of TGF-. beta.type I receptors. When the application is in particular, the desloratadine can be directly used as a therapeutic drug for treating pulmonary fibrosis or treating certain cancers.
3. The invention shows that the inhibition capacity of desloratadine is equivalent to that of the inhibitor SB431542 of TGF-beta type I receptor.
Drawings
FIG. 1A is the binding of ATP to TGF-. beta.type I receptors.
FIG. 1B is desloratadine binding to TGF-. beta.type I receptors.
FIG. 2A is a RMSD picture of kinetic modeling of SB431542 and desloratadine with TGF-. beta.type I receptors, respectively.
FIG. 2B is a graph of the binding patterns of ATP, SB431542 and desloratadine to TGF-. beta.type I receptors, respectively.
FIG. 3A shows the effect of SB431542 and desloratadine on the proliferative activity and morphology of HEK293 cells, respectively.
Fig. 3B shows that stimulation by SB431542(a) and desloratadine (B), respectively, results in a decrease in the proliferative activity of HEK293 cells.
Wherein, # #: TGF- β induced group was statistically different (p <0.01) compared to normal group; *: compared with the TGF-beta induced group, the drug group had statistical differences (p <0.05), n ═ 3mean ± SD; *: p < 0.05; **: p < 0.01; ***: p is less than 0.001.
FIG. 4A is a plasmid map of pGMSMAS-Lu.
FIG. 4B is a plasmid map of pGMR-TK.
FIG. 4C shows that stimulation by SB431542(a) and desloratadine (b), respectively, results in a decrease in TGF-. beta.type I receptor activity.
Wherein, # #: TGF- β induced group was statistically different (p <0.01) compared to normal group; *: the drug groups were statistically different (p <0.05) compared to the TGF- β induced group. n is 3mean ± SD; *: p < 0.05; **: p < 0.01; ***: p is less than 0.001.
FIG. 4D is the inhibition of TGF-. beta.type I receptors by SB431542 and desloratadine.
Detailed Description
The structural formula of SB431542 is shown as (II). The molecular formula of the compound is C22H16N4O3Pale yellow powder, molecular weight 384.39, CAS number 301836-41-9. It is a potent and selective inhibitor of TGF-beta type I receptor, IC50The value was 94 nM. The following experiment was performed using SB431542 as a positive control.
Example 1 virtual screening for ATP competitive inhibitors of TGF-. beta.type I receptors
TGF- β type I receptor structures (PDBID: 3TZM) were downloaded in PDB databases and optimized using Gromacs software, followed by molecular docking of ATP small molecule structures with optimized TGF- β type I receptor structures using AutoDock vina software to determine ATP binding pockets as active regions (see FIG. 1A). Virtual screening was performed using the active area and the screening results were recorded. The candidate drugs are filtered and screened according to three steps, and in the first step, the 60 small molecular structures which are ranked first are screened according to the sequence of the predicted binding energy of the AutoDock vina software. And in the second step, the AutoDock 4 and Autogrid 4 software are used for accurately docking 60 small molecules respectively and performing cluster analysis on the docking results. The ATP competitive inhibitor of TGF-beta type I receptor is screened by comprehensively considering the predicted binding energy, the predicted Ki value, the clustering analysis result, the drug related use information and the like (Table 1). And thirdly, accurately docking the screened drug small molecules with TGF-beta I type receptors for 5000 times by using AutoDock vina, and determining the initial conformation of each compound (shown in figure 1B).
TABLE 1 ATP, SB431542 and desloratadine binding energy and Ki values
From the data in FIG. 1A, FIG. 1B and Table 1, it was preliminarily determined that desloratadine has potential for inhibiting TGF- β type I receptor activity.
Example 2 mechanism of inhibition of TGF-. beta.type I receptor Activity by Desloratadine
The method comprises the following steps: molecular dynamics simulations were performed using NAMD and VMD software and RMSD analysis was performed, with the results shown in figure 2A. The results in FIG. 2A show that each composite system is stable after a 50ns kinetic simulation.
The MM-GBSA method using Ambertools 18 was used to calculate the free energy of receptor binding to the drug candidate in order to predict the binding potential of desloratadine to TGF- β type I receptors, and the results are shown in table 2.
TABLE 2 binding free energy of ATP, SB431542 and desloratadine to TGF-. beta.type I receptors
The results in Table 2 indicate that desloratadine may have a similar competitive inhibition of TGF-. beta.type I receptor binding to ATP as compared to SB 431542.
The binding pattern was co-analyzed using ligplot and PyMOL software, and the results are shown in fig. 2B. FIG. 2B shows that the substrate ATP interacts with the receptor via ILE13, VAL21, LYS15, GLY16, ARG17, ASP153, LYS139, LEU142, SER89, ASP92, GLY88, TYR84, and hydrogen bonds with SER89, ASP92, and LYS 139. From the interaction of SB431542 and desloratadine with TGF- β type I receptor binding, the range of residues and binding sites for hydrophobic and hydrogen bonding are essentially the same, suggesting that desloratadine binds to ATP and TGF- β type I receptor in a highly similar manner consistent with the center of binding, suggesting that it may have a strong competitive inhibition with ATP.
The results of the combined analysis of FIG. 2A, FIG. 2B and Table 2 show that desloratadine has strong binding potential with the ATP binding pocket of TGF-beta type I receptor.
Example 3 inhibition of TGF-. beta.induced proliferation of HEK293 cells by desloratadine
1. HEK293 cells with good growth status were inoculated into a 96-well plate and cultured, and after cells were stimulated for 4 hours with SB431542 and desloratadine, respectively, and induced for 24 hours with TGF- β, the cells were observed under a microscope, and the results are shown in fig. 3A. FIG. 3A shows that the number of cells in the TGF- β/DMSO group was significantly increased compared to the DMSO group in the solvent group, while the number of cells in the group to which different SB431542 or desloratadine was added was significantly decreased compared to the TGF- β/DMSO group, indicating that different concentrations of SB431542 or desloratadine both inhibited TGF- β induced cell proliferation.
2. Quantitative analysis of TGF-. beta.induced HEK293 cell activity by SB431542 and desloratadine using the CCK-8 assay is shown in FIG. 3B. FIG. 3B shows that the activity of cells added with TGF-. beta.was significantly enhanced compared to the control group, and that the positive control drug SB431542 showed significant inhibitory effects at concentrations of 1. mu. mol/L and 10. mu. mol/L. Similarly, after adding desloratadine, the cell activity was inhibited very significantly in the three dose groups of 0.1. mu. mol/L, 1. mu. mol/L and 10. mu. mol/L, and their inhibitory effect was stronger with increasing concentration, showing a significant dose-dependent pattern. These results suggest that they may influence proliferation of HEK293 cells induced by TGF- β by inhibiting the TGF- β signaling pathway.
Example 4 Effect of desloratadine on the level of TGF-beta type I receptor enzyme Activity
After co-transfection of pGMSMAS-Lu plasmid (FIG. 4A) and pGMR-TK plasmid (FIG. 4B) into HEK293 cells, cells were induced using two concentration gradients of SB431542 and desloratadine, 0.1. mu. mol/L and 1. mu. mol/L, and addition of TGF-. beta.to a final concentration of 10. mu.g/L. The dual-luciferase reporter gene kit is used for analyzing the inhibition strength of the drug on the activity of the TGF-beta type I receptor by detecting the relative expression condition of firefly luciferase. As shown in fig. 4C, the fluorescence of the firefly luciferase expression group of the TGF- β/DMSO group was significantly increased compared to the blank control group, indicating that the TGF- β/SMAD signaling pathway was significantly increased in activity, while the fluorescence signal of the firefly luciferase expression group was significantly attenuated after the addition of SB431542 and desloratadine. The inhibition rate of the TGF-beta type I receptor by the medicament is calculated according to the following formula:
inhibition ratio (%) ═ (RLU)Solvent set-RLUAdministration set)/RLUSolvent set×100％
The calculated inhibition rate is shown in FIG. 4D, and the inhibition rate of desloratadine on the fluorescence intensity of the firefly luciferase expression group at two concentrations of 0.1. mu. mol/L and 1. mu. mol/L is 38.9% and 51.3%, respectively. Overall, desloratadine has a similar TGF- β type I receptor inhibitory potency compared to SB 431542. The experimental results fully prove that the desloratadine can directly inhibit the TGF-beta/SMAD signal transduction pathway by inhibiting the TGF-beta type I receptor at the level of HEK293 cells.