阅读说明：本技术 达克利司在制备抗肺纤维化药物中的应用 (Application of dactylosin in preparation of anti-pulmonary fibrosis drugs ) 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：本发明提供了达克利司在制备抗肺纤维化药物中的应用。提供了达克利司在制药领域中的新用途。本发明首次发现达克利司是转化生长因子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,达到治疗肺纤维化的目的,具有重要应用前景。(The invention provides application of dactylosin in preparation of anti-pulmonary fibrosis drugs. Provides a new application of the dactylosin in the pharmaceutical field. The invention discovers that the dacrolimus is an ATP competitive inhibitor of a transforming growth factor beta (TGF-beta) type I receptor for the first time, and the invention can inhibit downstream signals by inhibiting the ATPase activity of the TGF-beta type I receptor, thereby achieving the purpose of treating pulmonary fibrosis and having important application prospect.)

1. The dacrolimus is used singly or combined with other medicines in the preparation of medicines taking TGF-beta type I receptor as a target.

2. The use according to claim 1, wherein the daclizine is used alone or in combination with other drugs in the manufacture of a medicament for the treatment of pulmonary fibrosis targeted at the TGF- β type I receptor.

3. The use according to claim 2, wherein the daclizine is used alone or in combination with other drugs in the manufacture of a medicament for pulmonary fibrosis targeting TGF- β type I receptors for the treatment of cancer complicated.

Technical Field

The invention relates to application of daculisib (Dactlisib) in preparing anti-pulmonary fibrosis 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-fibrotic factors at present, and is a key factor in pulmonary fibrosis. TGF-. beta.type I receptors have ATP binding domains 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 compounds which potentially inhibit the activity of a certain protein target from the existing drug database, thereby saving a great deal of time.

Dactlesin (English name, Dactlisib), the structural formula is shown as (I).

The molecular formula of the dacrolimus compound is C₃₀H₂₃N₅O, powder, molecular weight 469.55, CAS number 915019-65-7. It is currently known to be a dual ATP competitive PI3K and mTOR inhibitor, primarily useful as an antitumor agent.

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 dacrolimus is used singly or combined with other medicines in the preparation of medicines taking TGF-beta type I receptor as a target.

Furthermore, the daclizine is singly used or combined with other medicines to prepare the anti-pulmonary fibrosis medicine taking TGF-beta I type receptors as targets.

Further, the daclizine is used singly or combined with other medicines to prepare the lung fibrosis medicine which treats the combined cancer and takes TGF-beta I type receptor as a target.

The invention has the beneficial effects that:

1. the invention provides a new application of daclizine, namely the application of daclizine in preparing a medicament taking a TGF-beta I type receptor as a target belongs to the new application of old medicaments, and the medicament also has important application value and significance in the aspect of resisting pulmonary fibrosis.

2. According to the invention, through early drug screening research, the daclizine is found to be an enzyme inhibitor of TGF-beta I type receptor, so that the daclizine can be supposed to play a role as a novel anti-pulmonary fibrosis drug. In the invention, HEK293 cell experiments are utilized to prove that the daclizine can inhibit the 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. Thus, dacrolimus may act as a potent inhibitor of TGF- β type I receptors. In specific application, the daclizine can be directly used as a therapeutic drug for treating pulmonary fibrosis.

3. The invention shows that compared with the inhibitor SB431542 of TGF-beta type I receptor, the inhibition ability of the daclizine is equivalent to the inhibition ability of the TGF-beta type I receptor.

Drawings

FIG. 1A is the binding of ATP to TGF-. beta.type I receptors.

FIG. 1B is the binding of dacrolimus to TGF-beta type I receptors.

FIG. 2A is a RMSD graph of kinetic modeling of SB431542 and Dacrolix, respectively, with TGF-. beta.type I receptors. .

FIG. 2B is a graph of the binding patterns of ATP, SB431542 and dacrolimus, respectively, to TGF- β type I receptors.

FIG. 3A shows the effect of SB431542 and Dacleisin on the proliferative activity and morphology of HEK293 cells, respectively.

FIG. 3B shows that stimulation by SB431542 and Dacleisin, respectively, results in a reduction in the proliferative activity of HEK293 cells.

Wherein, # #: TGF- β induced group had statistical difference (p <0.01) compared to normal group: the drug groups were statistically different (p <0.05) compared to the TGF- β induced group. n-3 mean ± SD,: p <0.05, p <0.01, p: 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 is that stimulation by SB431542(a) and dacrolimus (b) results in a decrease in TGF-. beta.type I receptor activity.

Wherein, # #: TGF- β induced group had statistical difference (p <0.01) compared to normal group: the drug groups were statistically different (p <0.05) compared to the TGF- β induced group. n-3 mean ± SD,: p <0.05, p <0.01, p: p is less than 0.001.

FIG. 4D is the inhibition of TGF-. beta.type I receptors by SB431542 and dactylosin, respectively.

Detailed Description

SB431542 structural formulaAs shown in (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. Filtering the screened candidate drugs according to three steps, and screening the top 60 small molecule structures according to the sequence of the predicted binding energy of the AutoDock vina software in the first step. 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 Dakris binding energy and Ki values

From the data in FIG. 1A, FIG. 1B and Table 1, it was preliminarily determined that daclizine has the potential to inhibit TGF- β type I receptor activity.

Example 2 mechanism of inhibition of TGF-. beta.type I receptor Activity by Daclelix

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 calculates the free energy of receptor binding to the drug candidate in order to predict the binding potential of dacrolimus to TGF- β type I receptors, with the results shown in table 2.

TABLE 2 binding free energies of ATP, SB431542 and dacrolimus to TGF-beta type I receptors

The results in Table 2 indicate that daclizine may have a better competitive inhibition of TGF- β type I receptor binding to ATP than 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 daclizine with TGF- β type I receptor binding, the range of residues and binding positions for hydrophobic and hydrogen bonding are essentially the same, suggesting that daclizine binds to ATP and the binding center of TGF- β type I receptor in a highly similar manner, suggesting that it may have a strong competitive inhibition with ATP.

The results of fig. 2A, fig. 2B and table 2 were combined to show that dactylosin has a strong binding potential to the ATP-binding pocket of TGF- β type I receptors.

Example 3 inhibition of TGF- β induced proliferation of HEK293 cells by Dacrolix

1. HEK293 cells in good growth state were inoculated into a 96-well plate and cultured, and after adding daclizine and SB 431542-stimulated cells for 4 hours, respectively, the cells were induced with TGF- β for 24 hours and 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 Dacrolix or SB431542 group added at different doses was significantly decreased compared to the TGF- β/DMSO group, indicating that different concentrations of Dacrolix or SB431542 can inhibit TGF- β induced cell proliferation.

2. Quantitative analysis of TGF- β induced HEK293 cell activity by dacrolimus and SB431542 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 addition of dacrolimus, cell activity was very significantly inhibited in all 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, in a significantly dose-dependent manner. 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 Dacleisin 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 SB43152 and Darlins of 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 was significantly increased in the TGF- β/DMSO group compared to the blank control group, indicating that HEK293 cells were significantly enhanced in the activity of TGF- β/SMAD signaling pathway upon stimulation by TGF- β. Whereas the relative fluorescent signal intensity of the firefly luciferase expression panel decreased significantly after the addition of SB431542 and Daclex. 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}-RLU_{Administration set})/RLU_{Solvent set}×100％

The results of the calculation of the inhibition rate are shown in FIG. 4D, and the inhibition rates of Dakris at two concentrations of 0.1. mu. mol/L and 1. mu. mol/L for the fluorescence intensity expressed by firefly luciferase were 32.8% and 48.0%, respectively. Overall, daclizine has a similar TGF- β type I receptor inhibitory potency as compared to SB 431542. The experimental results fully demonstrate that daclizine can inhibit the TGF-beta/SMAD signaling pathway at the HEK293 cell level directly by inhibiting TGF-beta type I receptors.