The Preliminary Study On Screening Of Anti-pulmonary Fibrosis Nature Products Targeting TGF-β Type Ⅰ Receptor

In 2019,Corona Virus Disease 2019（COVID-19）swept the world.Lung infection by COVID-19 virus may lead to pulmonary fibrosis（PF）.Pulmonary fibrosis is a very serious lung disease.The main cause of the disease is the excessive or abnormal repair of the damaged alveolar tissue by the repair function of the human body.The clinical manifestations are dry cough and shortness of breath.At present,pulmonary fibrosis diseases are mainly divided into the following three categories:secondary pulmonary fibrosis,idiopathic or primary pulmonary fibrosis（IPF）and interstitial pneumonia（ILD）.One of the most dangerous is IPF.In recent years,the morbidity and mortality of pulmonary fibrosis have been increasing year by year.However,there is currently no specific drug that can cure or reverse pulmonary fibrosis in clinical practice.More and more studies have found that transforming growth factor-β（TGF-β）plays an irreplaceable role in pulmonary fibrosis by acting on TGF-β typeⅠ receptors.TGF-β typeⅠ receptor is regulated by ATP,that is,when it binds to ATP,it can show kinase activity,phosphorylate downstream SMAD protein,mediate downstream TGF-β/SMAD signaling pathway,and then play a role in the process of pulmonary fibrosis.However,there are currently no TGF-β typeⅠ receptor inhibitors for the treatment of pulmonary fibrosis.Based on this background,this study used virtual screening to screen natural products active ingredients with the potential to competitively inhibit the binding of ATP to TGF-β typeⅠ receptors in the Chinese Natural Product Database,select some of the top candidate natural products small molecules,evaluate their binding to TGF-β typeⅠ receptors in the simulated natural state through molecular dynamics simulation research methods,and screen out candidate components with good performance.Then,the inhibitory effect of candidate natural products active small molecules on the proliferation of human embryonic kidney cells（HEK293）induced by TGF-β was explored at the cellular level,and its inhibitory ability on the activity of TGF-β typeⅠ receptor was verified again at the enzyme activity level.Finally,the inhibitory effect of candidate small molecules on p-Smad protein in human lung cancer alveolar basal epithelial cells（A549）and the therapeutic effect on mice with pulmonary fibrosis were tested.First,we took TGF-β typeⅠ receptor as the target,and through virtual screening in the Chinese Natural Product Database,we found that when it binds to TGF-β typeⅠ receptor,the binding energy is lower than that of ATP（the absolute value of binding energy is larger than that of ATP）.And the top 543 natural products small molecules are ranked according to their binding energy.Then,according to the Lipinski drug-like principle,475 natural ingredients with the possibility of becoming medicines were screened,and then they were precisely docked.After comprehensive evaluation,5 candidate natural products small molecules were obtained.Then,the binding of these six small molecules to TGF-β typeⅠ receptors in the simulated natural state was evaluated by means of molecular dynamics simulation.The results of the root mean square deviation（RMSD）analysis showed that the kinetic simulation systems of the 5 candidate natural products components all tended to equilibrium at the end of the 50 ns simulation process.The MM-GBSA algorithm was used to calculate the ligand-receptor binding free energy.The results showed that the binding ability of CNPD 3 to TGF-β typeⅠ receptor was stronger,even better than that of the positive control group SB431542;The binding ability of CNPD 4 and CNPD 5 to TGF-β typeⅠ receptor is generally similar to SB431542;while the binding ability of CNPD 1 and CNPD 2 is weaker,and the absolute value of its binding free energy is even weaker than that of TGFβR1-ATP.Finally,the binding situation was judged by observing the binding positions of these 5 active components and TGF-βtypeⅠ receptors.The results showed that most of the small molecules were bound in the ATP-binding region of TGF-β typeⅠ receptors,and only CNPD 2 occurred deviation when binding in the system.The above results showed that the binding of CNPD 1 and CNPD 2 were not stable enough,while the other 3 candidate natural products active ingredients all have strong affinities with TGF-β typeⅠ receptors,which were in line with expectations.Next,we selected three natural products active small molecules CNPD3,4,5and the positive control group SB431542,and tested their inhibitory effects on TGF-β-induced HEK293 proliferation at different concentrations by CCK-8 kit.Comparisons were made to preliminarily judge the effect of candidate small molecules in inhibiting the TGF-β signaling pathway.We found that compared with SB431542,CNPD 3 and CNPD 5 had better inhibitory effect on cell proliferation,and the inhibitory ability was positively correlated with the added concentration.The inhibitory effect of CNPD 4 was poor.We further investigated the inhibitory effect of the three candidate-natural products active ingredients on TGF-β-induced TGF-β typeⅠ receptor activity in HEK293 at the enzymatic activity level using a dual-luciferase reporter gene assay.The results showed that the four candidate natural products could effectively inhibit the activity of TGF-β typeⅠ receptors,among which,compared with the positive control group SB431542,CNPD 3 and CNPD 5 had stronger inhibitory ability,showing a significant dose-dependent manner.The effect of CNPD4 is not as good as the former two.Finally,we conducted a preliminary study on the inhibition of TGF-β/SMAD signaling pathway and the anti-pulmonary fibrosis effect of the natural product CNPD5 at the cellular and animal levels.First,different concentrations of CNPD 5 were added to A549 induced by TGF-β,and the expression of p-Smad protein in the system was detected by Western Blot.The results showed that with the increase of CNPD 5 concentration,the expression of p-Smad protein The amount was significantly reduced,which proved that CNPD5 could significantly inhibit the TGF-β/SMAD signaling pathway at the cellular level.On this basis,we used the method of intratracheal instillation of bleomycin（BLM）to construct a pulmonary fibrosis model to study the anti-pulmonary fibrosis effect of CNPD5.We used the existing anti-pulmonary fibrosis drug Pirfenidone（PFD）as a positive control drug,and administered PFD and CNPD 5 by gavage.After that,the mice were sacrificed and the lungs were stained with HE and Masson to observe the degree of pulmonary fibrosis among different groups.The results showed that the degree of pulmonary fibrosis in the mice treated with CNPD 5 was significantly reduced,which was better than that of the positive control PFD-treated group.In summary,this study aimed to explore the development of new anti-pulmonary fibrosis drugs with TGF-β typeⅠ receptor inhibitors.First,the Chinese Natural Product Database was screened,and the cellular level,enzyme activity level and animal level were analyzed.The candidate small molecules were verified to inhibit the TGF-β typeⅠ receptor and thus inhibit the TGF-β/SMAD signaling pathway,and concluded that the natural product active small molecule CNPD 5 has the potential to become a new anti-pulmonary fibrosis drug.Our research provides an important reference for the research and development of new anti-pulmonary fibrosis drugs targeting TGF-β typeⅠ receptors,and lays a solid foundation for the development of new anti-pulmonary fibrosis drugs with my country’s independent intellectual property rights.