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

Pulmonary fibrosis(PF)is a progressive disease characterized by fibroblast proliferation and extracellular matrix deposition.It can be divided into two types:idiopathic pulmonary fibrosis(IPF)and secondary pulmonary fibrosis.Between them,idiopathic pulmonary fibrosis is the most serious and dangerous type.After intervention the patients are very poor and short life.Recent pathological studies have shown that patients with new coronary pneumonia generally have varying degrees of pulmonary fibrosis.Studies have shown that transforming growth factor-β(Transforming growth factor-β,TGF-β)is a multifunctional cytokine that participates in the synthesis and degradation of extracellular matrix,and is closely related to the direction of fibrosis induction and development.TGF-β can bind to TGF-β type II receptors in vivo and activate TGF-β type I receptors.The activated TGF-β type I receptors activate downstream signaling pathways and promote pulmonary fibrosis.The TGF-β type I receptor is an ATPase with a hydrophobic pocket that binds to ATP and requires ATP hydrolysis to activate downstream signals.At present,there is no ATP-competitive TGF-β type I receptor inhibitor as a drug for the treatment of pulmonary fibrosis.This experiment uses a virtual screening method based on molecular docking to screen potential TGF-β type I receptor ATP-competitive inhibitors in existing drug databases,and uses molecular dynamics simulation methods to calculate and predict the inhibitory effect of candidate drugs.At the cellular level,the CCK-8 method was used to detect the effect of candidate drugs on TGF-β-induced cell proliferation.At the level of enzyme activity,a dual luciferase reporter gene detection method is used to detect the inhibitory effect of candidate drugs on the activity of TGF-β type I receptors.This experiment is divided into the following four parts.1)Preliminary virtual screening of potential inhibitors of TGF-β type I receptors.First,we downloaded the TGF-β type I receptor protein structures from the PDB database to remove solvent molecules and ligand molecules and used molecular dynamics simulation to optimize the protein structures.The optimized receptor structures were docked with the original ligands and compared with the structure before optimization.The protein structure with a PDBID of 3TZM was selected as the receptor structure for virtual screening.After virtual screening,60 small molecule compounds were initially selected.Through precise docking and result analysis,6 small molecule compounds as candidate drugs were finally selected.2)Exploring the molecular mechanism of the binding of candidate drugs to TGF-βtype I receptors.We used NAMD 2.12 and VMD to perform 50 ns molecular dynamics simulation and related analysis on each candidate drug-receptor complex system.At 50 ns,each system tends to balance,and the MM-GBSA calculation results showed that five candidate drugs have good inhibitory effects.By analyzing the binding positions of small molecules after dynamics simulation,we found that the binding of DB02656 to the receptor structure is not stable enough.Compared with the positive control SB431542,DB00966 and DB11651 have stronger inhibitory effects,DB00967 and DB04690 have similar inhibitory effects,and the binding positions of the four drugs to the receptor are similar to ATP,suggesting that the candidate drugs have potential competitive inhibition of binding of TGF-β type I receptors to ATP.3)Exploring the effect of candidate drugs on the proliferation activity of HEK293 cells induced by TGF-β at the cellular level.After drug stimulation and TGF-β induction,we observed under the microscope,and the CCK-8 experiment quantitatively detected the inhibitory effect of candidate drugs on cell proliferation activity.The results showed that four drugs have strong inhibitory effects.Among them,DB00966 and DB11651 have more prominent effects,which are consistent with our predicted results.4)Exploring the effect of candidate drugs on the activity of TGF-β type I receptors at the level of enzyme activity.After co-transfection of firefly luciferase reporter gene plasmid and renilla luciferase reporter gene plasmid on HEK293 cells,DB00966 and DB11651 were used for stimulation and TGF-β induction.We used cell lysis buffer to lyse cells,added two types of luciferase substrates to detect the relative light unit and used statistical processing to calculate the inhibition rate of the drugs.The inhibitory rate of DB00966 on TGF-β type I receptors at concentrations of 0.1 μmol/L and 1 μmol/L can reach 27.3% and 54.9%.DB11651 has 32.8% and 48.0% inhibition rates on TGF-β type I receptors at concentrations of 0.1 μmol/L and 1 μmol/L.In summary,this study screened out two potential ATP-competitive inhibitors of TGF-β I receptors,DB00966 and DB11651.They may have therapeutic effects on pulmonary fibrosis.This study will provide a reference for the development of new TGF-β type I receptor inhibitors as anti-pulmonary fibrosis drugs.