Virtual Screening Of Cyclophilin D Inhibitors Based On Molecular Simulation

Background and aims: Acute pancreatitis（AP）is a common clinical emergency in digestive department with pain and heavy burden,at present,it has no specific therapeutic drugs approved for marketing.Cyclophilin D（CypD）,a mitochondrial matrix peptidyl proline enzyme,has been proved to be a drug target for the treatment of AP.At present,CypD peptide inhibitors such as cyclosporin A,sanglifehrin A have been reported,but their clinical efficacy is poor.Therefore,the development of new CypD small molecule inhibitors is of great significance for the treatment of AP.Methods: The pharmacophore model was constructed by using the reported CypD inhibitors and the ZINC and Durgbank database were screened by it.Then,the small molecules in ZINC database and the marketed drugs in Durgbank database were docking with CypD,the candidate molecules are determined according to the molecular docking results.The binding mode and stability of molecular candidate molecular with CypD was studied by molecular dynamics simulation,and the key amino acids of candidate molecular with CypD were analyzed by binding free energy calculation.The inhibitory activity of candidate drugs against CypD was evaluated by chymotrypsin-coupled assay.The protective effect of candidate drugs on mitochondria was evaluated by mitochondrial swelling test and JC-1 staining test.Sodium taurocholate（NAT）was used to induce pancreatic acinar cell necrosis and evaluate the protective effect of candidate drugs on acinar cells.Finally,we constructed mouse model of AP induced by cerulein（CER）to evaluate the therapeutic effect of candidate durg in mouse.Results: The established 3D-QSAR pharmacophore features include: two hydrogen bond acceptors,one hydrogen bond donor and one hydrophobic aromatic ring.The constructed pharmacophore was used to screen 206,724 small molecules in the ZINC database,1,399 molecules were hit,them were screened again by molecular docking.Finally,ZINC161011368,ZINC664707610,and ZINC65010466 were selected as candidate molecules.The 1026 marketed drug molecules in the Durgbank database were docked with CypD,and finally Nelfinavir（NF）was selected as the candidate drug.Molecular dynamics simulation in 150 ns showed that ZINC161011368,ZINC664707610,ZINC65010466 and Nelfinavir can stably binding to CypD.Further binding free energy results indicated that Asn144,Gln105,Ala143 and Ala145 were the key amino acids for CypD inhibition.The results of in vitro chymotrypsin-coupled assay showed that Nelfinavir at a concentration of 10μM had a78.04% inhibitory effect on CypD,1 μM Nelfinavir can inhibit mitochondrial MPTP opening,inhibit mitochondrial swelling,maintain mitochondrial membrane potential,and protect mitochondrial function.In primary pancreatic acinar cells,1μM Nelfinavir reduce cell necrosis induced by sodium taurocholate,and the necrosis rate was reduced from 32.16% in the model to 21.50%.In an animal model of CER-induced AP,75 mg/kg Nelfinavir ameliorated cerulein-induced necrosis,edema and inflammation in mouse pancreatic tissue,indicating that Nelfinavir has the potential to treat AP.Conclusion: In this paper,CypD was used as the target protein,and the ZINC and Durgbank databases were virtual screened by molecular simulation technology.The screened candidate drug Nelfinavir has the activity of inhibiting CypD and the potential to treat AP.The screening method of this study can be further applied to other databases to discover more CypD inhibitors for the treatment of AP and provide new research ideas for the development of potential therapeutic drugs for AP.