Drug Re-evaluation For APJ Receptor And Cobisistat Alleviates Apelin-mediated Platelet Aggregation

Purpose: APJ receptor has emerged as a potential therapeutic target in various diseases,and the agonist and antagonist binding to APJ is hopeful to be a new therapeutic drug for these diseases.The new biological function of the drug may be found by drug re-evaluation of the FDA-approved drug library and screening high affinity drug for APJ receptor binding site.Method: 1.The three-dimensional structure Model Ⅰ of APJ receptor was obtained by matching template protein with high homology and known tertiary structure in PDB database,then proceeding homologous modeling,using different units in Discovery Studio platform;2.Model Ⅰwas evaluated by Verify Score、Ramanchandran Plot and Profile-3D module;Side chain optimization,Loop area reconstruction and biological membrane simulation are completed by Side-Chain Refinement、Loop refinement and Create and Edit Membrane module,respectively.Then,the ideal three-dimensional structure of APJ receptor was obtained;3.APJ receptor activity site was searched by Find Sites from Receptor Cavities,and FDA drug database rating by Lib Dock Score review was screened by Lib Dock;4.The binding channel of APJ was searched by Multi-channel Surface module in Sy Byl;then cobisistat binds to APJ receptor active sites by Surflex Dock;5.APJ recombinant plasmid was transfected into HEK293 T cells,which stable expressed high quantity APJ protein;Apelin13-associated c AMP generation was detected by ELISA assays to identifycobisistat specially targetingto APJ receptor;6.To study novel biological function of cobisistat;has cobisistat played similar or inverse roles in apelin subtypes-related functions(e.g.platelet aggregation,thrombosis).Results: 1.The three-dimensional structure Model Ⅰ of APJ receptor was successfully constructed by matching template proteins(4ZUD,3EA3)with high homology(identity: 33%,34%)to APJ sequence;2.Reliability of APJ three-dimensional structure Model Ⅰis evaluated: four Loop areas(CYS19-LYS25,SER57-ARG62,MET183-ALA196 and ILE228-LYS242)existunreasonabletwist angle of amino acids in this areas(Verify Score<0);there are partialamino acids appearing outside the reasonable area in Ramanchandran Plot;Thus,APJ three-dimensional structure Model Ⅰ should to be further optimized;3.The scoring of post-optimized model,which the number of amino acids(Verify Score<0)have almost disappeared,is obviously moving upwards,indicated that twist angle of amino acids in Loop areaswere more reasonable.Then,the ideal three-dimensional structure of APJ receptor was obtained after added biomembrane structure to this model;4.extracellular domain exists ligands-binding surface wrapped in VAL164,GLU19,GLU198 and so on;there are 9 conformations of cobisistat which have the higher Screening hit(Lib Dock Score:199.008-175.278)than other drugs by screening FDA drug library;5.there are also 9 conformations of cobisistat docking with APJ binding site,which the rating of affinity between-7.4 and-6.8 using Autodock-vina;6.Cobisistat can dock into the activity site of APJ receptor;And,there are strong hydrogen bonding interactions between cobisistat and TYR299,TYR264,SER298 in APJ receptor detected by Sy Byl-Surflex Dock;7.In this assay,cobisistatacted as an inverse agonist on APJ,increases c AMP accumulation induced by forskolin with an EC50 of 0.1017 and 0.09704μM in HEK293T and APJ-HEK293 T cell;Similarly,Apelin-13 inhibits cobisistat-associated c AMP generation in dose dependent manner in HEK293 T and APJ-HEK293 T cell;8.Cobisistat cannot stimulate platelet aggregation rate on its own,while competitivelysuppresses Apelin12-,17-,36-and ELABELA-induced platelet aggregation in vitrowith an IC50 of0.0861、0.00337、0.00931 and 0.0261μM.Cobisistat also observably alleviates ADP-,AA-induced platelet aggregation in vitro,while cannot inhibites thrombin-,collagen-induced platelet aggregation;9.Cobisistat also dose-dependently inhibits Apelin12-,17-,36-and ELABELA-induced thrombosis in vitro.Moreover,cobisistat also markedly alleviates ADP-induced thrombosis in vitro.Conclusion:Cobisistat is the most affinity drug of APJ receptor in FDA drug library;Cobisistat also dose-dependently inhibits Apelin12-,17-,36-and ELABELA-induced platelet aggregationand thrombosis in vitro;Cobisistat may well be a novel APJ-targeted anti-thrombosis drug used in clinical treatment in future.