Screening Of ERp57 Small Molecule Inhibitors And Its Antiplatelet Effect

Objective:In this paper,using disulfide isomerase ERp57 as a novel target for antithrombotic therapy to screen small molecule inhibitors.For the first time,from the traditional Chinese medicine Danshen and the commercial compound library,use virtual screening,solid phase enzyme method and traditional small molecular inhibitor screening method to obtain the small molecule inhibitor.Then the active compounds were tested for antithrombotic activity and discussed the mechanism of action.Methods:1 Screening of ERp57 small molecule inhibitor from Danshen and its antiplatelet effectThe Danshen extract?DSE?were determined in anti-platelet test and inhibitory activity test of ERp57 in vitro.At the same time,the effect of DSE on the release of adenosine triphosphate?ATP?in platelets was measured.Screening ERp57 small molecule inhibitors from Danshen by virtual screening use ERp57 as the target and traditional small molecule inhibitor screening method.The small molecules with potential activity were determined in anti-platelet test and inhibitory activity test of ERp57 in vitro.Rosmarinic acid was assayed for the influence of releasing ATP in platelets,the rat tail bleeding time and the anti-Fe Cl₃-induced carotid artery thrombosis in rats.2 Screening of ERp57 small molecule inhibitors in traditional Chinese medicine extracts by solid phase enzyme method and its antiplatelet effectThe small molecule inhibitors of ERp57 from DSE were screened by solid phase enzyme method.The compounds were identified by LC-MS and HPLC.The small molecules with potential activity were tested for inhibition of ERp57 activity and anti-platelet aggregation in vitro.3 Screening of ERp57 small molecule inhibitors in commercial compound library and its antiplatelet effectIn the commercial compound library,the virtual screening was used to obtain potential small molecule inhibitors.Then the active small molecules were tested for inhibition of ERp57 activity and anti-platelet assay in vitro.Results:1 Screening of ERp57 small molecule inhibitor from Danshen and its antiplatelet effectThe DSE has a certain inhibitory effect on the platelet aggregation induced by ADP,AA and collagen in vitro.And the concentration of DSE was directly proportional to the effect of platelet aggregation inhibition.Among them,the inhibitory effect on AA-induced platelet aggregation was stronger than that on ADP and collagen-induced platelet aggregation.Furthermore,using ADP to induce the release of ATP from dense particles,DSE inhibited it in the platelets.At the same time DSE inhibited the catalytic activity of ERp57.With the increase of concentration and reaction time,the inhibitory effect was better.Also the inhibitory effect was irreversible.The ERp57 small molecule inhibitors daucosterol and rosmarinic acid were obtained by virtual screening.Rosmarinic acid has an inhibitory effect on the catalytic activity of ERp57 and it was irreversible,but there was almost no inhibitory effect on the daucosterol.Rosmarinic acid inhibited the platelet aggregation induced by AA,ADP and collagen in vitro,and the most obviously inhibitory effect on AA induced.At the same time,rosmarinic acid inhibited the release of ATP from the dense particles in the platelets induced by ADP.Animal studies showed that rosmarinic acid had inhibitory effect on tail bleeding time and anti-FeCl₃-induced carotid artery thrombosis in rats,but the effect was not obvious and had not statistical significant.The traditional method of screening small molecule inhibitors was used to screen ERp57inhibitors in Danshen.Through screening,salvianolic acid A,salvianolic acid B and salvianolic acid C were obtained,and the inhibitory effects of the three compounds on the ERp57 reduction activity were irreversible.At the same time,these had a certain inhibitory effect on platelet aggregation induced by AA,ADP and collagen in vitro.2 Screening of ERp57 small molecule inhibitors in traditional Chinese medicine extracts by solid phase enzyme method and its antiplatelet effectScreening the ERp57 inhibitors in Danshen by solid phase enzyme method,compounds obtained by experimental group were slightly more than the blank control group.And compounds were identified by LC-MS and HPLC.According to the results,there have dihydrotanshinone I.At the same time,dihydrotanshinone I inhibited ERp57 activity and the inhibitory effect was irreversible.Dihydrotanshinone I inhibited the platelet aggregation induced by AA,ADP and collagen in vitro,and the inhibitory effect on AA-induced platelet aggregation was the most obvious.3 Screening of ERp57 small molecule inhibitors in commercial compound library and its antiplatelet effectVirtual screening was used to screen ERp57 small molecule inhibitors from commercial compound library,and 16 potential small molecule compounds were obtained.The experiments showed that compound 41974954 and demethoxycurcumin inhibited the catalytic activity of ERp57,and the inhibitions were irreversible.The results of anti-platelet aggregation test showed that demethoxycurcumin and compound 41974954inhibited platelet aggregation induced by AA,ADP and collagen,but the effects were not obvious.Conclusion:1 DSE significantly inhibited platelet aggregation possibly by inhibiting ERp57.2 Rosmarinic acid was identified by structure-based virtual screening as one major active compound.It inhibited the reduction activity of ERp57 and platelet aggregation.3 Using traditional small molecule inhibitor screening method,salvianolic acid A,salvianolic acid B and salvianolic acid C as major active compounds of Danshen,inhibitd ERp57 reduction activity and platelet aggregation in vitro.4 Screening small molecule inhibitor in DSE by solid phase enzyme method,LC-MS and HPLC.In the product have dihydrotanshinone I.It significantly inhibited platelet aggregation possibly by inhibiting ERp57.5 Compound 41974954 and demethoxycurcumin were identified by structure-based virtual screening from commercial compound library.They inhibitd ERp57 reduction activity and platelet aggregation in vitro.