Discovery Of Acid-sensing Ion Channels (ASICs) Selective Antagonists And Initial Structure Relationship Research

Objective: Acid-sensing ion channels(ASICs)are the cation channels that can be activated by receiving extracellular protons,which are the members of epithelial sodium channel/degenerin(ENa C/DEG)family.ASICs protein assembled as homotrimers or heterotrimers.Till now,six isoforms of ASICs have been identified,which are ASIC1 a,ASIC1b,ASIC2 a,ASIC2b,ASIC3 and ASIC4.ASICs can sense the extracellular p H change and play important roles in several physiological and physiopathological processes,such as: pain,ischemic stroke,depression and so on.During the study of the function of ASICs,antagonists are necessary,and the study of antagonist could help the drug development to treat related disease as well.During this decade,several antagonists have been reported including small molecules and macromolecules.These antagonists promoted the study of the function of ASICs.The reported antagonists still have some shortcomings,including the poor selectivity and similar scaffold for the small molecules and difficult to obtain for the macromolecules.To get over these shortcomings,on one hand,we searched for the new small molecular antagonists by virtual screening and studied its activity,selectivity and structureactivity relationship,on the other hand,we tried to cut the peptide antagonist into short peptides to make it easy for synthesis.Method: We searched the new small molecular antagonists among the natural products library by virtual screen.We picked several compounds that got high ranks for further study.Subsequently,we expressed the ASIC protein on Chinese Hamster Ovary(CHO)cells and detected inhibitory activity of the compounds using patch-clamp techniques.During the test,(-)-epigallocatechin gallate(EGCG)exhibited inhibition of ASIC3 current.Then we studied the selectivity and initial SAR of EGCG.As for macromolecular antagonists,we chose Pc Tx1,a selective antagonist of ASIC1 a,as a lead compound.We designed to cut the peptide into three short peptides each one contains a disulfide bond.Then we tested the activities of those peptides on CHO cells.Result: During the study of small molecular antagonists,EGCG strongly inhibited the ASIC3 currents with an IC50 value of 13.2 ± 6.9 μM.During the SAR study of EGCG and its analogs,we found the following results:(1)The chirality of pyrogallol group is important for maintaining the inhibitory activity.(2)The 3-hydroxyl group of pyrogallol would notably furnish the inhibitory activity.(3)The absence of gallate would result in the loss of inhibitory activity.For the antagonists based on the short peptides,the result of activity tests showed: JAP-1 can inhibit the current of ASIC1 a to 64.0 ± 6.0% at a concentration of 20 μM.Though there is a large reduce on activity compared to Pc Tx1,its IC50 is still among 10-100 μM.Compared to Pc Tx1 witch was isolated from venom as a minor component(less than 1%),our synthesized short peptides are easier to obtain with a modest activity.The results gave us the confidence to do the further study.Conclusion: We found EGCG was a high selectivity,potent ASIC3 small molecular antagonist via visual screening study and electrophysiology test.The learned initial SAR results would guide our further medicinal chemistry modification of EGCG.And we got a short peptide JAP-1 with IC50 between 10-100 μM according our design by shortening the Pc Tx1.Compared to the neutral peptide antagonist,the short peptide can be synthesized easily.This study showed us a way to find peptide or peptide-like antagonists.