Jiang Tao Group and collaborators of Ocean University of China further elucidated the structure-activity relationship between α - conotoxin vc1.1 and human α 9 α 10 nicotinic acetylcholine receptor by using the strategy of amino acid side chain conservative modification

Nicotinic acetylcholine receptor (nAChR) is the first transmembrane pentamer composed of five subunits It is a ligand gated ion channel protein that mediates the rapid signal transmission between synapses and widely distributed in central and peripheral nervous systems and muscles It involves a series of nervous system diseases and disorders, including Parkinson's disease, Alzheimer's disease, schizophrenia, neuropathic pain, memory loss and stress regulation, so it is an important drug target α 9 α 10 nAChR is one of the newly discovered acetylcholine receptor subtypes in recent years It is mainly distributed in the cochlea of mammals and is related to the formation of the auditory system After a number of studies have proved that α 9 α 10 nAChR subtype is the target of chronic pain treatment, which has significant effect on the pain caused by trauma and chemotherapy Vc1.1 is a kind of α - conotoxin extracted from the venom of conus Victoria It can inhibit α 9 α 10 nAChR specifically and effectively, has strong analgesic activity, and has the potential to be a new drug for the treatment of neuropathic pain Through molecular dynamics simulation, site directed mutagenesis and electrophysiological experiments, the structure-activity relationship between α - conotoxin vc1.1 and α 9 α 10 nAChR was further elucidated and verified at the molecular level The development of vc1.1 analogues as molecular probes or analgesics has laid the foundation Up to now, the crystal of α - conotoxin vc1.1 combined with α 9 α 10 nAChR has not been resolved Model construction and site directed mutagenesis have become effective means to study its structure-activity relationship In view of the model analysis established before, the researchers found that S4 in vc1.1 was located near d166 and d169 of α 9 subunit, and formed hydrogen bond with d166 Therefore, the researchers introduced positively charged amino acids at four positions, which enhanced its activity by electrostatic interaction with the receptor, and also explored the influence of the length of its side chain on its activity In addition, in the molecular dynamics simulation, the researchers found that Y10 hydroxyl in vc1.1 formed two hydrogen bonds with n107 and d119 in α 9 In order to study the contribution of hydroxyl to the binding activity of vc1.1, the researchers introduced phenylalanine at position 10 At the same time, the hydroxyl group of Y10 in vc1.1 is replaced by f or Cl atom to explore the possibility of forming hydrogen bond or halogen bond with nearby residue due to the removal of hydroxyl group Finally, the researchers also found that D11 in vc1.1 interacts with n154 and r81 in α 9 subunit to form hydrogen bond and salt bridge Therefore, glutamate and γ - carboxyglutamate were introduced at position 11 to explore the influence of side chain length and charge on their activity by substituting D11 The activity of the synthesized vc1.1 analogues was tested by electrophysiological double electrode voltage clamp technique In order to further explain the interaction mechanism between α - conotoxin vc1.1 and α 9 α 10 nAChR at the molecular level, all the vc1.1 analogues were modeled and simulated by molecular dynamics The results of electrophysiological data analysis and molecular dynamics simulation show that S4 of vc1.1 forms hydrogen bond with d166 and d169 of α 9 subunit, and the introduction of amino acids with proper side chain length and positive charge at this position can improve its activity At the same time, it was confirmed that P6 was located near d119 of α 9 subunit, and the introduced hyp6 was close to d119 and formed hydrogen bond with it In addition, the hydrogen bond between the hydroxyl group on Y10 side chain and n107 and d119 of α 9 subunit plays an important role in its activity The length of the side chain and the number of negative charges are essential for the 11 residues of vc1.1 Finally, according to the results of activity and molecular dynamics simulation of the first generation of vc1.1 analogues, we designed the second generation of mutants [s4dab, n9a] vc1.1 and [s4dab, n9w] vc1.1, and tested their activity Their IC50 were 52.5 ± 3.2NM and 38.7 ± 2.8nm, respectively, which increased about 20 times compared with wild type vc1.1 In conclusion, this study proposed a method to accurately verify the binding mode of conotoxin by using the conservative modification of amino acid side chain, and obtained two variants with 20 times higher activity, which provided theoretical and technical guidance for the study of analgesic drugs and the explanation of the interaction between conotoxin and receptor from the molecular level The school of medicine of Ocean University of China is the first communication unit Chu Xin, a graduate student of the school of medicine, and Dr Han Shen Tae are the co authors of the research results Yu rilei, an associate professor, and Dr Han Shen Tae are the co authors Xu Qingliang, Professor Jiang Tao and Professor David J Adams are the co authors The research is supported by the central university fund, the National Laboratory Director fund, the Shandong Blue drug bank fund of Qingdao Marine National Laboratory, and the discovery project fund of the Australian Research Council (ARC) The research results were recently published online in ACS chemical Neuroscience journal (pharmaceutical chemistry area I, top journal, if = 3.861).