nAChRs are pentameric ligand-gated ion channels consisting of an extracellular domain, a transmembrane domain and an intracellular domain and are expressed in the central and peripheral nervous systems and non-neuronal cells. They have been implicated in a range of nervous system diseases and disorders, including Parkinson’s disease, Alzheimer’s disease, schizophrenia, neuropathic pain, memory loss, and stress mediation and are therefore important drug targets. The α9α10 nAChR has also been implicated in chronic pain and has been proposed to be a novel target for analgesics. α-Conotoxin Vc1.1 is a 16 amino acid, disulfide-bonded peptide identified from the venom of C. victoria and potently inhibits the α9α10 nAChR.
Professor Tao Jiang’s group collaborated with the David Adams team at the University of Wollongong, exquisitely elucidated the structure-activity relationship of Vc1.1 with the hα9α10 nAChR through molecular dynamics (MD) simulations, site-directed mutagenesis. This will contribute to the further development of Vc1.1 analogues as molecular probes or drugs.
To date, the crystal structure of Vc1.1 bound-α9α10 nAChR remains unavailable and computational modeling in combination with mutagenesis studies have been used as an effective method for understanding the structure-activity relationship of conotoxins. Researchers analyzed the previously established model ( Yu et al. 2018 J. Med. Chem. ) and found that Vc1.1 S4 was positioned nearby α9 D169 and D166, forming a hydrogen bond with D166, and introduction of a positively charged residue at position 4 was expected to strengthen the binding affinity of the Vc1.1 mutant. Thus, the hydroxyl group of S4 was replaced with amine to increase its electrostatic interaction with D169 and D166. Additionally, [S4K] and [S4Dab] mutants of Vc1.1 were synthesized to investigate the influence of side chain length on the activity. Residue P6 of Vc1.1 is located in the inner part of the binding site and is close to α9 D119, therefore, introducing a hydroxyl group to P6 could potentially increase or at least maintain its activity by forming a hydrogen bond with D119. Furthermore, researchers found that two hydrogen bonds were formed between the hydroxyl group of Vc1.1 Y10 and α9 residues N107 and D119 in the MD simulations. In order to validate the contribution of the hydroxyl group to the binding affinity of Vc1.1, phenylalanine was introduced to position 10 of Vc1.1. Besides, the hydroxyl group of Y10 in Vc1.1 was replaced with F (fluoride) or Cl (chloride) to explore the possibility of hydrogen or halogen bond formation with nearby residues due to the removal of the hydroxyl group. Furthermore, in previously built model, D11 in Vc1.1 forms hydrogen bond/salt-bridge interactions with the side chains of α9 N154 and R81. Here, researchers explore the influence of the length and charge of the side chain to the potency of the peptide by replacing D11 with glutamic acid or γ-carboxyglutamic acid.
The relative inhibitory activity of the designed Vc1.1 analogues was determined at the hα9α10 nAChRs heterologously expressed in Xenopus oocytes using the two-electrode voltage clamp technique. MD simulations of the designed Vc1.1 analogues binding to the α9α10 nAChR was performed for in-depth understanding of their interactions. The results of electrophysiological data analysis and molecular dynamics simulations indicate that Vc1.1 S4 forms hydrogen bonds with α9 D166 and D169, and introducing positively charged residues at this position can improve the potency. The P6 is nearby D119, and the introduced Hyp6 approaches D119 and forms a hydrogen bond. In addition, the hydroxyl group at Y10 side chain forms several hydrogen bonds with residues at the component of the α9 subunit. The side chain length and the number of negative charges are essential for residue at 11 position of Vc1.1. Finally, the researchers designed the second generation mutants [S4Dab, N9A]Vc1.1 and [S4Dab, N9W]Vc1.1 based on the results from activity testing and molecular dynamics simulations of the first generation of design and tested their activity. The IC50 of [S4Dab, N9A]Vc1.1 and [S4Dab, N9W]Vc1.1 were 52.5 ± 3.2 nM and 38.7 ± 2.8 nM, respectively. And the activity is about 20-fold higher than that of wild-type Vc1.1.
Overall, our findings provide valuable insights into the structure-activity relationship of Vc1.1 with the α9α10 nAChR and will contribute to further development of more potent and specific Vc1.1 analogues.
Article Link: https://pubs.acs.org/doi/10.1021/acschemneuro.9b00389
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