Conversed mutagenesis of an inactive peptide to ASIC3 inhibitor for active sites determination

Dmitry I Osmakov; Sergey G Koshelev; Yaroslav A Andreev; Igor A Dyachenko; Dmitry A Bondarenko; Arkadii N Murashev; Eugene V Grishin; Sergey A Kozlov

doi:10.1016/j.toxicon.2015.11.019

Conversed mutagenesis of an inactive peptide to ASIC3 inhibitor for active sites determination

Toxicon. 2016 Jun 15:116:11-6. doi: 10.1016/j.toxicon.2015.11.019. Epub 2015 Dec 11.

Authors

Dmitry I Osmakov¹, Sergey G Koshelev², Yaroslav A Andreev², Igor A Dyachenko³, Dmitry A Bondarenko³, Arkadii N Murashev³, Eugene V Grishin², Sergey A Kozlov²

Affiliations

¹ Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, ul. Miklukho-Maklaya 16/10, 117997 Moscow, Russia. Electronic address: osmadim@gmail.com.
² Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, ul. Miklukho-Maklaya 16/10, 117997 Moscow, Russia.
³ Branch of the Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, 6 Nauki Avenue, Pushchino, 142290 Moscow Region, Russia.

PMID: 26686983
DOI: 10.1016/j.toxicon.2015.11.019

Abstract

Peptide Ugr9-1 from the venom of sea anemone Urticina grebelnyi selectively inhibits the ASIC3 channel and significantly reverses inflammatory and acid-induced pain in vivo. A close homolog peptide Ugr 9-2 does not have these features. To find the pharmacophore residues and explore structure-activity relationships of Ugr 9-1, we performed site-directed mutagenesis of Ugr 9-2 and replaced several positions by the corresponding residues from Ugr 9-1. Mutant peptides Ugr 9-2 T9F and Ugr 9-2 Y12H were able to inhibit currents of the ASIC3 channels 2.2 times and 1.3 times weaker than Ugr 9-1, respectively. Detailed analysis of the spatial models of Ugr 9-1, Ugr 9-2 and both mutant peptides revealed the presence of the basic-aromatic clusters on opposite sides of the molecule, each of which is responsible for the activity. Additionally, Ugr9-1 mutant with truncated N- and C-termini retained similar with the Ugr9-1 action in vitro and was equally potent in vivo model of thermal hypersensitivity. All together, these results are important for studying the structure-activity relationships of ligand-receptor interaction and for the future development of peptide drugs from animal toxins.

Keywords: ASIC; Inflammation; Mutagenesis; Peptides; Sea anemone venom.

MeSH terms

Acid Sensing Ion Channel Blockers / chemistry*
Acid Sensing Ion Channels / chemistry*
Animals
Catalytic Domain
Cnidarian Venoms / chemistry*
Humans
Models, Molecular
Mutagenesis, Site-Directed
Protein Structure, Tertiary
Sequence Analysis, Protein
Structure-Activity Relationship
Xenopus laevis

Substances

Acid Sensing Ion Channel Blockers
Acid Sensing Ion Channels
Cnidarian Venoms