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

• Substitutions T9F and Y12H in inactive peptide Ugr 9-2 revealed their important role for the inhibition of ASIC3 channel.
• Two distinct clusters of basic-aromatic residues including either F9 or H12 residues were determined by structure analysis.
• Truncation of N- and C-termini of Ugr 9-1 did not change in vitro and in vivo potency.
• These results make important contribution to the understanding of ASIC3 channel-ligands structure–functional relationships.

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.