Full Antagonist of TRPV1 Receptor of Sea Anemone Heteractis crispa

Sea anemone venoms contain many peptides modulating biological targets such as ion channels or
receptors. The sequence of a new Kunitz-type peptide, HCRG21, belonging to the Heteractis crispa
RG (HCRG) peptide subfamily was deduced on the basis of the gene sequence obtained from the
Heteractis crispa cDNA. HCRG21 shares high structural homology with Kunitz-type peptides APHC1–
APHC3 from H. crispa, and clusters with the peptides from so named “analgesic cluster” of the HCGS
peptide subfamily but forms a separate branch on the NJ-phylogenetic tree. Three unique point
substitutions at the N-terminus of the molecule, Arg1, Gly2, and Ser5, distinguish HCRG21 from other
peptides of this cluster. The trypsin inhibitory activity of recombinant HCRG21 (rHCRG21) was
comparable with the activity of peptides from the same cluster. Inhibition constants for trypsin and α-
chymotrypsin were 2.0 × 10−7 and 7.0 × 10−7 M, respectively. Electrophysiological experiments
revealed that rHCRG21 inhibits 95% of the capsaicin-induced current through transient receptor
potential family member vanilloid 1 (TRPV1) and has a half-maximal inhibitory concentration of 6.9 ±
0.4 μM. Moreover, rHCRG21 is the first full peptide TRPV1 inhibitor, although displaying lower affinity
for its receptor in comparison with other known ligands. Macromolecular docking and full atom
Molecular Dynamics (MD) simulations of the rHCRG21–TRPV1 complex allow hypothesizing the
existence of two feasible, intra- and extracellular, molecular mechanisms of blocking. In this work, we
report the first venom derived peptide compound that is a full antagonist of the pain signal
transmission through TRPV1. HCRG21 represents a promising tool for studying TRPV1 channels
and, moreover, might be an interesting lead compound for the development of novel analgesics.
These data provide valuable insights in the structural and functional relationships and
pharmacological potential of bifunctional Kunitz-type peptides.