Poster Presentation The 16th Australian Peptide Conference 2025

The total chemical synthesis of HCRG21, a venom-derived inhibitor of TRPV1 with therapeutic Potential (#109)

Jinwei Sun 1 , Paul Harris 1 2 3 , Kerry Loomes 3
  1. School of Chemical Sciences, The University of Auckland, Auckland, New Zealand
  2. Maurice Wilkins Centre for Molecular Biodiscovery, The University of Auckland, Auckland, New Zealand
  3. School of Biological Sciences, The University of Auckland, Auckland, New Zealand

Venom peptides can bind a wide range of molecular targets and naturally act as ligands of ion channels. These properties have attracted significant interest as potential leads for developing novel therapeutics.1 Kunitz-domain-containing peptides are common serine protease inhibitors, characterized by a Kunitz motif comprising three disulfide bonds that confer high stability.2 HCRG21, a recently discovered Kunitz-type peptide from the venom of the sea anemone Heteractis crispa, comprising 56 amino acids and stabilized by three disulfide bonds.3 The peptide family that HCRG21 belongs to includes APHC1 and APHC3, also found in Heteractis crispa.3 

APHC1 and APHC3 have attracted interest as potential analgesics due to their partial inhibition of transient receptor potential vanilloid-type 1 (TRPV1), a calcium-permeable ion channel activated by heat and capsaicin.4 TRPV1 is part of the TRPV subfamily, responsible for multiple sensory responses such as heat, cold, pain and taste. Remarkably, HCRG21 is the first full antagonist of TRPV1.5 In addition, TRPV4, a related ion channel sharing 40% sequence homology with TRPV1 is involved in metabolic regulation.6 Inhibition of TRPV4 has been associated with increased muscle mass and increased energy expenditure.7,8 We hypothesise that HCRG21 might serve as a suitable template to develop novel peptides targeting TRPV4 to treat metabolic disease.

As HCRG21 has not been previously synthesised chemically, we pursued an Fmoc-based synthetic route by first synthesising the peptide as two fragments, then ligating them using native chemical ligation to form the linear HCRG21 peptide. Its chemical synthesis raises a potentially significant challenge due to the six cysteine residues that must be chemically folded from the linear peptide with 15 possible conformations into the correct disulfide connectivity. Disulfide bond connectivity was determined using enzymatic digestion and high-resolution mass spectrometry. This work represents the first reported synthesis of HCRG21, opening the possibility of developing novel analogues for therapeutic applications.

  1. V, V.; Achar, R. R.; M.U, H.; N, A.; T, Y. S.; Kameshwar, V. H.; Byrappa, K.; Ramadas, D. Venom Peptides – A Comprehensive Translational Perspective in Pain Management. Curr. Res. Toxicol. 2021, 2, 329–340. https://doi.org/10.1016/j.crtox.2021.09.001.
  2. Hernández-Goenaga, J.; López-Abán, J.; Protasio, A. V.; Vicente Santiago, B.; del Olmo, E.; Vanegas, M.; Fernández-Soto, P.; Patarroyo, M. A.; Muro, A. Peptides Derived of Kunitz-Type Serine Protease Inhibitor as Potential Vaccine Against Experimental Schistosomiasis. Front. Immunol. 2019, 10, 2498. https://doi.org/10.3389/fimmu.2019.02498.
  3. Monastyrnaya, M.; Peigneur, S.; Zelepuga, E.; Sintsova, O.; Gladkikh, I.; Leychenko, E.; Isaeva, M.; Tytgat, J.; Kozlovskaya, E. Kunitz-Type Peptide HCRG21 from the Sea Anemone Heteractis Crispa Is a Full Antagonist of the TRPV1 Receptor. Mar. Drugs 2016, 14 (12), 229. https://doi.org/10.3390/md14120229.
  4. Andreev, Y. A.; Kozlov, S. A.; Korolkova, Y. V.; Dyachenko, I. A.; Bondarenko, D. A.; Skobtsov, D. I.; Murashev, A. N.; Kotova, P. D.; Rogachevskaja, O. A.; Kabanova, N. V.; Kolesnikov, S. S.; Grishin, E. V. Polypeptide Modulators of TRPV1 Produce Analgesia without Hyperthermia. Mar. Drugs 2013, 11 (12), 5100–5115. https://doi.org/10.3390/md11125100.
  5. Zhang, M.; Ma, Y.; Ye, X.; Zhang, N.; Pan, L.; Wang, B. TRP (Transient Receptor Potential) Ion Channel Family: Structures, Biological Functions and Therapeutic Interventions for Diseases. Signal Transduct. Target. Ther. 2023, 8 (1), 1–38. https://doi.org/10.1038/s41392-023-01464-x.
  6. XING, R.; WANG, P.; ZHAO, L.; XU, B.; ZHANG, N.; LI, X. Mechanism of TRPA1 and TRPV4 Participating in Mechanical Hyperalgesia of Rat Experimental Knee Osteoarthritis. Arch. Rheumatol. 2017, 32 (2), 96–104. https://doi.org/10.5606/ArchRheumatol.2017.6061.
  7. Ye, L.; Kleiner, S.; Wu, J.; Sah, R.; Gupta, R. K.; Banks, A. S.; Cohen, P.; Khandekar, M. J.; Boström, P.; Mepani, R.; Laznik, D.; Kamenecka, T. M.; Song, X.; Liedtke, W.; Mootha, V. K.; Puigserver, P.; Griffin, P. R.; Clapham, D. E.; Spiegelman, B. M. TRPV4 Is a Regulator of Adipose Oxidative Metabolism, Inflammation and Energy Homeostasis. Cell 2012, 151 (1), 96–110. https://doi.org/10.1016/j.cell.2012.08.034.
  8. Gram, D. X.; Holst, J. J.; Szallasi, A. TRPV1: A Potential Therapeutic Target in Type 2 Diabetes and Comorbidities? Trends Mol. Med. 2017, 23 (11), 1002–1013. https://doi.org/10.1016/j.molmed.2017.09.005.