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Synthetic polypeptide crotamine: characterization as a myotoxin and as a target of combinatorial peptides


Crotamine is a rattlesnake-derived toxin that causes fast-twitch muscle paralysis. As a cell-penetrating polypeptide, crotamine has been investigated as an experimental anti-cancer and immunotherapeutic agent. We hypothesized that molecules targeting crotamine could be designed to study its function and intervene in its adverse activities. Here, we characterize synthetic crotamine and show that, like the venom-purified toxin, it induces hindlimb muscle paralysis by affecting muscle contraction and inhibits KCNA3 (Kv1.3) channels. Synthetic crotamine, labeled with a fluorophore, displayed cell penetration, subcellular myofiber distribution, ability to induce myonecrosis, and bind to DNA and heparin. Here, we used this functionally validated synthetic polypeptide to screen a combinatorial phage display library for crotamine-binding cyclic peptides. Selection for tryptophan-rich peptides was observed, binding of which to crotamine was confirmed by ELISA and gel shift assays. One of the peptides (CVWSFWGMYC), synthesized chemically, was shown to bind both synthetic and natural crotamine and to block crotamine-DNA binding. In summary, our study establishes a functional synthetic substitute to the venom-derived toxin and identifies peptides that could further be developed as probes to target crotamine.

Key messages

  • Synthetic crotamine was characterized as a functional substitute for venom-derived crotamine based on myotoxic effects.

  • A combinatorial peptide library was screened for crotamine-binding peptides.

  • Tryptophan-rich peptides were shown to bind to crotamine and interfere with its DNA binding.

  • Crotamine myofiber distribution and affinity for tryptophan-rich peptides provide insights on its mechanism of action.

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Availability of data and material (data transparency)

The datasets and materials generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

Code availability (software application or custom code)

Does not apply.


  1. 1.

    Radis-Baptista G, Kerkis I (2011) Crotamine, a small basic polypeptide myotoxin from rattlesnake venom with cell-penetrating properties. Curr Pharm Des 17:4351–4361

    CAS  Article  Google Scholar 

  2. 2.

    Rizzi CT, Carvalho-de-Souza JL, Schiavon E, Cassola AC, Wanke E, Troncone LR (2007) Crotamine inhibits preferentially fast-twitching muscles but is inactive on sodium channels. Toxicon 50:553–562.

    CAS  Article  PubMed  Google Scholar 

  3. 3.

    Koch Hansen L, Sevelsted-Moller L, Rabjerg M, Larsen D, Hansen TP, Klinge L, Wulff H, Knudsen T, Kjeldsen J, Kohler R (2014) Expression of T-cell KV1.3 potassium channel correlates with pro-inflammatory cytokines and disease activity in ulcerative colitis. J Crohns Colitis 8:1378–1391.

    Article  PubMed  Google Scholar 

  4. 4.

    Koshy S, Huq R, Tanner MR, Atik MA, Porter PC, Khan FS, Pennington MW, Hanania NA, Corry DB, Beeton C (2014) Blocking KV1.3 channels inhibits Th2 lymphocyte function and treats a rat model of asthma. J Biol Chem 289:12623–12632.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  5. 5.

    Marinovic MP, Campeiro JD, Lima SC, Rocha AL, Nering MB, Oliveira EB, Mori MA, Hayashi MAF (2018) Crotamine induces browning of adipose tissue and increases energy expenditure in mice. Sci Rep 8:5057.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  6. 6.

    Nascimento FD, Hayashi MA, Kerkis A, Oliveira V, Oliveira EB, Radis-Baptista G, Nader HB, Yamane T, Tersariol IL, Kerkis I (2007) Crotamine mediates gene delivery into cells through the binding to heparan sulfate proteoglycans. J Biol Chem 282:21349–21360

    CAS  Article  Google Scholar 

  7. 7.

    Campeiro JD, Marinovic MP, Carapeto FC, Dal Mas C, Monte GG, Carvalho Porta L, Nering MB, Oliveira EB, Hayashi MAF (2018) Oral treatment with a rattlesnake native polypeptide crotamine efficiently inhibits the tumor growth with no potential toxicity for the host animal and with suggestive positive effects on animal metabolic profile. Amino Acids 50:267–278.

    CAS  Article  PubMed  Google Scholar 

  8. 8.

    Pereira A, Kerkis A, Hayashi MA, Pereira AS, Silva FS, Oliveira EB, Prieto da Silva AR, Yamane T, Radis-Baptista G, Kerkis I (2011) Crotamine toxicity and efficacy in mouse models of melanoma. Expert Opin Investig Drugs 20:1189–1200.

    CAS  Article  PubMed  Google Scholar 

  9. 9.

    de Carvalho PL, Fadel V, D’Arc Campeiro J, Oliveira EB, Godinho RO, Hayashi MAF (2020) Biophysical and pharmacological characterization of a full-length synthetic analog of the antitumor polypeptide crotamine. J Mol Med 98:1561–1571.

    CAS  Article  Google Scholar 

  10. 10.

    Mambelli-Lisboa NC, Sciani JM, Brandão Prieto da Silva AR, Kerkis I (2018) Co-localization of crotamine with internal membranes and accentuated accumulation in tumor cells. Mol Basel Switz 23.

  11. 11.

    Chen P-C, Hayashi MAF, Oliveira EB, Karpel RL (2012) DNA-interactive properties of crotamine, a cell-penetrating polypeptide and a potential drug carrier. PLoS ONE 7:e48913.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  12. 12.

    Kerkis A, Kerkis I, Radis-Baptista G, Oliveira EB, Vianna-Morgante AM, Pereira LV, Yamane T (2004) Crotamine is a novel cell-penetrating protein from the venom of rattlesnake Crotalus durissus terrificus. Faseb J 18:1407-+ .

  13. 13.

    Ponce-Soto LA, Martins-de-Souza D, Marangoni S (2010) Structural and pharmacological characterization of the crotamine isoforms III-4 (MYX4_CROCu) and III-7 (MYX7_CROCu) isolated from the Crotalus durissus cumanensis venom. Toxicon 55:1443–1452.

    CAS  Article  PubMed  Google Scholar 

  14. 14.

    Ponce-Soto LA, Martins-de-Souza D, Novello JC, Marangoni S (2007) Structural and biological characterization of two crotamine isoforms IV-2 and IV-3 isolated from the Crotalus durissus cumanensis venom. Protein J 26:533–540.

    CAS  Article  PubMed  Google Scholar 

  15. 15.

    Toyama MH, Carneiro EM, Marangoni S, Barbosa RL, Corso G, Boschero AC (2000) Biochemical characterization of two crotamine isoforms isolated by a single step RP-HPLC from Crotalus durissus terrificus (South American rattlesnake) venom and their action on insulin secretion by pancreatic islets. Biochim Biophys Acta 1474:56–60

    CAS  Article  Google Scholar 

  16. 16.

    Toyama MH, Marangoni S, Novello JC, Leite GB, Prado-Franceschi J, da Cruz-Hofling MA, Rodrigues-Simioni L (2003) Biophysical, histopathological and pharmacological characterization of crotamine isoforms F22 and F32. Toxicon 41:493–500

    CAS  Article  Google Scholar 

  17. 17.

    Nedelkov D, Bieber AL (1997) Characterization of the two myotoxin a isomers from the prairie rattlesnake (Crotalus viridis viridis) by capillary zone electrophoresis and fluorescence quenching studies. Toxicon 35:689–698

    CAS  Article  Google Scholar 

  18. 18.

    Aird SD, Kruggel WG, Kaiser II (1991) Multiple myotoxin sequences from the venom of a single prairie rattlesnake (Crotalus viridis viridis). Toxicon 29:265–268

    CAS  Article  Google Scholar 

  19. 19.

    O’Keefe MP, Nedelkov D, Bieber AL, Nieman RA (1996) Evidence for isomerization in myotoxin a from the prairie rattlesnake (Crotalus viridis viridis). Toxicon 34:417–434

    Article  Google Scholar 

  20. 20.

    Ownby CL, Aird SD, Kaiser I (1988) Physiological and immunological properties of small myotoxins from the venom of the midget faded rattlesnake (Crotalus viridis concolor). Toxicon 26:319–323

    CAS  Article  Google Scholar 

  21. 21.

    Hindi SM, Shin J, Ogura Y, Li H, Kumar A (2013) Matrix metalloproteinase-9 inhibition improves proliferation and engraftment of myogenic cells in dystrophic muscle of mdx mice. PLoS ONE 8:e72121.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  22. 22.

    Daquinag AC, Zhang Y, Amaya-Manzanares F, Simmons PJ, Kolonin MG (2011) An isoform of decorin is a resistin receptor on the surface of adipose progenitor cells. Cell Stem Cell 9:74–86.

    CAS  Article  PubMed  Google Scholar 

  23. 23.

    Kolonin MG, Saha PK, Chan L, Pasqualini R, Arap W (2004) Reversal of obesity by targeted ablation of adipose tissue. Nat Med 10:625–632.

    CAS  Article  PubMed  Google Scholar 

  24. 24.

    Koivunen EE (1994) Peptides in cell adhesion research. Methods Enzymol 245:346–369

    CAS  Article  Google Scholar 

  25. 25.

    Peigneur S, Orts DJ, Prieto da Silva AR, Oguiura N, Boni-Mitake M, de Oliveira EB, Zaharenko AJ, de Freitas JC, Tytgat J (2012) Crotamine pharmacology revisited: novel insights based on the inhibition of KV channels. Mol Pharmacol 82:90–96.

    CAS  Article  PubMed  Google Scholar 

  26. 26.

    Azhdarinia A, Daquinag AC, Tseng C, Ghosh SC, Ghosh P, Amaya-Manzanares F, Sevick-Muraca E, Kolonin MG (2013) A peptide probe for targeted brown adipose tissue imaging. Nat Commun 4:2472.

    CAS  Article  PubMed  Google Scholar 

  27. 27.

    Staquicini FI, Cardó-Vila M, Kolonin MG, Trepel M, Edwards JK, Nunes DN, Sergeeva A, Efstathiou E, Sun J, Almeida NF et al (2011) Vascular ligand-receptor mapping by direct combinatorial selection in cancer patients. Proc Natl Acad Sci U S A 108:18637–18642.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  28. 28.

    Nie J, Chang B, Traktuev DO, Sun J, March K, Chan L, Sage EH, Pasqualini R, Arap W, Kolonin MG (2008) IFATS collection: combinatorial peptides identify alpha5beta1 integrin as a receptor for the matricellular protein SPARC on adipose stromal cells. Stem Cells Dayt Ohio 26:2735–2745.

    CAS  Article  Google Scholar 

  29. 29.

    Kolonin MG, Bover L, Sun J, Zurita AJ, Do K-A, Lahdenranta J, Cardó-Vila M, Giordano RJ, Jaalouk DE, Ozawa MG et al (2006) Ligand-directed surface profiling of human cancer cells with combinatorial peptide libraries. Cancer Res 66:34–40.

    CAS  Article  PubMed  Google Scholar 

  30. 30.

    Kolonin MG, Sun J, Do K-A, Vidal CI, Ji Y, Baggerly KA, Pasqualini R, Arap W (2006) Synchronous selection of homing peptides for multiple tissues by in vivo phage display. FASEB J Off Publ Fed Am Soc Exp Biol 20:979–981.

    CAS  Article  Google Scholar 

  31. 31.

    Kolonin MG, Pasqualini R, Arap W (2002) Teratogenicity induced by targeting a placental immunoglobulin transporter. Proc Natl Acad Sci U S A 99:13055–13060.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  32. 32.

    Ownby CL, Gutiérrez JM, Colberg TR, Odell GV (1982) Quantitation of myonecrosis induced by myotoxin a from prairie rattlesnake (Crotalus viridis viridis) venom. Toxicon 20:877–885

    CAS  Article  Google Scholar 

  33. 33.

    Hayashi MAF, Campeiro JD, Porta LC, Szychowski B, Alves WA, Oliveira EB, Kerkis I, Daniel MC, Karpel RL (2020) Crotamine cell-penetrating nanocarriers: cancer-targeting and potential biotechnological and/or medical applications. Methods Mol Biol 2118:61–89.

    CAS  Article  PubMed  Google Scholar 

  34. 34.

    Vu TT, Jeong B, Yu J, Koo BK, Jo SH, Robinson RC, Choe H (2014) Soluble prokaryotic expression and purification of crotamine using an N-terminal maltose-binding protein tag. Toxicon 92:157–165.

    CAS  Article  PubMed  Google Scholar 

  35. 35.

    Ziolkowski C, Murchison HA, Bieber AL (1992) Effects of myotoxin alpha on fusion and contractile activity in myoblast-myotube cell cultures. Toxicon 30:397–409

    CAS  Article  Google Scholar 

  36. 36.

    Hayes CE, Bieber AL (1986) The effects of myotoxin from midget faded rattlesnake (Crotalus viridis concolor) venom on neonatal rat myotubes in cell culture. Toxicon 24:169–173

    CAS  Article  Google Scholar 

  37. 37.

    Mebs D, Ehrenfeld M, Samejima Y (1983) Local necrotizing effect of snake venoms on skin and muscle: relationship to serum creatine kinase. Toxicon 21:393–404

    CAS  Article  Google Scholar 

  38. 38.

    Tu AT, Morita M (1983) Attachment of rattlesnake venom myotoxin a to sarcoplasmic reticulum: peroxidase conjugated method. Br J Exp Pathol 64:633–637

    CAS  PubMed  PubMed Central  Google Scholar 

  39. 39.

    Fletcher JE, Hubert M, Wieland SJ, Gong QH, Jiang MS (1996) Similarities and differences in mechanisms of cardiotoxins, melittin and other myotoxins. Toxicon 34:1301–1311

    CAS  Article  Google Scholar 

  40. 40.

    Yudkowsky ML, Beech J, Fletcher JE (1994) Myotoxin a reduces the threshold for calcium-induced calcium release in skeletal muscle. Toxicon 32:273–278

    CAS  Article  Google Scholar 

  41. 41.

    Furukawa K, Funayama K, Ohkura M, Oshima Y, Tu AT, Ohizumi Y (1994) Ca2+ release induced by myotoxin alpha, a radio-labellable probe having novel Ca2+ release properties in sarcoplasmic reticulum. Br J Pharmacol 113:233–239

    CAS  Article  Google Scholar 

  42. 42.

    Ohkura M, Furukawa K, Tu AT, Ohizumi Y (1994) Calsequestrin is a major binding protein of myotoxin alpha and an endogenous Ca2+ releaser in sarcoplasmic reticulum. Eur J Pharmacol 268:R1-2

    CAS  Article  Google Scholar 

  43. 43.

    Ohkura M, Furukawa K, Oikawa K, Ohizumi Y (1995) The properties of specific binding site of 125I-radioiodinated myotoxin a, a novel Ca++ releasing agent, in skeletal muscle sarcoplasmic reticulum. J Pharmacol Exp Ther 273:934–939

    CAS  PubMed  Google Scholar 

  44. 44.

    Coronado MA, Gabdulkhakov A, Georgieva D, Sankaran B, Murakami MT, Arni RK, Betzel C (2013) Structure of the polypeptide crotamine from the Brazilian rattlesnake Crotalus durissus terrificus. Acta Crystallogr Biol Crystallogr 69:1958–1964.

    CAS  Article  Google Scholar 

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We thank Alexes Daquinag for phage display training and general support. We would like to thank the Laboratory of Cellular Biology of the Butantan Institute and Alexsander F. Souza for their support in confocal microscopy (Confocal Leica TCS SP8—Project 175 FINEP—IBUINFRA 01.12.0175.00 by Dr. Carlos Jared). We would also like to acknowledge Dener Madeiro de Souza’s help with the cryostat and muscle section preparation.


Mikhail Kolonin was supported by the Bovay Foundation. Celine Pompeia’s work in this project was funded by the CNPq Conselho Nacional de Desenvolvimento Científico e Tecnológico, Science without Borders Project number 201949/2015–6. The project was also funded by Grant 2015/50040–4, São Paulo Research Foundation (FAPESP) and GlaxoSmithKline. Jan Tytgat was supported by grants G0E7120N, GOC2319N, and GOA4919N (FWO Vlaanderen). Steve Peigneur was supported by grant PDM/19/164 (KU Leuven).

Author information




C.P., I.K., and M.G.K. designed the experiments, analyzed data, and wrote the manuscript; C.P., E.O.F., S.P., J.T., A.P.S., E.B.O., and A. P. performed the experiments, analyzed data, and edited the manuscript.

Corresponding author

Correspondence to Mikhail G. Kolonin.

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Ethics approval

The animal studies were approved by the Ethical Committee of Animal Use of the Butantan Institute. All procedures for the use and handling of adult female Xenopus laevis frogs (CRB Xénopes, Rennes, France) were approved by the Animal Ethics Committee of the KU Leuven (Project No. P186/2019) following regulations of the European Union (EU) concerning the welfare of laboratory animals as declared in Directive 2010/63/EU.

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The authors declare no competing interests.

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Pompeia, C., Frare, E.O., Peigneur, S. et al. Synthetic polypeptide crotamine: characterization as a myotoxin and as a target of combinatorial peptides. J Mol Med (2021).

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  • Crotamine
  • Muscle
  • Peptide
  • Phage
  • Myofiber