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

Abstract

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.

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Acknowledgements

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.

Funding

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).

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Contributions

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). https://doi.org/10.1007/s00109-021-02140-9

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Keywords

  • Crotamine
  • Muscle
  • Peptide
  • Phage
  • Myofiber