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Biophysical and pharmacological characterization of a full-length synthetic analog of the antitumor polypeptide crotamine

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Abstract

Crotamine is a polypeptide isolated from the venom of a South American rattlesnake. Among the properties and biological activities of crotamine, the most extraordinary is its ability to enter cells with unique selective affinity and cytotoxic activity against actively proliferating cells, such as tumor cells. This peptide is also a cargo carrier, and anticipating commercial application of this native polypeptide as a potential theranostic compound against cancer, we performed here a side-by-side characterization of a chemically synthesized full-length crotamine compared with its native counterpart. The structural, biophysical, and pharmacological properties were evaluated. Comparative NMR studies showed structural conservation of synthetic crotamine. Moreover, similarly to native crotamine, the synthetic polypeptide was also capable of inhibiting tumor growth in vivo, increasing the survival of mice bearing subcutaneous tumor. We also confirmed the ability of synthetic crotamine to transfect and transport DNA into eukaryotic cells, in addition to the importance of proteoglycans on cell surface for its internalization. This work opens new opportunities for future evaluation of chimeric and/or point-mutated analogs of this snake polypeptide, aiming for improving crotamine properties and applications, as well as possibly diminishing its potential toxic effects.

Key messages

• Synthetic crotamine showed ex vivo and in vivo activities similar to native peptide.

• Synthetic crotamine structure conservation was demonstrated by NMR analysis.

• Synthetic crotamine is able to transfect and transport DNA into eukaryotic cells.

• Synthetic crotamine shows tumor growth inhibition in vivo.

• Synthetic crotamine increases survival of mice bearing tumor.

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Abbreviations

CPPs:

Cell-penetrating peptides

AMP:

Antimicrobial peptide

GMP:

Good manufacturing practice

NMR:

Nuclear magnetic resonance

IP:

Intraperitoneal

SC:

Subcutaneous

Synd-1:

Syndecan 1

WT:

Wild type

KD:

Knockdown

GFP:

Green fluorescent protein

DAPI:

4′,6-Diamidino-2-phenylindole

3D:

Three-dimensional

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Acknowledgments

We are grateful for the technical assistance of Marcela B. Nering and for the administrative assistance of Rosemary Oliveira.

Funding

This work was supported by the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, São Paulo, Brazil) (Grant Numbers: 2014/50891-1, 2017/02413-1, 2018/20014-0, and 2018/21381-6) and the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Brasília, Brazil) (Grant Numbers: 454234/2014-7, 455953/2014-7, 309428/2015-7). This study was also financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brazil (CAPES) - Finance Code 001. The authors acknowledge the Brazilian Biosciences National Laboratory (LNBio) for the NMR time under proposal RMN-9586.

Author information

Authors and Affiliations

  1. Departamento de Farmacologia, Escola Paulista de Medicina (EPM), Universidade Federal de São Paulo (UNIFESP), Rua 3 de maio 100, Ed. INFAR, 3rd floor, São Paulo, CEP 04044-020, Brazil

    Lucas de Carvalho Porta, Joana D’Arc Campeiro, Rosely Oliveira Godinho & Mirian Akemi Furuie Hayashi

  2. Universidade Estadual de São Paulo (UNESP), São José do Rio Preto, Brazil

    Valmir Fadel

  3. Departamento de Bioquímica e Imunologia, Universidade de São Paulo (USP-FMRP), Ribeirão Preto, Brazil

    Eduardo Brandt Oliveira

Authors
  1. Lucas de Carvalho Porta
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  2. Valmir Fadel
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  3. Joana D’Arc Campeiro
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  4. Eduardo Brandt Oliveira
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  5. Rosely Oliveira Godinho
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  6. Mirian Akemi Furuie Hayashi
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Contributions

Conceptualization, LCP and MAFH; formal analysis, LCP, VF, and JDC; investigation, LCP, VF, and JDC; resources, EBO, ROG, and MAFH; writing - original draft, LCP and MAFH; writing - review and editing, VF, ROG, and MAFH; visualization, LCP; supervision, MAFH; project administration, MAFH; funding acquisition, MAFH.

Corresponding author

Correspondence to Mirian Akemi Furuie Hayashi.

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The authors declare that they have no conflict of interest.

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This project was approved by the Animal Use and Ethic Committee (CEUA) of the UNIFESP/EPM (approval number: 7615290118).

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de Carvalho Porta, L., Fadel, V., D’Arc Campeiro, J. et al. Biophysical and pharmacological characterization of a full-length synthetic analog of the antitumor polypeptide crotamine. J Mol Med 98, 1561–1571 (2020). https://doi.org/10.1007/s00109-020-01975-y

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  • DOI: https://doi.org/10.1007/s00109-020-01975-y

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