Construction of Highly Stable Cytotoxic Nuclear-Directed Ribonucleases

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David Roura Padrosa1, Jessica Castro2,3, Alejandro Romero-Casañas4

  • 1Laboratori d'Enginyeria de Proteïnes, Departament de Biologia, Facultat de Ciències, Universitat de Girona, Campus de Montilivi, Maria Aurèlia Capmany 40, 17003 Girona, Spain. pcxdr1@exmail.nottingham.ac.uk.

Molecules (Basel, Switzerland)
|December 15, 2018
PubMed

Insights

Researchers enhanced the stability of cytotoxic human pancreatic ribonuclease variants for cancer therapy. Introducing disulfide bonds increased thermal stability and resistance to proteolysis, improving potential in vivo efficacy.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Oncology

Background:

  • Ribonucleases show promise in anticancer therapy.
  • Engineered human pancreatic ribonuclease variants exhibit selective tumor cell cytotoxicity via nuclear localization signals.
  • Previous modifications decreased protein stability, limiting in vivo application.

Purpose of the Study:

  • To enhance the thermal stability of cytotoxic human pancreatic ribonuclease variants.
  • To improve the in vivo behavior and therapeutic potential of these anticancer agents.

Main Methods:

  • Site-directed mutagenesis to introduce additional disulfide bonds.
  • Thermal stability assays.
  • Assessment of catalytic activity.
  • Evaluation of cytotoxic activity against tumor cells.
  • Proteolysis resistance assays using proteinase K and human sera.

Main Results:

  • Significant increase in thermal stability (up to 17 °C) achieved through disulfide bond introduction.
  • Catalytic and cytotoxic activities against tumor cells were preserved.
  • The most stable variant demonstrated enhanced resistance to proteolysis by proteinase K and human sera.

Conclusions:

  • Introducing additional disulfide bonds is an effective strategy to enhance the stability of cytotoxic ribonuclease variants.
  • Increased stability and proteolysis resistance suggest improved in vivo half-life and therapeutic potential.
  • These enhanced ribonuclease variants represent a promising advancement in anticancer therapy.
Keywords:
antitumor proteindisulfide bondnuclear-directed ribonucleaseproteolytic resistancethermal stability

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