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A diazirine-based photoaffinity etoposide probe for labeling topoisomerase II

Gaik-Lean Chee1, Jack C Yalowich, Andrew Bodner

  • 1Faculty of Pharmacy, University of Manitoba, Winnipeg, Manitoba, Canada R3E 0T5. lean_chee@umanitoba.ca

Bioorganic & Medicinal Chemistry
|December 17, 2009
PubMed

Insights

Researchers synthesized a novel photoaffinity probe to identify etoposide binding sites on topoisomerase II. This probe covalently labels the enzyme, aiding in understanding anticancer drug mechanisms and guiding future drug development.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Pharmacology

Background:

  • Etoposide is a crucial anticancer drug targeting topoisomerase II.
  • The precise binding site of etoposide on topoisomerase II remains unidentified despite extensive research.
  • Understanding this interaction is key to developing more effective cancer therapies.

Purpose of the Study:

  • To synthesize and characterize a diazirine-based photoaffinity etoposide analog probe.
  • To investigate the probe's photoreactivity and biological activity in identifying etoposide binding sites on topoisomerase II.
  • To establish a foundation for using proteomics and mass spectrometry to map etoposide interactions.

Main Methods:

  • Synthesis of a diazirine-based photoaffinity etoposide analog.
  • Characterization of the probe's photoreactivity upon UV irradiation.
  • Assessment of the probe's biological activity, including inhibition of K562 cell growth and topoisomerase II activity.
  • Evaluation of the probe's ability to induce DNA cleavage and protein-DNA complex formation.

Main Results:

  • The diazirine probe generates reactive carbenes upon UV irradiation, forming stable covalent adducts.
  • The probe exhibits similar anticancer and topoisomerase II inhibitory properties to etoposide.
  • The probe acts as a topoisomerase II poison, inducing DNA double-strand breaks.
  • Photoirradiation of probe-treated cells significantly enhanced protein-DNA covalent complex formation, indicating adduct formation with topoisomerase IIalpha.

Conclusions:

  • The synthesized diazirine-based photoaffinity etoposide analog is a valuable tool for studying etoposide-enzyme interactions.
  • The probe's ability to form covalent adducts suggests it can effectively label topoisomerase II.
  • Proteomics mass spectrometry is a viable strategy for identifying etoposide binding sites on topoisomerase II using this probe.

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