Methods for studying microtubule binding site interactions: zampanolide as a covalent binding agent

Methods Cell Biol. 2013:115:303-25. doi: 10.1016/B978-0-12-407757-7.00019-0.

Abstract

In this chapter, we describe the methods used to determine the binding site and binding profile of zampanolide, a novel microtubule-stabilizing agent (MSA) that binds covalently to tubulin. These methods can be applied to other novel MSAs in which the binding site and mechanism of binding are unknown. Using the described methods, we have shown that zampanolide binds to the taxoid site on β-tubulin, but unlike most other MSAs is able to covalently modify this site. The purpose of this chapter is to provide a step-by-step protocol for determining the binding site of a novel MSA.

Keywords: Anticancer drugs; Covalent binding drug; Flutax; Microtubule-stabilizing agent; Taxoid site; Zampanolide.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Binding Sites / physiology*
  • Brain / cytology
  • Brain / metabolism
  • Bridged Bicyclo Compounds, Heterocyclic / metabolism
  • Cattle
  • Lactones / metabolism
  • Macrolides / metabolism*
  • Microtubules / metabolism*
  • Paclitaxel / metabolism
  • Polycyclic Compounds / metabolism
  • Protein Binding / physiology*
  • Tubulin Modulators / metabolism

Substances

  • Bridged Bicyclo Compounds, Heterocyclic
  • FR 182877
  • Lactones
  • Macrolides
  • Polycyclic Compounds
  • Tubulin Modulators
  • laulimalide
  • peloruside A
  • zampanolide
  • Paclitaxel