Biology Reference
In-Depth Information
19.2.6 Mass Spectrometry ................................................................ 318
19.2.6.1 Protein Preparation and Digestion for MS Analysis.........319
19.2.7 Visualization of Covalent Binding in Cells ................................ 320
19.2.7.1 Adherent Cell Lines.......................................................322
19.2.7.2 Cells in Suspension.......................................................322
19.2.8 Further Experiments for Characterization ................................. 324
Summary ................................................................................................................ 324
Acknowledgments ................................................................................................... 324
References ............................................................................................................. 324
Abstract
In this chapter, we describe the methods used to determine the binding site and bind-
ing 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
-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.
b
INTRODUCTION
Microtubules (MTs) are one of the most successful targets for anticancer therapy
because of their essential role in mitosis. There are various MT-targeting drugs used
in the clinic today. However, mechanisms of resistance toward these drugs have
appeared, most importantly, the upregulation of drug efflux pumps, such as
P-glycoprotein (P-gp) (
Gottesman, Fojo, & Bates, 2002
). Because tumor cells can
become resistant to drugs with long-term chemotherapy, there is a need to find
new compounds with a similar mechanism of action but which avoid these mecha-
nisms of resistance. One way of circumventing resistance is to use an MSA that
binds covalently. Covalent binding agents are effective in P-gp overexpressing cells
because they cannot be pumped out of the cell once they covalently bind. It has
also been suggested that covalent binding agents would not be affected by upregula-
tion of different tubulin isotypes or mutations in tubulin (
Singh, Petter, Baillie, &
Whitty, 2011
).
One of the first steps in characterizing a novel MSA is to determine the site where
it binds. Currently, there are two known MSA binding sites, the well-characterized
taxoid site and the laulimalide/peloruside site. The taxoid site is located in the lumen
of the
b
-tubulin subunit in a pocket where
a
-tubulin has eight extra amino acids
(
Nogales, Whittaker, Milligan, &Downing, 1999
). There is also evidence that taxoid
