Methods for Studying Microtubule Binding Site Interactions: Zampanolide as a Covalent Binding Agent - Methods in Cell Biology - page 296

site compounds interact with the pore type I of MTs on their way to the taxoid site
( Buey et al., 2007 ). The laulimalide/peloruside site is biochemically distinct from the
taxoid site; however, its exact location is unknown, with evidence suggesting it could
be present in both subunits. A number of different assays can be performed to deter-
mine the binding site of a novel MSA, and competition methods appear to be the
most popular. The mechanism by which an MSA settles into its binding site is an
important factor in its characterization. It is well known that MSAs may bind in a
reversible manner, like the taxanes for example, or in an irreversible manner, like
cyclostreptin ( Buey et al., 2007 ). Covalent adduct formation causes permanent dis-
ruption of the target's biological function, increasing the compound's potency, and
prolonging its effect. This can lead to a more desirable clinical profile and therapeu-
tic window as this gives potential for less-frequent dosing and lower drug concen-
trations ( Singh et al., 2011 ).
Zampanolide (ZMP), isolated from a marine sponge, is a potent MSA but is not
susceptible to the P-gp efflux pump ( Field et al., 2009 ), thus it evades the main mech-
anism of resistance presented toward these drugs in the clinic. Its enantiomer, dac-
tylolide, also stabilizes MTs, but with much lower potency ( Zurwerra, Gertsch, &
Altmann , 2010 ). Both compounds bind in a covalent manner, but the binding kinetics
of dactylolide is much slower. Interestingly, ZMP is unique to both cyclostreptin and
dactylolide as it binds covalently to tubulin with high affinity but, unlike the other
two drugs, is a potent inducer of MT polymerization ( Field et al., 2012 ).
In this chapter, we describe how the stabilizing activity of a “test” compound can
be confirmed and the location of its binding site determined. It is possible that a li-
gand can bind to one of the known MSA binding sites or to a novel site. We then
probe the mechanism of binding to ascertain whether the compound binds in a re-
versible or irreversible manner. The methods described have been used to establish
the stabilizing activity, the location of the binding site, and the mechanism of binding
of ZMP ( Field et al., 2012 ).

19.1 MATERIALS
19.1.1 Tubulin and stabilized microtubules
Prepare purified calf brain tubulin for in vitro experiments as described by Andreu
(2007) using a modified Weisenberg procedure. Freeze the tubulin in 5-20 mg
aliquots in liquid nitrogen and thaw when required. Alternatively, purified bovine
brain tubulin can be purchased ( [e-mail] ).

1. Take an aliquot and remove sucrose using a Sephadex G-25 medium-size column
(25

0.9 cm) (GE Healthcare Bioscience) equilibrated with two volumes of
cold buffer ( Section 1.2 ).
2. Collect the tubulin and using the buffer as the blank, measure the absorbance at
295 nm, pooling all fractions with an absorbance greater than 1.0.