Biology Reference
In-Depth Information
epothilone A all compete with Flutax-2; whereas peloruside acts as a negative con-
trol as it binds to a different site, showing no change in anisotropy as its concentration
is increased. The majority of taxoid site ligands bind exothermically to preformed
glutaraldehyde-stabilized MTs, and the drug binding reaction is enthalpy driven,
with the ligands binding reversibly to the taxoid binding site. The binding constants
therefore decrease with temperature (as stabilized MTs are being used, the true bind-
ing constant is being measured not an apparent one). However, for ZMP, the apparent
binding constants measured increased with temperature (
Table 19.2
), the opposite of
what is expected for an enthalpy-driven reaction. This indicates that either there is a
strong entropic effect or that the reaction observed is not reversible but an irrevers-
ible covalent reaction. In this case, the binding constants measured are apparent, not
true, given the nature of the binding reaction. With covalent binding, the observed
reaction is only kinetically controlled, as with any other irreversible reaction. Thus,
the extension of the reaction (apparent displacement) increases as temperature
increases and is limited by the rate at which ZMP binds to the MTs. The same rela-
tionship was seen previously with cyclostreptin, which also binds irreversibly to the
taxoid site (
Calvo et al., 2012
).
19.2.3
Laulimalide/peloruside competition assay
If the compound of interest does not bind to the taxoid site, it is possible that it
may bind to the laulimalide/peloruside site. Like the taxoid site, this site can be
probed using a number of competition techniques. HPLC techniques are useful to
quantify the bound ligand.
1.
Treat stabilized cross-linked MTs with equimolar concentrations of test ligand
and either DMSO or a 5- to 10-fold excess of competitor ligand in GAB-0.1 mM
GTP buffer for 45 min at 37
C. Alternatively, purified tubulin can be assembled
into MTs in GAB buffer (1 mM GTP, 6 mM MgCl
2
), and the competition
experiments carried out.
2.
Separate the supernatant and pellet by ultracentrifugation (10 min, 37
C),
resuspend the pellet in 10 mM NaPi, and determine the presence of ligands in
both pellets and supernatants by HPLC.
3.
Add 10
M docetaxel, although any
other ligand with a detectable HPLC trace can be used).
4.
Extract the ligands from the samples with three volumes of dichloromethane, dry
in a vacuum, and resuspend in solvent for analysis.
5.
Analyze the samples by HPLC. We use an Agilent-1100 Series instrument
employing a Supercosil, LC18 DB, 250
m
M internal standard to the samples (e.g., 10
m
4.6 mm, 5 mm bead diameter column
at a flow rate of 1 mL/min. Follow the absorbance of the compounds at their
respective wavelengths.
6.
Run solvent blanks at the start of each experiment and quantify the concentration
of ligands in each of the samples by comparison of the integrated areas of the
HPLC peak with that of the internal standard.
