REDOR NMR of Tubulin-Bound Paclitaxel
A R T I C L E S
prepared a series of fluoro- and deuterio-labeled derivatives of
PTX suitable for measurement of interatomic distances by solid-
state 2H{19F} REDOR NMR. Samples of ligand-doped micro-
tubules were subjected to three weeks of spectrometer time to
measure each of three pairwise distances. The latter were
compared to the same quantities for five PTX conformers, each
of which has been proposed to be the bioactive form at the
tubulin-binding pocket.
on the level of protein assembly, the taxane-binding site
sequestered within â-tubulin appears to be faithfully retained
in the two situations. This underscores the necessity that
alternative bioactive conformations for paclitaxel pass the three
tests outlined in the previous paragraph.
Experimental Section
Synthesis of Labeled Paclitaxels. Full experimental details of the
syntheses of compounds 4 and 5 are provided as Supporting Informa-
tion.
The outcome definitively eliminates the polar conformer from
consideration. The nonpolar conformer is also eliminated by
the combination of the present data with our previous REDOR
data.15 The PTX-NY structure fits the REDOR data well, though
there are irregularities in its molecular geometry, as illustrated
by Figures 6 and 7 and Table 3. The latter alone, however, do
not disqualify the conformer from bioactive candidacy. Critical
is the observation that the shape of PTX-NY’s C13 side chain
is in conflict with the experimental EC density. On the other
hand, the T-Taxol structure passes three fundamentally important
tests. First, this conformation is within 0.7 Å of four well-defined
REDOR distances and fully consistent with the fifth. Second,
it is the only conformer in Table 2 that is compatible with the
EC density. Third, it is unique in being a template for synthesis
of bridged taxanes that match or surpass both the binding affinity
and cytotoxicity characteristics of the paclitaxel parent. For these
reasons, the T-Taxol conformation proposed by a combination
of structure and modeling techniques and by synthesis and
bioassay20 is confirmed by REDOR measurement of interatomic
distances.
ED50 Determination. Pure tubulin was obtained from bovine brain
and used in polymerization and REDOR experiments. Polymerization
activity of paclitaxel and derivatives was evaluated in the following
manner. GTP-tubulin (5 µM) in a buffer containing 0.1 M PIPES, 1
mM MgSO4, 2 mM EDTA, and 0.1 mM GTP (pH 6.90) was
equilibrated to 37 °C in a quartz cuvette in a Hewlett-Packard model
8453 diode array spectrophotometer, and a baseline was recorded.
Polymerization was initiated by addition of the compound to be tested
in DMSO, and the extent of assembly was measured as the change in
apparent absorption at 350 nm. In all the solutions, the percentage of
DMSO was maintained as 4% v/v to avoid any effect of DMSO on
tubulin assembly. A plot of change in apparent absorption at 350 nm
versus the concentration of the compound was fit to a rectangular
hyperbola to obtain the ED50 value.
NMR Samples. Paclitaxel analogues 4 (0.7 µmoles) and 5 (0.1
µmoles) were bound 1:1 to tubulin to form microtubule complexes
following the protocol described earlier14 with the following modifica-
tions: GMPCPP rather than GTP was employed as the nucleotide, and
20 mM sucrose and 1% PEG (Mn 8000) were used as the lyo- and
cryoprotectants, respectively. A small amount of the freeze-dried
material was reconstituted in 10 mM phosphate buffer (pH 7.0) and
examined by electron microscopy; normal microtubules were observed.
The complexes were packed into rotors under a dry atomosphere. A
reference sample for testing 2H{19F} REDOR dephasing was prepared
by dissolving D-[2-2H,3-19F]alanine (10 mg) with HEPES buffer (100
mg), flash freezing the solution, and then lyophilizing at low temper-
ature. A control sample that contained no 19F was prepared by physically
mixing 5 mg of D-[3-2H3]alanine (5 mg) with sulfur powder (100 mg).
This mixture was used to confirm that a null REDOR difference signal
was observed (Figure S3 of the Supporting Information) from a 32-Tr
2H{19F} REDOR experiment after 1 million scans.
Finally, with respect to evaluating prospective bioactive
taxane conformations at the binding site of â-tubulin, it must
be noted that the EC binding model that led to the T-Taxol
proposal was derived from Zn+2-stabilized tubulin sheets5,7 and
not genuine microtubules. It is thus necessary to determine
whether this model is suitable as a guide to the 3D-shape of
the bioactive form at the tubulin-binding site. It is no coincidence
that virtually every recent synthetic program attempting to
prepare more potent and less resistant taxanes based on
biostructural principles has employed the EC structure.18,20,29,37
Furthermore, the tubulin dimer structure derived from Zn+2
-
Spectrometer. Deuterium NMR spectra were obtained using a
spectrometer equipped with a four-frequency (HFCD) transmission-
line probe operating at an 1H frequency of 500.5 MHz and an 2H
frequency of 76.83 MHz. Tune and match circuits for each frequency
were external to the magnet. The probe had a 12-mm long, 6-mm inside-
diameter analytical coil and a Chemagnetics/Varian magic-angle
spinning ceramic stator. Five-millimeter thin-wall ceramic rotors were
spun at 8000 Hz with the speed under active control to within (2 Hz.
A 12-T static magnetic field was provided by an 89-mm bore Magnex
superconducting solenoid. The spectrometer was controlled by a
Tecmag Apollo pulse programmer. Levels of all radio frequency pulses
were under active control during data acquisition by sampling of test
pulses fired on each channel in use immediately after each scan.
REDOR. The version of REDOR used in these experiments (Figure
S1) had a single 2H 180° pulse (5.6 µs) and 16n (n ) 2, 3, ...) 19F 180°
pulses (5.8 µs). The phases of the 19F pulses followed the xy8 phase-
alternation scheme.39 This combination gave the best echo refocusing
and most complete dephasing (Figure S2). Internuclear distances were
calculated from the REDOR dephasing by spectral simulations per-
stabilized sheets has been modeled into the 8 Å resolution
microtubule structure determined by cryoelectron microscopy38
and shown to be entirely consistent with the MT structure.
Finally, as we have pointed out previously,17,20,29 not only is
the REDOR geometry derived from lyophilized MTs co-incident
with the T-Taxol form (Table 2), but also bioactivities in
situations where MTs are present as soluble dynamic entities
can be significantly improved by exploiting the EC structure
and bound ligand from stabilized tubulin sheets. The implication
is clear. While the details of tubulin polymerization may vary
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