Chemistry Reference
In-Depth Information
slurry) is pipetted into each cell one at a time, allowed to settle by gravity and packed
at a pressure of 0.5 bar. Once packed, the cell can be connected to the sample delivery
system via PEEK capillary tubes and inserted into the magnet using an aluminum arm. By
attaching the cell to the aluminum arm, we can readily orient it such that the plane that
bisects each of the two cylindrical cells is parallel to one of the transverse gradients in our
triple-gradient flow-injection probe.
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In this way, optimization of the NMR experiment for
each screen is minimized. All that is necessary is to perform routine tuning and matching
and shim, which we do using the FID of water. When known ligands are available, initial
tests are performed to insure the integrity of the immobilized sample. This same experiment
is repeated 4-5 times throughout and after the screen to detect possible target degradation
(Figure 6.3A.) Once prepared, the mixes are placed in the Gilson autosampler in deep
96-well plates and the Bruker HyStar software is programmed for each.We also use standard
ICON NMR in Topspin to acquire the TINS data. A complete screen of about 1500 unique
compounds (including some replicates for quality assurance) requires about 7 days and
runs without human intervention. Having evaluated a variety of different spatially selective
NMR experiments, we have settled on the Hadamard sampling approach. The quality of
the data using this experiment with carefully designed mixes is fairly high, as can be seen
in Figure 6.3B.
We have now screened a number of different targets, both soluble and membrane bound,
using TINS. The hit rate for targets has varied from a low of 3% to a high of about 10%,
where we define a hit as having at least a 30%difference in amplitude between the reference
(a)
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Experiment number
Figure 6.3
(A) Determination of target integrity during a TINS ligand screen. A known ligand
was applied to both the target and reference cells and the reduction in peak amplitude was
measured ('TINS effect'). This experiment was carried out serially after the indicated number
of mixes had been applied to the immobilized target. (B) Direct determination of ligand iden-
tity using TINS. A mix of five compounds was applied to the dual sample holder containing
immobilized target and the pH domain of AKT, both at 100 μM solution equivalent. The indi-
vidual spectra of each cell, acquired with a 30min measuring time, are overlaid at the bottom
of the figure. The
1
H
spectra of four of the five compounds are shown above for reference. The
identity of the ligand (fourth spectrum identifier 1059) is readily obtained by simple inspection.
