binding and unbinding, a significant fraction of the free population
of the ligand has been bound during the course of the experiment
and so is labeled with a signal indicative of binding. The observed
signal is substantially larger than the bound fraction of the ligand
might indicate [28, 29], and the sensitivity of these experiments is
dramatically increased when a large molar excess of the ligand over
the protein is used. Also, since a large molar excess of the ligand is
used, relatively little protein is required, resulting in reduced protein demands compared to PO-NMR. Additionally, since only the
free population of the ligand is observed, there is no limit on the
size of the protein that can be used; indeed, these experiments work
best with large, slowly tumbling macromolecules. These factors
have contributed strongly to the prevalence of LO-NMR as a
fragment screening technique [17].
A limitation of LO-NMR is that these experiments only indicate an interaction between the protein and the ligand; unlike
PO-NMR, no information is obtained as to the location of the
binding site (or even whether the interaction is specific to a single
site or nonspecific). A simple solution to this is to perform a
competition experiment using a potent ligand known to bind to
the site of interest; if the fragment is displaced by the competitor,
the signal in the LO-NMR experiment will be reduced or abolished
[16, 30]. However, this requires prior knowledge of a potent
competitor that binds to the site of interest; while this is the case
for many proteins, a significant number remain where this is not
applicable.
A third class of NMR experiments widely used for FBS is
19 F-observed NMR. While this usually refers to a ligand-observed
experiment, the considerations and requirements are sufficiently
different from
1
H observed LO-NMR that
19 F LO-NMR is often
considered as a separate technique.
19
F-observed NMR has been
reviewed extensively elsewhere [21, 31, 32], and only a brief overview will be given here. The chemical shift of a
19 F nucleus is
exquisitely sensitive to changes in the chemical environment; consequently, the large spectral width together with the low number of
resonances in each fragment allows the use of mixtures containing
large numbers of compounds and the simple analysis of these
spectra using automated software. Thus, the differential relaxation
rates of
19
F in the free and bound states of a fragment can be
maximally exploited to rapidly identify ligands. However, the high
sensitivity of
19
F to changes in the chemical environment also
results in a high false-positive rate, and this can reduce the reliability
of the data from the screen. This can be mitigated through the use
of orthogonal methods (such as PO-NMR or SPR), but this in turn
has implications for the amount of time and resource required for
the FBS.
Because both LO-NMR and PO-NMR use large amounts of
protein compared to other FBS techniques such as SPR or DSF
Fragment Screening by NMR
251
of the ligand has been bound during the course of the experiment
and so is labeled with a signal indicative of binding. The observed
signal is substantially larger than the bound fraction of the ligand
might indicate [28, 29], and the sensitivity of these experiments is
dramatically increased when a large molar excess of the ligand over
the protein is used. Also, since a large molar excess of the ligand is
used, relatively little protein is required, resulting in reduced protein demands compared to PO-NMR. Additionally, since only the
free population of the ligand is observed, there is no limit on the
size of the protein that can be used; indeed, these experiments work
best with large, slowly tumbling macromolecules. These factors
have contributed strongly to the prevalence of LO-NMR as a
fragment screening technique [17].
A limitation of LO-NMR is that these experiments only indicate an interaction between the protein and the ligand; unlike
PO-NMR, no information is obtained as to the location of the
binding site (or even whether the interaction is specific to a single
site or nonspecific). A simple solution to this is to perform a
competition experiment using a potent ligand known to bind to
the site of interest; if the fragment is displaced by the competitor,
the signal in the LO-NMR experiment will be reduced or abolished
[16, 30]. However, this requires prior knowledge of a potent
competitor that binds to the site of interest; while this is the case
for many proteins, a significant number remain where this is not
applicable.
A third class of NMR experiments widely used for FBS is
19 F-observed NMR. While this usually refers to a ligand-observed
experiment, the considerations and requirements are sufficiently
different from
1
H observed LO-NMR that
19 F LO-NMR is often
considered as a separate technique.
19
F-observed NMR has been
reviewed extensively elsewhere [21, 31, 32], and only a brief overview will be given here. The chemical shift of a
19 F nucleus is
exquisitely sensitive to changes in the chemical environment; consequently, the large spectral width together with the low number of
resonances in each fragment allows the use of mixtures containing
large numbers of compounds and the simple analysis of these
spectra using automated software. Thus, the differential relaxation
rates of
19
F in the free and bound states of a fragment can be
maximally exploited to rapidly identify ligands. However, the high
sensitivity of
19
F to changes in the chemical environment also
results in a high false-positive rate, and this can reduce the reliability
of the data from the screen. This can be mitigated through the use
of orthogonal methods (such as PO-NMR or SPR), but this in turn
has implications for the amount of time and resource required for
the FBS.
Because both LO-NMR and PO-NMR use large amounts of
protein compared to other FBS techniques such as SPR or DSF
Fragment Screening by NMR
251