3.3.2 Displacement
of the Tool Compound by
a Potent Competitor
Once binding of a low-affinity ligand has been observed, a potent
competitor can be used to displace the ligand. The concentration of
competitor required for full displacement of the low-affinity ligand
should be determined by titration.
If no potent competitor molecule is available, the screen can
still be acquired. However, the rate of false positives is likely to be
significantly higher than is the case where a competition step is
possible. This will increase the overall hit rate and will also increase
the burden on subsequent activities such as singleton validation and
validation using orthogonal biophysical techniques. Where no
competitor ligand is available, the stringency of subsequent validation assays must be high in order to reduce the number of false
positives diluting the pool of fragment hits (see Subheading 3.8).
3.3.3 Stability of Binding
and Competition
Having determined the concentration of competitor required in
order to observe displacement of the low-affinity tool compound, a
number of samples (typically 4 or 5) containing protein and the
low-affinity tool compound should be prepared. The competitor
should then be added to different samples over time in order to
observe the stability of binding of the low-affinity ligand and degree
of displacement by the competitor ligand. Typically, time intervals
of 0, 4 h, 8 h, 24 h, and 48 h are used, although this depends on the
total expected duration of the NMR-observed fragment screen.
If stable binding is not observed, it may be necessary to optimize buffer conditions (or possibly modify the protein construct)
in order to increase the stability of the system. It is also possible to
screen the fragment in library in subsections such that each experiment duration is relatively short; although this increases the overall
duration and workload of the screen, the investment is often worthwhile for the increased quality of the data.
If stable competition is not observed, the stability of the competitor molecule should be examined—this is the most commonly
observed cause of variable degrees of competition. Degradation
over time of the competitor molecule can readily lead to decreased
levels of displacement, and once identified, this is readily overcome
by preparation of fresh samples of the competitor.
3.3.4 Trial Screen
After identifying conditions where the binding of low-affinity fragments can be readily observed, along with displacement by a potent
competitor molecule, it is productive to run a “trial” screen of
100–200 fragments. This screen is run in order to identify any
issues regarding the stability of protein or fragments to optimize
experimental parameters and to determine an approximate hit rate
for the target. Issues identified with the trial screen should be
rectified before proceeding to the full fragment screen.
Fragment Screening by NMR
259
of the Tool Compound by
a Potent Competitor
Once binding of a low-affinity ligand has been observed, a potent
competitor can be used to displace the ligand. The concentration of
competitor required for full displacement of the low-affinity ligand
should be determined by titration.
If no potent competitor molecule is available, the screen can
still be acquired. However, the rate of false positives is likely to be
significantly higher than is the case where a competition step is
possible. This will increase the overall hit rate and will also increase
the burden on subsequent activities such as singleton validation and
validation using orthogonal biophysical techniques. Where no
competitor ligand is available, the stringency of subsequent validation assays must be high in order to reduce the number of false
positives diluting the pool of fragment hits (see Subheading 3.8).
3.3.3 Stability of Binding
and Competition
Having determined the concentration of competitor required in
order to observe displacement of the low-affinity tool compound, a
number of samples (typically 4 or 5) containing protein and the
low-affinity tool compound should be prepared. The competitor
should then be added to different samples over time in order to
observe the stability of binding of the low-affinity ligand and degree
of displacement by the competitor ligand. Typically, time intervals
of 0, 4 h, 8 h, 24 h, and 48 h are used, although this depends on the
total expected duration of the NMR-observed fragment screen.
If stable binding is not observed, it may be necessary to optimize buffer conditions (or possibly modify the protein construct)
in order to increase the stability of the system. It is also possible to
screen the fragment in library in subsections such that each experiment duration is relatively short; although this increases the overall
duration and workload of the screen, the investment is often worthwhile for the increased quality of the data.
If stable competition is not observed, the stability of the competitor molecule should be examined—this is the most commonly
observed cause of variable degrees of competition. Degradation
over time of the competitor molecule can readily lead to decreased
levels of displacement, and once identified, this is readily overcome
by preparation of fresh samples of the competitor.
3.3.4 Trial Screen
After identifying conditions where the binding of low-affinity fragments can be readily observed, along with displacement by a potent
competitor molecule, it is productive to run a “trial” screen of
100–200 fragments. This screen is run in order to identify any
issues regarding the stability of protein or fragments to optimize
experimental parameters and to determine an approximate hit rate
for the target. Issues identified with the trial screen should be
rectified before proceeding to the full fragment screen.
Fragment Screening by NMR
259