Samples for the trial screen are prepared as relatively small
cocktails of 4–8 compounds per mixture, and the LO-NMR experiments acquired on each sample. The competitor molecule is then
added to each sample, and the LO-NMR experiments reacquired
on the samples containing competitor. Analysis of the trial screen is
then performed (see Subheading 3.5), and the number of putative
hits (which show binding and displacement) is calculated.
Typically, a hit rate of 2–10% is observed for most targets. If the
hit rate is below 2%, conditions and/or experimental parameters
should be optimized using a low-affinity tool compound; at this
point, a preliminary fragment hit can be used as a low-affinity tool,
although further biophysical validation is advised if this is to be the
case in order to reduce the risk of being misled by experimental
artifacts. If the hit rate is above 10%, it may be worthwhile increasing the stringency of the screen (for example, by reducing the
concentration of protein and/or fragments) so as to identify only
apparently higher affinity hits and avoid creating a large pool of
fragments requiring additional downstream validation.
It is important to consider at this point where the putative hits
are located in the course of the screen. If the rate of identified
binders is higher at the beginning of the screen than the end, this
can be a strong indication that the system is not stable and further
experimental optimization is required. Similarly, an increased rate
of noncompetitive binders toward the end of a screen can be an
indication of protein degradation or other instability.
The trial screen is also useful for determining the number of
compounds that should be present in each mixture in the screen. If
the hit rate is high, relatively few compounds (for example, 4–6)
should be included in each mixture in order to reduce the number
of samples that contain more than one putative hit. If the hit rate is
low, the number of compounds per mixture can be increased (for
example, to 8–12).
3.4 Screening
the Library
After running the trial screen, the following points should be
confirmed:
1. Low-affinity fragments can be observed to bind.
2. These fragments are (in some or all cases) displaced by a potent
competitor.
3. The degree of binding and competition is stable over time.
4. The hit rate is constant over time.
5. The mixture size is appropriate for the observed hit rate.
If these points are valid, the full library should be screened
under these conditions.
Samples should be prepared (see Note 1), and NMR experiments acquired under automation. Details of the experimental
acquisition will obviously vary according to both the specific
260
Ben J. Davis
cocktails of 4–8 compounds per mixture, and the LO-NMR experiments acquired on each sample. The competitor molecule is then
added to each sample, and the LO-NMR experiments reacquired
on the samples containing competitor. Analysis of the trial screen is
then performed (see Subheading 3.5), and the number of putative
hits (which show binding and displacement) is calculated.
Typically, a hit rate of 2–10% is observed for most targets. If the
hit rate is below 2%, conditions and/or experimental parameters
should be optimized using a low-affinity tool compound; at this
point, a preliminary fragment hit can be used as a low-affinity tool,
although further biophysical validation is advised if this is to be the
case in order to reduce the risk of being misled by experimental
artifacts. If the hit rate is above 10%, it may be worthwhile increasing the stringency of the screen (for example, by reducing the
concentration of protein and/or fragments) so as to identify only
apparently higher affinity hits and avoid creating a large pool of
fragments requiring additional downstream validation.
It is important to consider at this point where the putative hits
are located in the course of the screen. If the rate of identified
binders is higher at the beginning of the screen than the end, this
can be a strong indication that the system is not stable and further
experimental optimization is required. Similarly, an increased rate
of noncompetitive binders toward the end of a screen can be an
indication of protein degradation or other instability.
The trial screen is also useful for determining the number of
compounds that should be present in each mixture in the screen. If
the hit rate is high, relatively few compounds (for example, 4–6)
should be included in each mixture in order to reduce the number
of samples that contain more than one putative hit. If the hit rate is
low, the number of compounds per mixture can be increased (for
example, to 8–12).
3.4 Screening
the Library
After running the trial screen, the following points should be
confirmed:
1. Low-affinity fragments can be observed to bind.
2. These fragments are (in some or all cases) displaced by a potent
competitor.
3. The degree of binding and competition is stable over time.
4. The hit rate is constant over time.
5. The mixture size is appropriate for the observed hit rate.
If these points are valid, the full library should be screened
under these conditions.
Samples should be prepared (see Note 1), and NMR experiments acquired under automation. Details of the experimental
acquisition will obviously vary according to both the specific
260
Ben J. Davis