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However, the use of a biochemical assay in the primary fragment screen is likely to lead
to a comparatively high rate of false positives. The control of the assay system is much
lower than with a method such as NMR and also the assay system in itself is more complex.
There is no information on the state of the assay components, such as solubility and integrity
of the compounds, or on the state of the protein. Further, the risk of compound-induced
fluorescence artifacts increases with the very high fragment concentrations necessary. On
the other hand, conjugated unsaturated systems are present to a lesser extent in fragments
than larger compounds. Another drawback is that fragments binding in the vicinity of the
active site without modulating the function or displacing the tagged ligand will not be
detected. Such fragments are very interesting since they provide information on how to
expand fragments binding to the active site.
4.3.5 X-ray Cystallography
Recent technological advances have led to automation of most steps in the protein crys-
tallographic process. The development of crystallization robotics and the automation of
data collection, processing and analysis have enabled X-ray crystallography to be used as a
primary screening technique.
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Cocktails of fragments are soaked into preformed crystals
of the target protein. The fragments in a cocktail should preferably be distinguishable by
shape in order to avoid deconvolution. The fragment concentration must be very high, not
only because the fragments bind weakly, but also since the protein concentration in the
crystal typically is on the order of 10 mM. The advantage of using X-ray crystallography as
the primary screening technique is that detailed structural information is obtained directly.
However, large amounts of target protein are necessary and the throughput is lower than
with the other techniques. It is much more efficient to use another method, such as NMR,
to 'prescreen' fragment cocktails. The soaking attempts will then be performed on binding
fragments only.
4.3.6 Choice of Technique for Primary Fragment Screening
In our opinion, the most reliable and versatile primary screening techniques are NMR
methods. X-ray crystallography will in general be necessary in a fragment screening cam-
paign, but is much more efficient to employ after the initial fragment screening. Also, a
biochemical activity assay to measure potency will be needed at some stage in the devel-
opment of the fragment hits. When used for primary screening, the comparatively high
number of false positives will make the subsequent crystallography work less efficient,
unless a biophysical binding assay such as NMR is employed as a filter in between. It may
be more suitable to apply a biochemical activity assay when the primary fragments have
been developed to slightly larger compounds with a higher affinity.
4.4 Practical Aspects of NMR-based Fragment Screening
4.4.1 Fragment Library
In our experience, typical hit rates for fragment libraries when using ligand-detected NMR
techniques are on the order of 2-10%. Consequently, the fragment library used for the
