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is usually optimized by testing close analogues with no substantial increase in molecular
weight. In the following iterations, the fragments become larger either by expansion of the
fragment or by linking or merging different fragments.
The expansion strategy is the most generally applicable. Optimized fragments are expan-
ded with features (ring systems, functional groups) that interact with the target protein in
a favorable way without disrupting the binding of the core fragment. Selection of appro-
priate expansions is based on affinity (from a biophysical method) and/or potency (from a
biochemical activity assay) and on structural information (from X-ray crystallography or
NMR), and also binding hypotheses frommodeling. The SAR obtained from each iteration
is used for the selection of the next generation of molecules, which subsequently is tested
for affinity and/or potency to the target protein in order to confirm or reject the direction of
the design. In a successful iterative design cycle supported by detailed structural inform-
ation, the process converges rapidly to more potent and complex molecules with
r
etained
high ligand efficiencies. There are many published examples of this approach.
[
4, 20 31
]
An elegant approach is to identify fragments binding to separate but adjacent sites on
the target protein and then link the fragments into one high-affinity compound. For the full
potential of the linking to be fulfilled in terms of potency, the relative orientations of the
bound fragments that are to be linked must be determined and the linker must be designed
so that it preserves the relative orientations of the bound fragm
e
nts. This is far from a trivial
task but several successful cases have been published.
[
4, 29, 32 39
]
An interesting variant is
so-called 'self-assembly'where reactive fragments link together to form an active inhibitor
in the presence of the protein target.
[
40, 41
]
Instead of linking, fragments with overlapping
but not identical binding sites can be merged into one hybrid molecule.
[
42 44
]
A variation of the schematic workflow in Figure 4.2 is to combine virtual screening
of compou
n
d databases and experimental verification by a biophysical method, usually
NMR.
[
21, 45 47
]
An additional use of fragment screening is to identify fragments that can
replace part of an existing lead compound responsible for a non-desired characteristic such
as low solub
i
lity, poor bioavailability, high albumin binding, metabolic instability or lack
of novelty.
[
48 50
]
4.3 Techniques for the Primary Fragment Screen
The function of the primary screening is to identify good starting points, i.e. compounds
that are chemically attractive (in terms of 'synthetic handles' and/or with many analogues
available) and have a high likelihood of success in co-crystallization experiments (or struc-
ture determination of ligand-target complexes by NMR) with the target macromolecule.
The requirements of the assay to be used in the primary fragment screen differ substantially
from the requirements of an assay used in HTS. Most importantly, the assay should be
able to reliably detect weakly binding fragments. Further, it should be possible to apply
the assay (or a selection of assays) to a wide range of target proteins with a minimum of
assay development. Due to the relatively small sizes of fragment libraries, questions of
throughput and reagent consumption are much less important compared with HTS. Differ-
ent types of assays for the primary fragment screen along with their respective strengths and
weaknesses are listed below. The amount of protein necessary for the primary screening
differs between the techniques. However, in many cases the success of fragment-based
