Chemistry Reference
In-Depth Information
drug discovery projects is critically dependent on structures of ligand-target complexes to
drive the chemistry. If a successful development of fragments to potent inhibitors is defined
as reaching a potency
<
100 nM, statistics fromAbbott Laboratories show that the success
rate increases from 33% to 93% with the aid of structure-based design.
[
4
]
Thus, milligram
amounts of target protein will in principle always be required, which renders the differences
in protein consumption between the primary screening techniques insignificant in practice.
Different techniques require different degrees of ligand occupancy of the target protein
for a binding event to be reliably detected. One-dimensional (1D) ligand-detected NMR
techniques are very sensitive in this respect, where the binding fragment needs to bind to
only a very small fraction of the target protein molecules for binding to be detected. For
example, a fragment concentration of 20-40 M in a transverse relaxation-filter experi-
ment (see below) is sufficient to reliably detect a weak binding event with a
K
d
of several
hundred M, depending on the rotational correlation time of the target protein. This trans-
lates to a required ligand occupancy of
<
10% with the ligand occupancy (
p
B
) defined
as
p
B
K
D
).
[
6
]
Two-dimensional (2D) protein-detected NMR methods and a
biochemical assay require higher ligand occupancies for reliable detection and X-ray crys-
tallography requires nearly 100% ligand occupancy. Hence the fragment concentrations
and consequently the solubility requirements of the fragments will be much higher when
using a biochemical assay or X-ray crystallography compared with ligand-detected NMR
techniques for the detection of fragments with a given affinity. This partly explains why it
is usually not possible to obtain crystal structures of fragment-target complexes from all
fragment hits detected by ligand-detected NMR techniques.
=
[L]/([L]
+
4.3.1 NMR
NMR techniques have dominated fragment screening from the beginning. The wide choice
of different ligand-detected NMR techniques is especially suited to primary fragment
screening. Very weak binders are reliably detected and these generic binding assays can
be applied to a wide array of different therapeutic targets. Further, there are no serious
limitations on buffer choice and information on compound solubility and integrity, and
also on the state of the target protein, is obtained as a part of the experiment. The main
disadvantage is that nonspecific binders will also be picked up, but they can be filtered out
by competition experiments using known, specific binders. The greatest advantage with
protein-detected NMR techniques is that it is possible to obtain direct information on the
binding site. The drawbacks include that it is necessary to produce large amounts of isotop-
ically labeled target protein and the difficulties associated with target proteins of molecular
weights above 30 kDa. A survey of NMR techniques for fragment screening is given later
in this chapter.
4.3.2 Nondenaturing Electrospray Ionization Mass Spectrometry (ESI-MS)
Reversible (noncovalent) binding events can be detected with nondenaturing ESI-MS.
[
51 54
]
A considerable advantage of the method is that the stoichiometry of the binding event is
measured directly. As a consequence, it is possible to observe directly if two fragments bind
simultaneously. There is also direct information on compound solubility and integrity, in
addition to the state of the target protein. The method suffers, however, from a few major
