Chemistry Reference
In-Depth Information
It is a boon because the chip technology of SPR lends itself to miniaturization and thus
great increases in throughput and decreases in material consumption.
X-ray cry
s
tallography is the most commonly used indirect biophysical tech-
nique.
[
61, 62, 70 73
]
It has the potential to yield very high-resolution atomic data of the
ligand-target complex, but that is reliant upon obtaining high-quality crystals; this is
not a trivial task. Certain classes of targets, such as membrane proteins, are simply not
amenable to routine analysis by X-ray crystallography. If a ligand-targe
t
complex can
be obtained, the resultant data are a crucial feed for the SBDD process,
[
74 77
]
which also
includes the multitude of
in silico
methods. As noted earlier, sometimes the high-resolution
structure of a surrogate target can be used as an entrée into the SBDD process. Iso-
thermal titration calorimetry (ITC) is the other popular indirect biophysical method.
[
78, 79
]
It is especially powerful for FBDD because it measures the thermodynamic paramet-
ers of the system. This information must then be used to infer ligand-target binding
information. However, this can be vital in hit confirmation and SAR evaluation.
[
65, 80
]
In hit confirmation, ITC can discriminate 'bad actors', especially those that aggreg-
ate. During SAR evaluation, ITC can monitor enthalpic and entropic changes in the hit
series and determine if the line of SAR is proceeding in the right direction. The most
commonly used indirect screening technique is the biochemical screen.
[
81
]
Discussion
of this topic is well beyond the scope of this chapter; we simply point the reader to a
recent review.
[
82
]
An interesting, new method to screen for ligand-target interactions involves protein
stability measurements.
[
83
]
The ability of ligand binding to enhance protein stability is a
well-recognized phenomenon. The degree of stabilization can be systematically probed
by observing the increased resistance of a protein to chemically or physically denaturing
conditions, such as urea or temperature, in the presence of compounds. Many methods are
able to monitor the extent of denaturation, e.g. specific enzyme activity, NMR, circular
dichroism (CD) and, most recently, extrinsic fluorescence using a probe that binds select-
ively to unfolded protein.
[
83, 84
]
Care still must be exercised here as there are cases in which
ligand biding actually causes protein stability to decrease.
[
85, 86
]
Alex and Flocco, in a superb paper, have reviewed the pipeline of fragment-derived
molecules.
[
87
]
In their analysis, they divide the FBDD approaches into three broad categor-
ies: linking, expansion and assembly. In terms of the three types of fragment approaches
we define (see below), linking and assembly are the same and expansion corresponds
to our 'anabolic' approach. There were data for 51 projects that we further analyzed for
increase/decrease in ligand efficiency, the type of target (kinase, protease, other) and the
primary method used (NMR, X-ray, other) (Tables 2.2 and 2.3). Of these 51 projects,
23 used linking/assembly as their hit-to-lead strategy and 28 used the anabolic (expansion)
approach. Of the 23 linking/assembly projects, most (88%) resulted in a compound that
upon optimization was less ligand efficient. For the 28 anabolic projects, slightly more than
half (54%) resulted in less ligand-efficient compounds. From this, it can be concluded that
the anabolic approach is better at maintaining ligand efficiency through the LG process
compared with linking/assembly. Breaking down the hit-to-lead (H2L) strategy by screen-
ing method reveals the two main biophysical methods (NMR and X-ray) and a catch-all
category (other). In both H2L strategies, NMR has the highest rate of producing more effi-
cient lead compounds. In Table 2.2, we survey the rate of projects that maintain or increase
ligand efficiency in going from hit to lead. NMR again has the highest rate of maintaining or
