Chemistry Reference
In-Depth Information
affinities (sometimes in the millimolar range), they are difficult to detect in typical HTS
assays where compound libraries are usually screened at low micromolar concentrations.
Additionally, with increasing targeting of nonactive site compounds, the possibility of easily
read activity is low. In addition, functional screening (especially at high concentrations)
is subject to a number of pitfalls.
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However, biochemical data are inherently orthogonal
to biophysical data and hence is an extremely useful type of data to have and, whenever
possible, should be collected for all fragment-based screens. The greatest downside is that
one must know the function of the target to develop the screen.
Biophysical techniques.
In order to use fragments effectively in drug design, experimental
methods are required that can rapidly and reliably screen up to thousands of low-MW test
compounds for weak binding to the target protein. The most robust screens for fragments
are binding assays (Table 2.1). Detection of binding to the target by fragments is the key to
successful FBDD. The initial stages of FBDD can efficiently proceed without functional
activity, but to exit from the LG phase functional activity must be correlated with binding
activity. X-ray crystallography,
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NMR, surface plasmon resonance (SPR),
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fluores-
cence spectroscopy,
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MS
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and isothermal titration calorimetry
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have also been used
alone or as part of fully integrated FBDD effort to deliver initial fragment hits. These meth-
ods can be divided into two groups: indirect methods which include low-resolution X-ray
crystallography,
3
various fluorescence methods and isothermal titration calorimetry and
direct methods which include SPR, MS, high-resolution X-ray crystallography and NMR
spectroscopy. Direct methods (i.e. those that directly measure ligand-target interactions)
are obviously superior. NMR is the most utilized direct method in FBDD because the pro-
tein does not need to be modified for analysis (like SPR), the samples can mimic the native
environment as closely as needed (unlike MS), either ligand-based or target-based NMR
experiments can be used to deliver data
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and the throughput can be relatively high
(hundreds of compounds or more a day). NMR, however, is not inexpensive, spectrometers
cost portions or multiples of millions of dollars, upkeep can be costly and it requires expert
users for both analysis and maintenance. Also, in many instances, there is little institutional
knowledge of how to interpret and utilizeNMRdata to progressmedicinal chemistry efforts.
This institutional inertia can be very difficult to overcome and quantitative measurement of
NMR's contribution is impossible. Once all of these hurdles are overcome, NMR proves
to be a most powerful drug discovery tool.
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The other two main direct methods (SPR and MS) have been shown to be vital tools in
FBDD.
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These are less commonly utilized as each method has its own caveats. MS
needs to have the sample in a volatile buffer (commonly ammonium acetate). The use of
adjuncts and co-solvents is thus severely curtailed for MS; proteins that require them for
stability will not be amenable to MS screens. SPR has its own needs,
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most notably the
need to have the target immobilized. This is both a bane and a boon. It is a bane because,
depending on the immobilization technique chosen, the target could be adversely affected.
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X-ray crystallography is both an indirect and direct method because the quality of the model used to fit the electron density
depends on the resolution of the data. At lower resolution, the fit between the model and the density may be good (in R-free
terms), but lacking details in interactions between target and ligand due to poor or lacking electron density. Hence any interactions
are inferred and not directly measured. At high resolution, the electron density is of such high resolution that side-chains can be
unambiguously placed in the structure, in addition to the ligand. In this case, X-ray crystallography is a direct method.
