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on our previous studies with residue type-specifically
1
H-labeled kinaseX samples. In total,
20 ligand-protein peaks have been observed in this spectrum.
In the examples above, lead-like inhibitors of kinaseX with sub-micromolar affinities
were studied.We have recently combined residue type-specific labeling (
1
H/
13
C/
15
N-labeled
amino acids incorporated into a
2
H/
15
N background) with 3D X-filtered NOESY studies at
elevated temperatures to characterize a fragment-like compound bound to kinaseX. Protein-
ligand NOEs have also been observed in this case (data not shown). Due to the extreme
conformational plasticity of protein kinases,
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adequate sampling of protein conforma-
tional space is crucial for applying pose generation and NOE matching to kinase-inhibitor
complexes, including those involving kinaseX. As described earlier in this chapter, pose
sampling (including protein conformational sampling) is an ongoing area of research in our
group and elsewhere. Flexibility is known to be a significant challenge for kinase-inhibitor
docking procedures.
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As we have shown for kinaseX, NOEs between kinases and inhib-
itors can readily be observed. Protein NMR assignments can be obtained for some of these
interactions without undertaking a full sequential assignment of the protein. NOE data
can provide detailed information on the location and orientation of inhibitor moieties (e.g.
hinge-binding cores) that interact with the relatively rigid regions of kinases. In general, an
accurate pose should be consistent with all of the observed NOE data; therefore, we expect
enhanced NOE measurements and NOE matching to play significant roles in evaluating
models of fragments and leads bound to large, flexible proteins.
5.10 Conclusion
In the pharamaceutical industry, NMR spectroscopy has demonstrated itself to be a power-
ful, highly versatile tool that has impact throughout the drug discovery process. NMR is
frequently used as an assay to screen compound collections, to facilitate the assessment
of hits, and to provide detailed structural and dynamical characterization of protein-ligand
complexes. Because NMR can provide information in discrete units, the spectroscopist can
“fine tune'' data collection strategies.
The application of NMR to the characterization of biomolecular structures has, in most
cases, followed bottom-up approaches
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wherein discrete pieces of information (reson-
ance assignments, NOE contacts, specific dihedral angle restraints, inter-atomic vector
orientations within some reference frame, etc.) are gathered and finally used to define a
consistent ensemble of structures. In some situations, this aspect of biomolecular NMR
is a great advantage, since minimal information may be all that is needed to answer the
specific question at hand, e.g. one may want to know if a particular aromatic ring on the
ligand interacts with an aromatic ring from the protein. In other situations (complete struc-
ture determination, binding pose determination, etc.), the piecewise aspect of NMR is a
disadvantage since, even with automation (reviewed in ref. 53), the bottom-up process of
NMR-based structure determination is very time and resource consuming.
There have been efforts to utilize NMR data in top-down approaches for structure
determinations. Perhaps the most ambitious protocol, and the one that is most closely
analogous to X-ray crystallography, is the CLOUDS method.
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In this approach, an
unassigned 2D NOESY spectrum is transformed into a 'proton density' via relaxation
