Biomedical Engineering Reference
In-Depth Information
chemical environment of a nucleus is a ubiquitous probe that is readily and
simply available. In practice, the chemical shift is most often used in
conjunction with NMR spectroscopy of the target (protein- or target-observed
NMR), where it can not only differentiate specific, reversible binding from
artefactual interactions, but, given the availability of the sequential assign-
ment, can define an approximate binding site. Protein-observed methods
typically require isotopic labelling placing certain limitations on the applic-
ability of the method. The very large difference in relaxation behavior between
a small molecule and a protein is typically exploited to characterise binding by
observing changes in the NMR spectrum of the small molecule (ligand
observed NMR). Ligand-observed methods require substantially less protein,
which need not be labelled, and may be more sensitive than protein observed
methods, but may be more prone to artefacts.
Due to the demonstrated utility of NMR for drug discovery, there continues
to be developments in the field that are both evolutionary and revolutionary.
In this review we highlight selected recent accomplishments in the use of NMR
for finding and characterising small molecule ligands primarily during the very
early stages of pre-clinical drug discovery. In addition, exciting developments
in detecting molecules binding to targets inside living cells will also be
presented. In this chapter, the emphasis is made on the newer technical
development and functional insights obtained in recent years.
11.2 NMR for Ligand Discovery
At present, ligand discovery and characterisation comprise the major use of
NMR in the drug-discovery process. Despite the fact that NMR has been used
for more than 15 years in this role, interesting new methods continue to be
developed and exciting results continue to flow. Interestingly, a recent poll on
Practical Fragments (http://practicalfragments.blogspot.com/) suggests that,
including both protein- and ligand-observed techniques, NMR is the most
frequently used method of fragment screening.
11.2.1 Protein-Observed NMR
Protein-observed screening is the only NMR method which provides
information on the ligand binding site as well as the mode of binding to the
protein. Despite the fact that it was introduced 15 years ago,
2
protein-observed
NMR remains the gold standard for identifying weak, yet specific binding of
fragments to targets and differentiating these from non-specific or artefactual
interactions. The very nature of the technique introduces some limitations such
as the requirement for isotopic labelling and the restriction to small-medium
sized proteins. However, advances such as the introduction of TROSY,
3
particularly in conjunction with selective labelling schemes that introduce
NMR-visible isotopes only at e.g., methyl groups,
4
have allowed complete
backbone assignment and ligand screening of considerably larger proteins.
