Chemistry Reference
In-Depth Information
labelling of protons from methyl groups of valin, leucin and isoleucine. This
approach significantly reduces the complexity of the resulting HSQC spectra and
increases the sensitivity due to the presence of three protons in methyl groups
versus
a single proton in the NH groups. In addition, the favourable relaxation
properties of methyl groups also provide the application of this methodology to
larger proteins [3]. It is important to note that HSQC-NMR can also contribute to
understand the kinetics of complex formation, once the line shapes of peaks in an
HSQC spectrum corresponding to the free and bound states are sensitive to on and
off rates, especially
k
off [18, 22].
In general, NMR screening methods that monitor the macromolecular target are
known as “SAR by NMR” (Structure-Activity Relationship by Nuclear Magnetic
Resonance) technique, which is based on the use of chemical shift changes to
screen low-affinity ligands, in combination with structural information to direct a
linked-fragment approach for achieving binding affinity enhancement [15].
The utility of the SAR by NMR methodology for pharmaceutical research has
been demonstrated in a number of systems, including FK506 binding protein
(FKBP), metalloproteinase stromelysin [23], and DNA-binding E2 protein from
papilloma virus. In the first two cases, compounds with nanomolar affinity were
developed by covalently linking fragment molecules with micromolar affinities.
The fragment molecules were identified as leads using the SAR by NMR method.
The tethering of these two compounds was based on the NMR determined binding
sites and known structural information from protein. As an example, a 15 nM
inhibitor of stromelysin was discovered by the covalent joining of two weak
binders, acetohydoxamic acid (K
D
= 17 mM) and 3-(cyanomethyl)-40-
hydroxybiphenyl (K
D
= 0.02 mM) [24].
Ligand-Based NMR Methods
Ligand-based NMR methods are based on the main difference that exists between
protein and ligand,
i.e.
size and volume. Small molecules commonly used as
protein inhibitors have molecular masses below 1 kDa, whereas macromolecular
proteins have masses usually larger than 10 kDa [25, 26]. These clear differences
suggest that it is possible to watch for changes in the ligand NMR relaxation
parameters in order to investigate binding.
