Biomedical Engineering Reference
In-Depth Information
NMR relaxation can also be enhanced by use of paramagnetism, i.e., the
interaction between unpaired electrons and the nuclear spin. Two types of
interaction can be defined depending on the nature of the paramagnetic center.
Isotropic centres give rise to a purely distance-dependent increase in the
transverse relaxation rates of nuclear spins (paramagnetic relaxation enhance-
ment or PRE) where anisotropic centres tend to cause a shift in the resonance
frequency of spins (pseudo-contact shift or PCS). If a paramagnetic centre(a
spin-label) is attached to a protein at a defined site, it is possible to measure
distance-dependent and/or angle-dependent effects on the NMR spectrum ofa
ligand that is bound in the vicinity of the spin-label. The first ligand-screening
technique to take advantage of a spin-label was called SLAPSTIC (Spin Labels
Attached to Protein Side chain as a Tool to identify Interacting
Compounds).
16
The use of a spin-label for screening not only enhances the
sensitivity of the method, it enhances the specificity and is selective for a small
binding site in proximity to the spin-label. This latter property was put to use
to find ligands specific for the hydrophobic binding pocket of the HIV-1 fusion
protein gp41.
17
Here the spin-label was attached to the N-terminus of an
engineered a-helical peptide derived from the C-terminus of gp41. The peptide
was truncated such that the hydrophobic binding site on the target remained
open for small-molecule binding. By measuring both PRE and PCS effects, the
authors could determine structural constraints to include in-docking proce-
dures leading to a model of the ternary complex. Similarly, a lanthanide-
binding peptide was fused to the SH2 domain of Grb2 where proof-of-concept
data demonstrated the feasibility of the system for screening for weakly
binding, small molecules.
18
The authors then used PCS to elucidate the
structure of a complex of a phosphotyrosine peptide in fast exchange with the
SH2 domain and a non-peptidic inhibitor in slow exchange. In both cases the
correspondence between the structures determined using paramagnetic effects
and one based either on X-ray crystallography or NOEs was reasonable. The
power of the system lies in the specificity of the screen and the ability to rapidly
get structural information from the same system.
The Pellecchia group has recently published
19
an interesting twist on
paramagnetic labelling that is reminiscent of the earlier work from Novartis.
16
The group aimed to develop inhibitors of the protein tyrosine phosphatase of
Yersina pestis. A spin-labelled phosphotyrosine mimetic was developed and a
library of approximately 500 low molecular weight compounds was screened
for proximal binding. Eight positives from the screen were then linked to the
phosphotyrosine mimetic to yield a series of novel bidentate compounds with
low or even sub-mMIC
50
s against the YopH phosphatase and with activity in a
cell-based assay.
11.2.2.2 Magnetisation-Transfer Methods
Methods based on the transfer of magnetisation from or via the target are the
most
popular
in
drug
discovery
as
they
are
applicable
to
large
protein
