Biomedical Engineering Reference
In-Depth Information
intra-ligand NOE information can then be used to determine the conformation
of the ligand in the bound state. This method is applicable to large proteinsof
which only small amounts are available, but requires relatively high compound
concentrations and therefore the compounds must be quite soluble. TrNOE
has been used to determine the conformation of the bound form of weak
inhibitors of MurD ligase, an enzyme that contributes to the formation of
peptidoglycan.
49
Epitope mapping was carried out using STD and the NMR
information was used to restrain molecular dynamics calculations generating
models of the protein-ligand complex. Although crystal structures of the
complex were available, they did not explain the weak protein-ligand
interaction. The NMR experiments suggested that the ligands are highly
dynamic in the bound state, potentially explaining the unexpectedly low
potency.
If the protein can be isotopically labelled, group-selective STD can provide
information on the types of
1
Hs on the protein that are close to
1
Hs of the
ligand. This approach has been elegantly used to map the carbohydrate
binding site of galectin 1.
26
Protein amide resonances can be directly and
efficiently saturated, while ligand resonance can be eliminated, if necessary,
through a half-filter. However, due to the relative size of the STD signal and
the natural abundance of
13
C, carbon-attached
1
Hs must be selectively
saturated to avoid artefacts. In principle, if the structure of the target is known,
restraints derived from such a study could be used to guide molecular-docking
efforts even in the absence of the sequential assignment.
The chemical shift is a readily available parameter that contains information
on not only the chemical environment, but also the conformation about a
nucleus. As noted, perturbations to the NMR spectrum of a protein have long
been used for detecting binding of e.g., small molecules. Returning to the work
on Abl kinase, differences in the chemical shift of Val 525 were correlated with
the presence of myristate in its binding pocket and the attendant activation of
the enzyme.
34
By selectively
15
N-labelling valine residues of Abl, the authors
developed a conformation-specific assay that could be used to detect allosteric
ligands and differentiate agonists from antagonists. The assay was used to
screen hits for their ability to induce the same active conformation as
myristate. This assay confirmed the activity of the known allosteric modulator,
GNF-2. However, GNF-2 had liabilities related to non-specificity.
34
The assay
was then used to discover novel allosteric agonists of Abl.
Unfortunately, despite numerous efforts, it is not yet possible to directly
convert chemical shift information into structural constraints. However, there
have been significant achievements in using protein CSPs to guide docking.In
one example, amide CSPs from an [
15
N,
1
H] HSQC were used to guide the
docking of compounds to the b2subunit of the transcription factor CBF.
50
Thirty five compounds had been selected from in silico screening amongst
which four were validated in the HSQC experiment. After directed-library
synthesis two, more potent, compounds were selected for detailed experimen-
tally guided docking. The docking suggested two possible orientations for the
