Biomedical Engineering Reference
In-Depth Information
bound to a hinge region between the dimers.
61
The use of perdeuterated
protein and assignment of the intermolecular NOEs gave a well-defined
solution structure which consisted of a 1 : 1 complex of protein and inhibitor.
61
The solution structure suggested vectors for elaboration/improvement of this
non-phosphate containing ligand. The protein Mesencephalic astrocyte-
derived neurotrophic factor (MANF) provides a second interesting example
of a significant difference between the crystal and solution structure.
62
Where
the crystal structure indicated a disordered C-terminal domain,
62
the NMR
structure clearly demonstrated the existence of a SAP domain.
62
Interestingly,
the SAP domain of the protein Ku70 inhibits the pro-apoptotic activity of
Bax.
62
A similar role for the SAP domain of MANF would be consistent with
the demonstrated ability of MANF to protect neurons of rats.
62
When a
crystal structure is not sufficient to observe loops due to high disorder, NMR
can become an asset, especially when important residues for activity are
located in these loops. In one example, two inhibitors of the proline-rich kinase
(PYK2) were thought to stabilise the relatively rare DFG-out, inactive form of
the kinase.
63
As with most kinases, the DFG loop was not visible in the crystal
structure of the protein-ligand complex.
15
N-labelling of phenylalanine
residues was used in conjunction with TROSY-HSQC to demonstrate changes
in the spectrum consistent with the expected rigidification of the DFG loop
upon stabilisation in the out conformation.
11.5 In-Cell NMR Spectroscopy
In-cell NMR spectroscopy holds great potential as a tool to discover and
validate the interaction of small molecules with pharmaceutical targets in a
natural milieu. We make no attempt to comprehensively review this field here
(we refer the reader to a recent review on the subject
64
), but rather focus on
two recent developments that we feel are important for drug discovery.
STINT-NMR was developed as a method to study protein-protein
interactions inside living bacterial cells using NMR spectroscopy.
65
This
method uses sequential expression of the two proteins whose interaction isto
be studied. The target protein is first expressed using
15
N-labelled media, in
order to obtain a [
15
N,
1
H] HSQC spectrum inside the living cell. The growth
medium is subsequently changed and the interacting protein is overexpressed
without labelling. The HSQC spectrum of the target changes upon increasing
concentration of the interacting protein. Screening small-molecule interactor
libraries (SMILI-NMR) uses STINT-NMR to screen libraries of small
molecules for protein-protein interaction inhibitors with activity in the cell.
66
SMILI-NMR represents the first effort to use in-cell methods to screen
compound libraries. Combined with the methods described below, this could
prove
to
be
a
powerful
method
to
combine
biophysical
sensitivity
with
biological activity.
While the initial in-cell NMR experiments used isotope-labelled proteinsin
E. coli,
67
for drug discovery it is critical to assess small-molecule functions in
