Chemistry Reference
In-Depth Information
reconstituting milligram amounts of therapeutically interesting integral membrane pro-
teins, e.g. GPCRs, membrane-bound enzymes (e.g. tyrosine kinases) and ion channels. In
recent years, the number of crystal structures of integral membrane proteins has increased
dramatically; especially notable is a second high-resolution GPCR structure.
[
135
]
This is
mainly a consequence of the impressive progress in the protein production process
[
136
]
and
leaves good hope that integral membrane protein targets will be subjected to fragment-based
screening by NMR and structure-based drug design in the very near future.
The integral membrane proteins that will prove most suitable for fragment screening by
NMR can be predicted (i) to be possible to produce in milligram quantities and reconstitute
in liposomes and (ii) not to contain large amounts of detergents in the binding pocket of
interest, i.e. the binding pocket should be hydrophilic. If the binding pocket is lipophilic,
the fragments will probably not be able to displace the detergent molecules that could bind
with relatively high affinity. Amethod that appears to be very promising for target proteins
that are difficult to produce or that are insoluble, such as membrane proteins, is called
TINS (target-immobilized NMR screening).
[
137, 138
]
Binding is detected by comparing
1
H
1D spectra in the presence and absence of the target protein that is immobilized on a solid
support. The compounds are pumped through a dual flow cell and binding is detected as a
simple reduction in ligand peak height in the cell with target protein present. The binders
are then washed off and the experiment is repeated with the next fragment cocktail using
the same immobilized protein. Hence only a single protein sample is required to screen the
fragment library. The use of paramagnetic spin labels, attached either to the target protein
in the vicinity of the binding pocket of interest or to a known ligand, can also be predicted
to play a role in fragment screening of membrane proteins.
4.8 Acknowledgements
I am grateful to many colleagues, past and present, for providing a stimulating and fun
environment for the daily work. Thanks are due to everyone at iNovacia and all those
from the former Structural Chemistry Department at Biovitrum. I would, however, like to
especially acknowledge the following individuals: the NMRers, Maria van Dongen, Johan
Weigelt, Tomas Åkerud, Mats Wikström and Toshiaki Nishida; mass spectrometry, Agneta
Tjernberg; dynamic light scattering, analytical ultracentrifugation, microcalorimetry and
other biophysical techniques, Natalia Markova, Carina Norström and Dan Hallén; X-ray
crystallography, Jonas Uppenberg, Stefan Svensson and Derek Ogg; medicinal chem-
istry, Jan Vågberg, Styrbjörn Byström, Wei Berts, Annika Jenmalm and Katarina Roos;
computational chemistry, Micael Jacobsson, Mats Kihlén, Anna-Lena Gustavsson, Evert
Homan, Jerk Vallgårda and René Avontuur; and biochemical assays, Thomas Lundbäck
and Eva-Maria Axén.
References
[1] Rees, D. C.,
et al
., Fragment-based lead discovery.
Nat Rev Drug Discov
, 2004,
3
, 660-672.
[2] Erlanson, D. A.,
et al
., Fragment-based drug discovery.
J Med Chem
, 2004,
47
, 3463-3482.
[3] Carr, R. A.,
et al
., Fragment-based lead discovery: leads by design.
Drug Discov Today
, 2005,
10
, 987-992.
