Chemistry Reference
In-Depth Information
soluble targets. On the other hand, the attraction of membrane proteins as pharmaceutical
targets has been well documented,
[
4
]
with approximately 60% of all current targets being
membrane proteins. Hence it would be a significant advantage to be able to apply FBDD
to the class of targets that includes integral and membrane-associated proteins.
We have developed a technology called target-immobilized NMR screening (TINS)
[
5, 6
]
that in principle can be applied to screening of membrane proteins. In TINS, the target to be
screened is immobilized on a commercially available chromatography resin in a simple and
efficient process. The immobilized target, along with a second, reference sample, is placed
in a flow-injection, dual-cell sample holder in the magnet and the compounds to be screened
are injected in mixes of about five compounds each.
[
6
]
Spatially selective spectroscopy
[
1
]
is then used to acquire independently a 1D
1
H spectrum of the compounds in the presence
of the target or the reference. Comparison of the two spectra directly yields the identity of
any compound that binds the target due to the simple reduction in peak amplitude of all
resonances from the ligand. This configuration yields a number of advantages for ligand
screening. The combination of effective
T
2
relaxation and chemical exchange endows the
method with great sensitivity with specific binding as weak as 5-10mM (
K
D
) being readily
detected. On the other hand, the presence of a reference sample in routine use cancels the
weak, nonspecific interactions typically observed between many of the compounds to be
screened and the target. Thus the presence of artifacts in TINS screens is greatly reduced,
as is the false positive rate. The sensitivity can also be used to reduce the concentration of
immobilized target to as low as 5 μM solution equivalent, which combined with the fact
that the entire compound collection is routinely screened with a single sample, means the
screening can be carried out with as little as 5 nmol of the target.
TINS has been applied to a variety of soluble proteins and in this chapter we will present
some of these results. In principle, immobilization should allow an extension of the range
of targets to which TINS can be applied to include insoluble membrane proteins. This idea
is not new and others have attempted to apply biophysical methods for detecting ligand
binding to immobilized membrane proteins.
[
7
]
In particular, surface plasmon resonance
(SPR) has been used for this application. Membrane proteins represent difficult targets for
in
vitro
ligand screening studies, however, since they are insoluble, often require the presence
of specific lipids for proper function, are highly challenging to purify and rarely amenable to
high-resolution structural analysis. Furthermore, a general limitation that has always been
encountered is the difficulty of functionally immobilizing membrane proteins in a form
appropriate for the assay. SPR for instance requires a flat surface with an underlying metal
layer (to provide the material with dielectric constant opposite that of water). Although a
few cases of successful immobilization of membrane proteins have been reported under
these conditions, a widely applicable method is still lacking. Here we will report on our
initial efforts in two areas, the ultimate goal of which is to allow routine
in vitro
fragment
screening of a wide variety of membrane proteins.
6.2 General Considerations for Fragment Screening
6.2.1 Fragments
Since an entire chapter of this topic is devoted to fragment library design, it is not our
intention to recapitulate this information here. Instead we will focus on the principles and
