LC-NMR and Mass Spectrometry at Bristol
University for fragment screening.
Dr Matthew Crump (NMR),
Dr John Crosby (Mass Spectrometry)
Dr Richard Taylor (NMR, Celltech)
The current proposal is to apply a number of new NMR
and Mass Spectrometry techniques for the screening of
small molecule libraries. Of special interest are proteinligand interactions that play a key role in the development
of severe disease states such as cancer. The process of
identifying and optimizing such drug candidates is a very
time consuming task and consists of several steps. One
major step comprises the identification of substances with
binding affinity from which to generate so-called leads.
Classically, binding assays such as ELISA have been
performed. Recently several novel NMR spectroscopic
techniques have emerged as powerful tools in monitoring
binding processes. Importantly these techniques are twotiered in their information content, allowing a simple
‘yes’/’no’ answer for binding activity and at the same time
revealing at atomic resolution the interaction site on the
target protein.
NMR Techniques.
In collaboration with Celltech we have previously applied
SAR by NMR to the analysis of small molecule inhibitors
binding to LFA-1. We utilised 1H/15N HSQC experiments
to monitor chemical shift changes on LFA-1 using selected
compounds identified as LFA-1 ligands. This proved
successful for mixtures of compounds (say 5 at once) in
which only one target had affinity for the protein and could
be extended to solving a structure of the bound compound.
Figure 1. Titration of a small molecule inhibitor into the
target protein, LFA-1 shows peak shifts in orange and
where annotated shows the mapping of the binding site.
For library screening it is estimated that mixtures of 10
compounds could be screened at the rate of 5-10 mixtures
per hour. Successes would have to be deconvoluted from
the mixtures and this places a high demand on protein
supply. Per mixture, 7.5mgs of a 15kDa protein is typically
required (0.5mL of a 1mM sample). For 100 samples (a
1000 molecule library), 750 mgs of sample would be
required. To reach the limits of high throughput screening
and libraries of 5000 compounds, gram quantities of
protein would be required. However a significant increase
in sensitivity is achieved by applying cryoprobe
technology and the concentration of 15N-labelled protein
can be dropped to 50 M. More importantly this now
allows testing of up to 100 compounds per mixture, where
the overall ligand mixture is only 5mM. Deconvolution is
achieved by testing mixtures of 10 and then individually if
a hit is found. It is estimated that using a cryo-probe up to
200,000 compounds could be tested per month. Currently
we are seeking funding for a cryoprobe on the Bristol
biological 600 MHz NMR and it is our aim to have this
installed and running in 2005.
MASS Spectrometry Techniques
The introduction of electrospray ionisation mass
spectrometry (ESI-MS) in 1984 and matrix-assisted laser
desorption ionisation mass spectrometry (MALDI-MS) in
1988 has led to rapid advances in the analysis of the
intrinsic properties of biological molecules (molecular
weight, subunit composition, and post-translational
modification). More recent advances have allowed
investigations of extrinsic properties of these molecules.
Intact, non-covalent protein-protein, protein-DNA, as well
as protein/DNA-ligand interactions are capable of being
investigated. The rapid screening of biomacromoleculeligand non-covalent interactions is of particular
importance, as rapidly and efficiently identifying
compounds, which bind under physiological conditions
should lead to the development of novel therapeutic agents.
A pulsed ultrafiltration binding chamber can be used to
simultaneously screen hundreds of ligands which can noncovalently bind to a specific target. Bound ligands eluted
from the cell can be rapidly identified, and the use of
modified targets can give some indication of the exact
binding site, or be used to exclude non-specific binding.
Alternatively, the high resolution, high precision mass
measurement afforded by Fourier transform-ion cyclotron
resonance mass spectrometry (FT-ICR) can be used to
simultaneously identify from complex libraries noncovalently bound ligand, target molecule, as well as
determine the specific binding affinity in one set of
experiments.
The Mass Spectrometers necessary will become available
by Spring 2004 and go into refurbished space currently
being modified in the School of Chemistry. An operational
biological 600 MHz NMR facility will perform initial
small library screens to be followed up by cryoprobe
driven screening of much larger libraries.
Plan for CASE
As an extensive testable library of ligands is not yet
available, we anticipate a series of proof of concept
experiments. These could be undertaken during the first
year of the CASE studentship at Bristol. To this end we
will take a small library of 10 compounds one of which
is a known tight binder to an identified target, in this
instance LFA-1. The compounds will be selected
randomly from the Organic Section and the mixture
spiked with a known concentration of CT-8081, that
binds LFA-1 with a nM affinity. A series of
experiments that we envisage is the use of NMR and
pulsed ultrafiltration binding to see if we can detect
binding and then deconvolute a simple mixture. We
will then increase the number of compounds in the
mixture and determine our limits of detection for both
techniques. We wish to also explore the possibility of
using a mutated protein target where mutations around
the binding site or covalently linking a ligand within the
active site knock out any specific binding. This nonligand binding target will be used to identify nonspecfically binding compounds, i.e. those that weakly
bind at positions other than active site.
This approach will then be repeated using the
simultaneous screening ability of the FT Mass
spectrometer which is already available in the
Department of Biochemistry. We hope to have an
instrument in-house in 2004. In addition as the
cryoprobe becomes available we will extend our new
expertise to the use of smaller amounts of target and
larger libraries.
During the first year of the PhD we will also identify
suitable targets of interest to Celltech and begin to
prepare protein for NMR and Mass Spectrometry.