One of the fun aspects of fragment-based lead discovery is
the number of ingenious biophysical methods for finding low-affinity fragments.
In a recent issue of J. Biomol. Screen.,
Carol Austin and colleagues at Selcia describe their approach, capillary
electrophoresis, which they term CEfrag.
Capillary electrophoresis itself has been around for quite a
while. It involves applying a high voltage across a thin capillary filled with
liquid; a solution to be analyzed is injected, and the voltage causes migration
of analytes (for example, proteins or small molecules). Analyte movement
through the capillary depends on charge and “hydrodynamic radius,” which is a
function of molecular size and shape. In the case of CEfrag, the idea is to
start with a reporter molecule that can be readily detected, for example via UV
absorbance. Under a standard set of conditions, this “probe ligand” will have a
characteristic mobility. If an excess of protein that binds to the probe ligand
is present in the running buffer, the migration time will shift. If an
inhibitor is also present in the running buffer, this will prevent the probe
ligand from binding to the protein, also causing the migration time to change.
By running different concentrations of inhibitor and measuring the changes in
mobility, the inhibition constant can be determined.
The researchers demonstrated their approach using that old
work-horse of FBLD, the cancer target Hsp90. The known Hsp90 inhibitor
radicicol was used as the probe ligand. A total of 609 fragments were screened
individually at an initial concentration of 0.5 mM, yielding 42 fragments that
reproducibly inhibited radicicol mobility by 20% or more. This ~7% hit rate is
similar to that found by others for this target.
Only 12 of the 42 hits identified by CEfrag were also detected
in a confirmatory fluorescence polarization (FP) assay, of which only 5 gave
measurable IC50 values. However, FP is not ideal for evaluating
fragments. In fact, one of the CE hits that didn’t reproduce by FP was
ethamivan, the starting fragment for the program that ultimately led to Astex’s
AT13387, now in a phase 2 clinical trial for GIST.
To get a better sense of the quality of the CE hits, the
researchers put 6 fragments into crystallography trials: 3 hits from both CE
and FP, two from CE alone, and one that hit neither. The negative control didn’t
produce a structure, whereas two of the FP-confirmed hits produced co-crystal
structures (the one that did not had solubility issues). One of the two CE-only
hits (ethamivan) also did.
With a throughput of 100 compounds per day per instrument,
this is not a high-throughput method, but it is comparable to many other
biophysical approaches. Also, the low protein consumption and ability to use
unmodified protein are selling points. Have you tried CEfrag? If so, what do
you think?
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