12 June 2012

Capillary Electrophoresis

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?


Dr. Teddy Z said...

So with the plethora of HSP90 data out there, and assays, and so forth, why did they choose a sub-optimal confirmatory assay?
I am left wondering wether CEFrag is highly prone to false positives (probably not based on what we know of FP). is 0.5mM the highest they can go (that would affect the composition of the library you could run.

Carol Austin said...

Hi Teddy,

thank you for your comment. FP assays generally cannot detect the weak affinity compounds (ie high uM, low mM), they can also show fluorescent interference with some compounds. We have other fragment screening campaigns that have higher orthogonal hit rates using methods that are able to pick up the weaker affinity hits, the best we have obtained was 80% confirmation of the CEfrag hits, so we believe it does not pick up a lot of false positives! The screening conc depends on the stock conc of the fragment library and DMSO tolerability of your target under the assay conditions. This conc using our fragment library (stocks of 30mM) corresponded to 1.7% DMSO. Cheers!

Unknown said...

I also have some questions. What about the baseline drift that appears with increasing concentrations of competitor? Is the competitor present at the inlet/outlet? What about viscosity and ionic strength changings? Is there any correction?
Is there any requirement of Kon/Koff?