Capillary electrophoresis revisited

Among the various fragment-finding methods, capillary electrophoresis (CE) seems to be among the least-used, at least according to our polls. Indeed, we last wrote about CE in 2012, and since then the company that was popularizing the technique seems to have quietly dropped it from its website. A new paper by Marianne Fillet and collaborators at the University of Liege and the University of Namur in Analytica Chimica Acta presents a how-to guide for CE.

As we previously discussed, the most general CE assay involves filling a capillary with a protein solution as well as a “probe ligand” with affinity for the target protein. Interactions between the probe ligand and the protein will increase the migration time of the probe ligand compared to its progress through a capillary without protein (i.e., the probe ligand will move more slowly through the capillary in the presence of protein).

If a “test ligand” is introduced into the capillary and displaces the probe ligand, the migration time of the probe ligand will again decrease. By changing the concentration of test ligand and measuring the shift in migration time of the probe ligand, the affinity of the test ligand can be determined.

The researchers applied CE to thrombin, a drug target that is often used for validating fragment-finding methods. The low nanomolar inhibitor NAPAP was chosen as the probe ligand due to its strong chromophore (simplifying detection) and positive charge (allowing it to move in the electric field of the capillary). They tested three literature compounds with inhibition constants ranging from high nanomolar to high micromolar and found good agreement with published results.

Next, the researchers applied CE to a small library of fragments, generating several hits. They also describe a method for detecting irreversible binding: this involves screening the protein with an even higher concentration of probe ligand to see whether the test ligand itself can be displaced.

This is a nice study, but it perhaps also illustrates why the technique hasn’t caught on. First and most importantly is throughput; the runs shown are on the order of 14 minutes. Second, it does require a probe ligand. (Screening test ligands directly only works if they are positively charged.) On the other hand, CE can work with native protein, unlike immobilization-based techniques such as SPR and WAC.

Have you tried CE yourself – and if so how did it perform?