Playing the ACE Card in LBAs

Ligand binding assays (LBAs) are indispensable for biological understanding – and to develop new pharmaceuticals. Due to their tremendous importance, LBAs need to be biologically relevant, precise and accurate. Unfortunately, sometimes they are not. In fact, the data obtained often vary so strongly that only variations by orders of magnitude are considered as real changes. This questionable data quality still limits progress in drug discovery.

Therefore, several different ligand binding assays always need to be combined to (hopefully) confirm each other, and also to reveal various properties of a certain binding process. Different molecules can bind at different positions at a binding site, and conditions such as the solvent (or vacuum), the pH and the temperature obviously influence the binding behavior. Frequently used LBAs hence rely on various techniques, including surface plasmon resonance, ultraviolet, circular dichroism, nuclear magnetic resonance and Fourier transform infrared spectrometry, immunoassays, fluorescence assays, mass spectrometry as well as isothermal titration calorimetry.

But what about affinity capillary electrophoresis (ACE)? ACE is an excellent extension of the LBA toolbox, in particular when charge interactions are involved. When a charged ligand binds to any macromolecule, its charge-to-mass ratio is altered. This can be measured very precisely by electrophoresis.

ACE is applied in aqueous solution, in contrast to mass spectrometry, which requires a vacuum – highly artificial in relation to biological systems. ACE is particularly suited to measure weak and medium binding constants, so for screening in earlier stages of drug development, for example. ACE requires only a few microliters of sample volume; probably even only nanoliters if optimized for this purpose. Pure samples are not required, since ACE comes with an electrophoretic separation process.

There are already a number of ACE success stories (1). A platform to investigate metal ion protein interactions has been developed. Because all proteins interact with metal ions, at least weakly on their surface – ACE allows the screening of these interactions efficiently, in aqueous solution (2). Recently, heparinoids came into focus again and are considered valuable pharmaceuticals, not only as anticoagulants and fibrinolytic agents, but also in the treatment of HIV, certain kinds of cancer and inflammatory diseases. Heparinoids are highly sulfated polysaccharides, and therefore highly charged in aqueous solutions. ACE was successfully used in our research group to characterize various targets and also to differentiate between the different members of this class of pharmaceuticals.

Molecular modeling rounds up this success story. Results from computer models help us to think, but these models still do not have the prediction power that we desire. The best predictions are always obtained with charge interactions – as with ACE, making it an ideal combination.