Contribution of capillary electrophoresis at the early stage of drug discovery

With the emergence of novel pharmaceutical targets, there is an imperative need for fast, reliable and cost-effective analytical techniques able to shorten the long and expensive process of drug development.
In this context, we demonstrated that capillary electrophoresis (CE) could be a robust and efficient analytical tool, notably for the quantification of weak ligand-target interactions, for the investigation of ligands impact on targeted enzyme activity and for drug metabolism monitoring.
To perform our proof of concept studies, the pathology targeted was thrombosis, since it remains one of the most important causes of morbidity and mortality worldwide.
Two affinity CE approaches were designed to quantitate the interaction between thrombin (Thr) and low-affinity ligands in near physiological conditions. The direct binding approach consisted of monitoring the ligand electrophoretic mobility (µep) modification upon binding, while the indirect binding approach was based on the competition of the ligand with a known Thr inhibitor (PL). The modifications of PL µep as a consequence of the competition was in this case monitored. The developed affinity CE approaches allowed not only the accurate determination of complex dissociation constants (Kd)/half maximal inhibitory concentration (IC50) values, but they additionally allowed the conduction of highly discriminant and robust screening campaigns. It should be mentioned that the direct binding approach was suitable for the affinity determination only for cationic molecules that absorb in UV-VIS, while the indirect binding assay was extended to all kind of molecules, no matter their UV-VIS absorbance, or charging state.
The information obtained during the affinity CE analyses was completed with the results generated by fast activity CE assays (analysis time less than 3 min). In this case, the capillary was employed as a multifunction nanoreactor on which the reagents were injected, mixed, incubated and the reaction product (the reporter molecule) was electrophoresed and detected. Two different mixing procedures, namely electrophoretically mediated microanalysis (EMMA) and transverse diffusion of laminar flow profiles (TDLFP) were investigated and the reaction parameters were optimized. In the context of molecules screening TDLFP approach proved to be the mixing procedure of choice, especially when the assayed molecules were charged species. The screening of our small library was performed in the optimized conditions and the results obtained were in good concordance with the affinity screening results. Additionally, a monolithic-functionalized GFP capillary column was investigated for method sensitivity enhancement.
The usefulness of CE for metabolism studies realization was also demonstrated. A fully automated in-capillary system was developed to monitor the activity of CYP1A1 in physiological conditions. Ethoxycoumarin, the selected substrate, underwent an in-line bioreaction in the presence of CYP1A1 giving rise to hydroxycoumarin. The optimization of the experimental conditions was supported by the application of a design of experiment, providing a better understanding of electrophoretic mixing parameters that influence the metabolic reactions.
The developed approaches demonstrate that CE is a powerful and robust tool to be considered in the early stages of drug discovery, due to its minimal sample requirements, ease of automation, its ability to detect and quantify weak interactions and to study drug metabolism in a fully-automated fashion and in near-physiological conditions.