Automated workflow for the study of drug-protein interactions based on capillary electrophoresis-frontal analysis combined with in-capillary mixing of interacting partners


Zdenek Glatz, Biochemistry, Faculty of Science, Masaryk University, Brno, Czech Republic (glatz@chemi.muni.cz)

Biomolecular interactions are at the base of all biological events occurring in living cells. The understanding of interactions between protein-protein, protein-nucleic acids, protein-sugars, nucleic acid-nucleic acids and protein-small molecules represents a high-priority research area in the life science – interactomics and subsequently provides essential knowledge that may help in disease diagnosis, prognosis and therapeutics. Therefore, biomolecular interaction analyses thus do not only give fundamental insights into the biological processes in the cells but also paves the way to modify them by therapeutic molecules – drugs towards improved disease treatment that is the main goal of the research done by pharmaceutical industry in a new drug development.

An essential part of this process is a study of the plasma protein-drug binding, since according to the widely accepted free drug hypothesis it can limit the drug concentration available to act at the target, as well as in its final impact, drug disposition and efficacy. Quite a lot of approaches differing in their basic principle were developed over time to characterize these interactions. The capillary electrophoresis‑frontal analysis (CE‑FA) is together with the mobility shift affinity CE most frequently CE mode used for this purpose. Whereas in the classic CE-FA setup the sample is prepared by off-line mixing of the interaction partners in the sample vial  outside the CE instrument and after short incubation period is loaded in the capillary and analysed, in this work a new methodological approach combining the CE‑FA with the mixing of interacting partners directly inside the capillary has been developed. Their combination gives rise to a fully automated and versatile methodology for characterization of binding interactions besides the substantial reduction of the sample compound amounts.

The in-capillary mixing is based on transverse diffusion of laminar flow profile methodology introduced by S. Krylov et al. in 2009  using its multi‑zone injection modification presented by Řeminek at al. in 2013. The apparent binding parameters of BSA for propranonol and lidocaine, drug differing both in the physico-chemical properties and binding sites on the albumin molecule, and in the binding strength were determined by on-line and off-line approaches. The values obtained by a new on‑line CE‑FA methodology are in agreement with values estimated by classic off‑line CE‑FA, as well as with literature data obtained using different techniques. According the best author knowledge the in-capillary sample mixing for the CE‑FA has not been published so far.

Acknowledgements

This work was supported by the Czech Science Foundation (No. 19-08358S).


Abstract Reference & Short Personal Biography of Presenting Author

Prof. RNDr. Zdeněk Glatz, CSc

Education
1993    –  CSc - PhD equivalent Thesis Title “Analytical Biochemistry of Quinoproteins” Masaryk University, Faculty of Science, Department of Biochemistry 

Affiliation
2007–present  Full Professor, Masaryk University Brno, Faculty of Science, Department of Biochemistry

Scholarships and longer visits
1998, 2003, 2013        University of Lund, Sweden – prof. B. Matiassson, KU Leuven, Belgium – prof. A. Van Schepdael, University Bern, Swiss – prof. W. Thormann
1992–1993      Postdoctoral stay Indiana University, Bloomington, USA - prof. Novotny

Membership and activities in professional societies

  • Board of Scientific Group for Chromatography and Electrophoresis, Czech Chem. Society
  • Institute Council of Institute of Analytical Chemistry of the ASCR, v. v. i. Brno
  • Member of Scientific Board Faculty of Science, Palacký University Olomouc.
  • Member of International Editorial Board Journal od Separation Science, Wiley

Research interests in last 5 years

  • Application of CE in pharmacology, clinical chemistry, enzymology and metabolomics
  • Analytical biochemistry
  • Metabolomics

Research publication activities

120 scientific papers in scientific journals with or proceeding abstract in WOS (all databases), 3 chapter in the book, 160 presentations at  scientific conferences, 3 patents, ∑ citation cca 1127 without self-citations, H-index 20.


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