Capillary electrophoresis as a fragment screening tool to cross-validate hits from chromogenic assay: Application to FXIIa

C Davoine; M Fillet; L Pochet

doi:10.1016/j.talanta.2021.122163

Capillary electrophoresis as a fragment screening tool to cross-validate hits from chromogenic assay: Application to FXIIa

Talanta. 2021 May 1:226:122163. doi: 10.1016/j.talanta.2021.122163. Epub 2021 Feb 2.

Authors

C Davoine¹, M Fillet², L Pochet³

Affiliations

¹ Namur Medicine & Drug Innovation Center (NAMEDIC - NARILIS), University of Namur, Rue de Bruxelles 61, 5000, Namur, Belgium; Laboratory for the Analysis of Medicines (LAM), Department of Pharmacy, CIRM, University of Liege, Place du 20 Août 7, 4000, Liège, Belgium.
² Laboratory for the Analysis of Medicines (LAM), Department of Pharmacy, CIRM, University of Liege, Place du 20 Août 7, 4000, Liège, Belgium.
³ Namur Medicine & Drug Innovation Center (NAMEDIC - NARILIS), University of Namur, Rue de Bruxelles 61, 5000, Namur, Belgium. Electronic address: lionel.pochet@unamur.be.

PMID: 33676706
DOI: 10.1016/j.talanta.2021.122163

Abstract

In this study, a partial-filling affinity capillary electrophoresis (pf-ACE) method was developed for the cross-validation of fragment hits revealed by chromogenic factor XIIa (FXIIa) assay. Chromogenic assay produces false positives, mainly due to spectrophotometric interferences and sample purity issues. pf-ACE was selected as counter-screening technology because of its separative character and the fact that the target does not have to be attached or tagged. The effects of protein plug length, applied voltage and composition of the running buffer were examined and optimized. Detection limit in terms of dissociation constant was estimated at 400 μM. The affinity evaluation was performed close to physiological conditions (pH 7.4, ionic strength 0.13 mol L^-1) in a poly (ethylene oxide)-coated capillary of 75 μm internal diameter x 33 cm length with an applied voltage of 3 kV. This method uncovered chromogenic assay's false positives due to zinc contamination. Moreover, pf-ACE supported the evaluation of compounds absorbing at 405 nm.

Keywords: Capillary electrophoresis; Chromogenic assay; Factor XIIa; False positive; Fragment-based lead discovery; Serine proteinase inhibitors.