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Optimization of an electrophoretic approach for the screening and the development of new antithrombotic drugs
Farcas, Elena; Bouckaert, Charlotte; Servais, Anne-Catherine et al.
2017The 45th International Symposium on High Performance Liquid Phase Separations and Related Techniques (HPLC)
 

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Keywords :
Fragment screening; affinity capillary electrophoresis bioassay; antithrombotic agent
Abstract :
[en] The discovery of lead compounds that can modulate the activity of a biological target is essential to provide efficient pharmacological tools and to serve as starting points for new drug generations. Fragment-based drug discovery (FBDD) approach is an attractive tool for the identification of new selective inhibitors of a target of interest, but its success largely depends on the ability to develop screening bioassays capable to detect and gauge weak affinity binders. To achieve this goal, we investigated capillary electrophoresis (CE) for identifying and ranking fragments from an initial library. Indeed, due to its ability to evaluate weak interactions, CE seems to be promising for fragment-based screening. This technique is a powerful analytical tool with a unique separation mechanism, speed, efficiency and versatility. Its main advantages are low protein consumption, higher throughput compared to NMR and X-ray crystallography and the fact that screening can be carried out using native protein in physiological solution without the need of immobilization. We developed a proof of concept study on thrombin, a serine protease implicated in the coagulation cascade using affinity capillary electrophoresis (ACE) for ranking fragments from an initial library. For this study, we followed a probe ligand, benzamidine, and we investigated interactions with the target by monitoring the changes of its electrophoretic mobility upon binding. The first step of this study consisted in the optimization of the experimental conditions suitable for the CE method (target and probe ligand concentrations, separation buffer composition, voltage, separation effective length, target partial filling…). Then, numerous thrombin inhibitors with a wide range of inhibitory potency (i.e. Ki 200 µM – 5 nM) were tested to validate our system demonstrating the possibility to fish binders in the optimized conditions. We also checked the absence of non-specific binding with the target using the inactivated enzyme at the binding site. It is noteworthy that in this operating system (ACE assay), binding occurs in free solution using physiological buffers, thus preventing artifacts that may result from target immobilization, which is a requirement for some techniques such as SPR.
Research center :
CIRM - Centre Interdisciplinaire de Recherche sur le Médicament - ULiège
Disciplines :
Pharmacy, pharmacology & toxicology
Author, co-author :
Farcas, Elena ;  Université de Liège > Département de pharmacie > Analyse des médicaments
Bouckaert, Charlotte;  Université de Namur - UNamur > Pharmacie > Chimie Pharmaceutique
Servais, Anne-Catherine  ;  Université de Liège > Département de pharmacie > Analyse des médicaments
Crommen, Jacques ;  Université de Liège > Département de pharmacie > Département de pharmacie
Pochet, Lionel;  Université de Namur - UNamur > Pharmacie > Chimie Pharmaceutique
Fillet, Marianne ;  Université de Liège > Département de pharmacie > Analyse des médicaments
Language :
English
Title :
Optimization of an electrophoretic approach for the screening and the development of new antithrombotic drugs
Publication date :
20 June 2017
Event name :
The 45th International Symposium on High Performance Liquid Phase Separations and Related Techniques (HPLC)
Event organizer :
University of Prague
Event place :
Prague, Czechia
Event date :
du 18 juin au 22 juin 2017
Audience :
International
Funders :
F.R.S.-FNRS - Fonds de la Recherche Scientifique [BE]
Fonds Léon Fredericq [BE]
Available on ORBi :
since 28 June 2017

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