Affinity capillary electrophoresis as a powerful tool for fragment-based screening: application to the inhibition of S1A serine proteases implicated in the coagulation cascade

Fragment-based drug discovery (FBDD) proved its efficacy and gained an increasing interest in the pharmaceutical industry and academia.[1] In contrast to the traditional medicinal chemistry approach, FBDD leads to small chemical templates that should be easier to tune into compounds displaying a high inhibitory potency associated with adequate selectivity and pharmacokinetic properties. However, the use of fragments during the screening implies that the affinity with the target is very weak. In consequence, the success of the FBDD approach strongly depends on the ability to develop bioassays that are sensitive enough to detect and gauge affinity in the µM-mM range. NMR methods, X-ray screening and surface plasmon resonance (SPR) are generally the most popular approaches reported in the literature. Nevertheless, these valuable techniques are rather expensive, time-consuming or unable to reflect the physiological environment. Generally, they also require highly purified reagents as well as large sample amounts. In this context, we investigated the ability of affinity capillary electrophoresis (ACE) for FBDD screening. ACE has the advantages to do not need highly purified, modified or immobilized species.[2]

In this communication, we will report the development of the ACE approach for the screening of fragments towards two S1A serine proteases namely thrombin and FXIIa. We performed a direct and competitive ACE approach for the screening. The direct ACE method was designed to screen positively charged fragments. Indeed, in S1A serine proteases, the specificity pocket S1 is characterized by an aspartate (Asp189) at its bottom and a cationic group is expected to guide the fragment inside the active site [3]. Competitive ACE-binding is suitable for any fragments regardless of their charge, but the assay requires a higher concentration of ligands. [4]

[1] D.A. Erlanson, W. Jahnke, Wiley-VCH2016; b) G. Siegal, E. Ab, J. Schultz, Drug Discov Today (2007),12 (23/24), 1032; c) CW. Murray, ML. Verdonk, DC. Rees, Trends Pharmacol Sci (2012), 33(5), 224.
[2] E. Farcas, L. Pochet, J. Crommen, A.C. Servais, M. Fillet, JPBA (2017), 144, 195–212
[3] E. Farças, C. Bouckaert, A.C. Servais, J. Hanson, L. Pochet, M. Fillet, Anal Chim Acta (2017), 211-222.
[4] E. Farças, J. Hanson, L. Pochet, M. Fillet, Anal Chim Acta (2018), 214-222