An automated analysis method enabling the screening of covalent thrombin and factor XIIa inhibitors via liquid chromatography‐mass spectrometry

Abstract

AbstractAn automated sample preparation and separation method for the analysis of various enzyme‐inhibitor combinations using liquid chromatography (LC) coupled to mass spectrometry (MS) is presented. As conventional anticoagulants have several drawbacks, the most severe being the elevated risk of internal bleedings, it is necessary to develop new‐generation anticoagulants with reduced side effects. Therefore, the screening of potential inhibitors against anticoagulation targets like thrombin and FXIIa is important to design a potent and selective inhibitor. To facilitate the analysis of numerous enzyme‐inhibitor covalent complexes, automation of the analysis using an LC system with a user‐defined injection sequence is helpful. The developed method ensures comparable reaction conditions like reaction time and temperature for all enzyme‐inhibitor complexes. Furthermore, it prevents time‐consuming manual sample preparation and potential manual errors. To achieve good reproducibility with relative standard deviation of approximately 3% for three‐fold determination, multiple cleaning steps were added to the automated sample preparation. Subsequently, this method was applied to screen a variety of 15 aminopyrazole‐ and aminotriazole‐based inhibitors with a covalent mechanism of action against thrombin and to test two covalent inhibitors for FXIIa. Successful complex formation and acylation of the catalytic center of the enzymes was monitored using deconvoluted mass spectra and the matching mass shifts of the acyl moiety of the analyzed inhibitors. The inhibitors' structure directly influenced reaction yields. Sterically demanding aminotriazoles and acyl moieties both affected the product formation negatively. However, the screening yielded several promising candidates for new covalent thrombin inhibitors, which might find their application as prospective anticoagulants.