The aim of this master's thesis was to identify new competitive inhibitors of the recombinant enzyme paraoxonase 1 (rePON1) that could replace the known inhibitor 2-hydroxyquinoline (2HQ) with higher affinity. PON1 is a calcium-dependent mammalian hydrolase involved in protection against oxidative stress and in the hydrolysis of various esters. Initially, we successfully isolated rePON1 through expression in E. coli and purification using immobilized metal affinity, ion exchange, and size exclusion chromatography. Subsequently, we conducted a spectrophotometric screening of 1,409 clinically approved compounds from the National Medical Products Administration (NMPA) library and identified 25 compounds with at least 10% inhibition based on reduced enzymatic activity. The ten most promising inhibitors were further evaluated kinetically. The most efficient inhibitors were efavirenz (Ki = 0.93 ± 0.01 µM) and prulifloxacin (Ki = 1.39 ± 0.02 µM), both showing 2- to 3-fold higher affinity for rePON1 than 2HQ (Ki = 2.54 ± 0.03 µM). The results confirm the structural promiscuity of the PON1 active site. Due to their favorable spectroscopic properties, both novel founded inhibitors could be incorporated into the development of more sensitive luminescent assays, thereby contributing to improved quantification of PON1 in biological samples.
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