Ligand screening assay for the enzyme kallikrein immobilized on NHS-activated Sepharose

Human tissue kallikreins (KLKs) are serine proteases involved in various physiological and pathological conditions, including cancer and neurological disorders. These enzymes constitute attractive drug targets, which has stimulated the search for new KLK inhibitors. In this study, we have covalently immobilized porcine pancreas KLK on an NHS-activated Sepharose matrix, to obtain KLK-Sepharose-NHS. The immobilized enzyme showed high recovered activity and maintained the ability of free KLK to recognize the synthetic substrate Z-Phe-Arg-AMC (K_Mapp = 10.3 ± 0.9 μM). As proof of concept, we used leupeptin as a reference inhibitor to perform inhibition studies for KLK-Sepharose-NHS and to determine the half-maximal inhibitory concentration (IC₅₀ = 0.13 ± 0.01 μM), the inhibition constant (K_i = 0.06 μM), and the leupeptin inhibition mechanism. We evaluated several complex matrixes (plant crude extract) by the same bioassay, to demonstrate their applicability. The species Solanum lycocarpum, Stryphnodendron adstringens, and Psychotria carthagenensis gave the best results. KLK-Sepharose-NHS was fully active after six consecutive reaction cycles and retained about 60 % of its initial activity after being used for at least five months, so the bioassay developed herein is a promising strategy to screen and to identify KLK ligands.