The tetrapeptide IEPD has been the main scaffold reported for the preparation of covalent inhibitors as well as fluorogenic substrates targeting human GzmB (hGzmB)22,23,24,25,26,27,28,29. First, we assessed the reactivity of the commercial Ac-IEPD-AMC (i.e., a substrate that releases 7-amino-4-methylcoumarin upon reaction with hGzmB) against concentrations of an enzyme that would be applicable in clinically relevant assays. We observed slow enzymatic cleavage rates of Ac-IEPD-AMC (i.e.,
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