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M. Lopez, G. Nowak
Chromogene Substrate als Basis zur Enwicklung neuer Thrombininhibitoren: Bestimmung von Dissoziationskonstanten
The structure-activity relationship of dipetalogastin II, the strongest thrombin inhibitor isolated and cloned from the bug Dipetalogaster maximus, was examined by introducing gradual changes into the molecule by means of molecular biological methods. The effect upon its inhibition equilibrium constant was determined after each change by a chromogenic assay. This structural information was fundamental to design new dipetalogastin II-derived inhibitors. Our results suggested that the acidic sequence DEHDHDFEDT corresponding to amino acid residues 49 to 58 of dipetalogastin II reacts with the anion binding exosite (ABE) 1 of thrombin. Based on this finding, we constructed a chimeric molecule consisting of the active site blocking segment of dipetalogastin II (amino acid residues 1 to 48) and the ABE 1 blocking segment of hirudin. This construct showed better thrombin inhibitory activity than both separated segments only after the introduction of a glycine linker between both blocking segments. We thus obtained a thrombin inhibitor called dipetarudin with an inhibition equilibrium constant comparable to that of dipetalogastin II and a molecular mass below that of dipetalogastin.
Thrombosis and Haemostasis, Schattauer
Print ISSN: 0340-6245
Volume: 25, 01/2005
Pages: 267 - 271