Chromogenic substrates as fundamental tool to design new thrombin inhibitors

Summary

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.

Zusammenfassung

Die Struktur/Aktivitätsbeziehungen von Dipetalogastin II, dem stärksten bisher isolierten und klonierten Thrombininhibitor aus der Raubwanze Dipetalogaster maximus, wurden durch graduelle Veränderungen seiner molekularen Struktur und anschließender Messung der Dissoziationskonstanten mittels chromogener Substratmethode untersucht. Das war die Grundlage für die Synthese weiterer, von Dipetalogastin II abgeleiteter Thrombininhibitoren. Unsere Ergebnisse zeigten, dass die saure Sequenz DEHDHDFEDT (49.-58. Aminosäurerest von Dipetalogastin II) mit der anionischen Bindungsstelle 1 (ABE-1) des Thrombins interagiert.

Deshalb wurde eine chimäre Verbindung entworfen, die zum einen aus dem das Aktivitätszentrum blockierenden Segment von Dipetalogastin II und zum anderen aus dem ABE-1 blockierenden Segment von Hirudin besteht. Das chimäre Molekül zeigte bereits eine akzeptable Thrombinhemmung, die durch Einfügen eines Glycin-Linkers zwischen beide Teilbereiche verstärkt wurde. Dieser Dipetarudin genannte Thrombininhibitor weist bei einem deutlich geringeren Molekulargewicht als Dipetalogastin II eine vergleichbare Dissoziationskonstante auf.

Keywords

Direct thrombin inhibitor - Ki-value - chromogenic thrombin substrate

Schlüsselwörter

Direkter Thrombininhibitor - Ki-Wert - chromogenes Thrombinsubstrat

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