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Structure-based design of a potent chimeric thrombin inhibitor
R Morenweiser1, E A Auerswald, A van de Locht
1Abteilung für Klinische Chemie und Klinische Biochemie in der Chirurgischen Klinik und Poliklinik, Klinikum Innenstadt der Ludwig-Maximilians-Universität München, D-80336 München, Germany.
The Journal of Biological Chemistry
|August 8, 1997
Summary
Researchers modified a leech-derived inhibitor to block thrombin, achieving picomolar inhibition. This breakthrough offers a potent new thrombin inhibitor comparable to hirudin for therapeutic applications.
Area of Science:
- Biochemistry
- Structural Biology
- Pharmacology
Background:
- Thrombin is a key enzyme in blood coagulation.
- Leech-derived tryptase inhibitor (LDTI) does not inhibit thrombin.
- Understanding thrombin's active site is crucial for developing inhibitors.
Purpose of the Study:
- To engineer LDTI variants that inhibit thrombin.
- To investigate modifications for enhancing thrombin inhibition.
- To compare engineered inhibitors with existing anticoagulants.
Main Methods:
- Utilized 3D structures of thrombin and LDTI.
- Modified LDTI's reactive site loop to fit thrombin's active site.
- Added an acidic C-terminal peptide mimicking hirudin's tail.
- Assessed inhibition constants (Ki) and plasma clotting assays.
Main Results:
- Engineered LDTI variants demonstrated significant thrombin inhibition.
- Trimming the loop yielded Ki in the nanomolar range.
- Adding a hirudin-like tail also resulted in nanomolar inhibition.
- Combined modifications achieved picomolar Ki, indicating very strong inhibition.
- The best inhibitor matched hirudin's efficacy in plasma assays.
Conclusions:
- Thrombin's narrow active site cleft dictates substrate specificity.
- Strong binding at the fibrinogen recognition exosite can overcome active site constraints.
- Engineered LDTI variants represent potent thrombin inhibitors with therapeutic potential.