Structure-based design of a potent chimeric thrombin inhibitor.
Using the three-dimensional structures of thrombin and the leech-derived tryptase inhibitor (LDTI), which does not inhibit thrombin, we were able to construct three LDTI variants inhibiting thrombin. Trimming of the inhibitor reactive site loop to fit thrombin's narrow active site cleft resulted in inhibition constants (Ki) in the 10 nM concentration range; similar values were obtained by the addition of an acidic C-terminal peptide corresponding to hirudin's tail to LDTI. Combination of both modifications is additive, resulting in very strong inhibition of thrombin (Ki in the picomolar range). On the one hand, these results confirm the significance of the restricted active site cleft of thrombin in determining its high cleavage specificity; on the other, they demonstrate that sufficient binding energy at the fibrinogen recognition exosite can force thrombin to accept otherwise unfavorable residues in the active site cleft. The best inhibitor thus obtained is as effective as hirudin in plasma-based clotting assays.
Morenweiser
R
R
Abteilung für Klinische Chemie und Klinische Biochemie in der Chirurgischen Klinik und Poliklinik, Klinikum Innenstadt der Ludwig-Maximilians-Universität München, D-80336 München, Germany.
Auerswald
E A
EA
van de Locht
A
A
Fritz
H
H
Stürzebecher
J
J
Stubbs
M T
MT
D000818:Animals; D001665:Binding Sites; D003001:Cloning, Molecular; D015195:Drug Design; D006801:Humans; D019476:Insect Proteins / Q000378:metabolism; D007865:Leeches; D008958:Models, Molecular; D011506:Proteins / Q000138:chemical synthesis* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000138:chemical synthesis* / Q000235:genetics; D011994:Recombinant Proteins / Q000138:chemical synthesis / Q000235:genetics; D015842:Serine Proteinase Inhibitors / Q000138:chemical synthesis* / Q000235:genetics; D013917:Thrombin / Q000378:metabolism
D019476:Insect Proteins; D011506:Proteins; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D015842:Serine Proteinase Inhibitors; C090901:leech-derived tryptase inhibitor C; C082506:rhodniin protein, Rhodnius; D013917:Thrombin