N-terminal requirements of small peptide anticoagulants based on hirudin54-65.
C-terminal fragment analogues of the leech anticoagulant peptide hirudin represent a unique class of thrombin inhibitors that blocks thrombin's cleavage of fibrinogen but does not block the catalytic site of thrombin. In this paper, a series of synthetic peptides were prepared by solid-phase methodology to determine the optimal N-terminal and position 56 functionalities for these C-terminal fragment analogues of hirudin. Inhibition of fibrin clot formation by thrombin in vitro was used as a measure of anticoagulant activity. In the minimal C-terminal sequence necessary for anticoagulant activity, hirudin56-64, an L aromatic amino acid is required at position 56. Phe56----Tyr substitution retained potency, whereas p-Cl-Phe56 and phenylglycine56 substitutions resulted in decreased potencies. Removal of the cationic amino functionality from the vicinity of Asp55 results in increased potency (e.g., hirudin54-65, Ac-hirudin55-65) and [desNH2-Asp55]hirudin55-65 has a marked increase in potency over hirudin55-65. [DesNH2-Phe56]hirudin56-65 and related analogues show no detectable anticoagulant activity. The sensitivity of position 56 to modification demonstrates the significance of this residue in the interaction between the C-terminal region of hirudin and thrombin.
Owen
T J
TJ
Merrell Dow Research Institute, Cincinnati, Ohio 45215.
Krstenansky
J L
JL
Yates
M T
MT
Mao
S J
SJ
D000596:Amino Acids / Q000032:analysis; D000925:Anticoagulants / Q000138:chemical synthesis*; D055598:Chemical Phenomena; D002621:Chemistry; D005260:Female; D006629:Hirudins / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000494:pharmacology; D006801:Humans; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000494:pharmacology; D013329:Structure-Activity Relationship
D000596:Amino Acids; D000925:Anticoagulants; D006629:Hirudins; D010446:Peptide Fragments