Incorporation of noncoded amino acids into the N‐terminal domain 1‐47 of hirudin yields a highly potent and selective thrombin inhibitor

• DOI: 10.1110/ps.8.10.2213
• Publication Date: 2009-02-17T21:23:00Z
• Authors: Vincenzo De Filippis; Ilaria Russo; Alessandro Vindigni; Enrico Di Cera; Stefano Salmaso; Angelo Fontana
• Keywords: Hirudin; Dissociation constant

Abstract Hirudin is an anticoagulant polypeptide isolated from a medicinal leech that inhibits thrombin with extraordinary potency ( K d = 0.2‐1.0 pM) and selectivity. composed of compact N‐terminal region (residues 1‐47, cross‐linked by three disulfide bridges) binds to the active site thrombin, flexible C‐terminal tail 48‐64) interacts exosite I enzyme. To minimize sequence hirudin able bind also improve its therapeutic profile, several fragments have been prepared as potential anti‐coagulants. However, practical use these has impaired their relatively poor affinity for enzyme, given increased value dissociation constant ) corresponding complexes 30‐400 nM). The aim present study obtain derivative domain 1‐47 displaying enhanced inhibitory compared natural product. In this view, we synthesized analogue fragment HM2 in which Val1 replaced tert ‐butylglycine, Ser2 Arg, Tyr3 β‐naphthylalanine, give BugArgNal analogue. results chemical conformational characterization indicate synthetic peptide fold efficiently correct topology (Cys6–Cys14, Cys16–Cys28, Cys22–Cys37), while retaining properties fragment. Thrombin inhibition data effects amino acid replacements are perfectly additive if singly substituted analogues (De Filippis V, Quarzago D, Vindigni A, Di Cera E, Fontana 1998, Biochemistry 37 :13507‐13515), yielding molecule fast or slow form 2,670‐ 6,818‐fold more effectively than fragment, exclusively at enzyme d,fast 15.4 pM, d,slow 220 comparable full‐length 0.2 5.5 pM). Moreover, displays absolute selectivity over other physiologically important serine proteases trypsin, plasmin, factor Xa, tissue plasminogen activator, up highest concentration inhibitor tested (10 μM).