DESIGN OF POTENT BIVALENT THROMBIN INHIBITORS BASED ON HIRUDIN SEQUENCE - INCORPORATION OF NONSUBSTRATE-TYPE ACTIVE-SITE INHIBITORS

Hirudin from medicinal leech is the most potent and specific thrombin inhibitor from medicinal leech with a K-i value of 2.2 x 10(-14) M. It consists of an active site blocking moiety, hirudin(1-48), a fibrinog en-recognition exo-site binding moiety, hirudin(55-65), and a linker, hirudin(49-54), connecting these inhibitor moieties. Synthetic inhibit ors were designed based on the C-terminal portion of hirudin. The bulk y active site blocking moiety, hirudin(1-48), was replaced by small no nsubstrate-type active site inhibitors of thrombin, e.g., dansyl-Arg-( D-pipecolic acid). The linker moiety was replaced by omega-amino acids of (12-aminododecanoic acid)-(4-aminobutyric acid), and hirudin(55-65 ) was used as a fibrinogen-recognition exo-site binding moiety in most of the inhibitors. The crystal structure of the inhibitor in complex with human alpha-thrombin showed that dansyl, Arg, and D-pipecolic aci d of the active site blocking moiety occupy S3, S1, and S2 subsites of thrombin, respectively, and were therefore designated as P3, P1, and P2 residues. The use of dansyl-Arg-(D-pipecolic acid) improved the aff inity (K-i) of the inhibitor 10-100-fold (down to 1.70 x 10(-11) M) co mpared to that of the similar compounds having D-Phe-Pro-Arg as their substrate-type inhibitor moiety (K-i = 10(-9) - 10(-10) M). The linker connected to P2 residue eliminated the scissile peptide bond. The inh ibitor was also stable against human plasma proteases. Further inhibit or design revealed that the toxic dansyl group could be replaced by 4- tert-butlybenzenesulfonyl group and 1- or 2-napthalenesulfonyl group f or in vivo studies. In addition, the replacement of hirudin(55-65) wit h [Tyr(56), Pro(58), Ala(63), Cha(64), D-Glu(65)]hirudin(55-65) improv ed the affinity of the inhibitors (K-i = 2.0 x 10(-12) M) to the level 10-fold less potent than recombinant hirudin (Ki = 2.3 x 10(-13) M).