Design of potent bivalent thrombin inhibitors based on hirudin sequence: incorporation of nonsubstrate-type active site inhibitors
Research article published in Biochemistry (1994)
Abstract
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, hirudin1-48, a fibrinogen-recognition exo-site binding moiety, hirudin55-65, and a linker, hirudin49-54, connecting these inhibitor moieties. Synthetic inhibitors were designed based on the C-terminal portion of hirudin. The bulky active site blocking moiety, hirudin1-48, was replaced by small nonsubstrate-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 hirudin55-65 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 acid 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 affinity (K(i)) of the inhibitor 10-100-fold (down to 1.70 x 10(-11) M) compared 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 inhibitor was also stable against human plasma proteases. Further inhibitor design revealed that the toxic dansyl group could be replaced by 4-tert-butylbenzenesulfonyl group and 1- or 2-naphthalenesulfonyl group for in vivo studies.(ABSTRACT TRUNCATED AT 250 WORDS)
Abstract sourced from PubMed (NCBI) for the cited record. See the original publication for the authoritative version.
Summary
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.
Why This Matters for Hirudotherapy
This study describes the rational design of bivalent thrombin inhibitors modeled on hirudin—the most potent and specific thrombin inhibitor from the medicinal leech (Ki = 2.2 × 10⁻¹⁴ M)—by replacing the bulky active-site blocking moiety (hirudin1-48) with smaller nonsubstrate-type inhibitors and retaining the C-terminal exosite-binding fragment (hirudin55-65). Crystallographic analysis confirmed that the synthetic inhibitors occupied thrombin's S1, S2, and S3 subsites, achieving Ki values down to 1.70 × 10⁻¹¹ M, with improved plasma protease stability and identification of less toxic group replacements for potential in vivo studies. This work is directly relevant to ASH's domain as it leverages the structural biology of the leech secretome's flagship molecule for drug design. However, the study is purely in vitro with no leeches or hirudotherapy involved, and clinical relevance remains undemonstrated.
Citation
Design of potent bivalent thrombin inhibitors based on hirudin sequence: incorporation of nonsubstrate-type active site inhibitors
Tsuda Y et al. · Biochemistry, 1994
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