From haemadin to haemanorm: Synthesis and characterization of full-length haemadin from the leech Haemadipsa sylvestris and of a novel bivalent, highly potent thrombin inhibitor (haemanorm)
Basic science published in Protein Sci (2023)
Abstract
Hirudin from Hirudo medicinalis is a bivalent α-Thrombin (αT) inhibitor, targeting the enzyme active site and exosite-I, and is currently used in anticoagulant therapy along with its simplified analogue hirulog. Haemadin, a small protein (57 amino acids) isolated from the land-living leech Haemadipsa sylvestris, selectively inhibits αT with a potency identical to that of recombinant hirudin (KI = 0.2 pM), with which it shares a common disulfide topology and overall fold. At variance with hirudin, haemadin targets exosite-II and therefore (besides the free protease) it also blocks thrombomodulin-bound αT without inhibiting the active intermediate meizothrombin, thus offering potential advantages over hirudin. Here, we produced in reasonably high yields and pharmaceutical purity (>98%) wild-type haemadin and the oxidation resistant Met5 → nor-Leucine analogue, both inhibiting αT with a KI of 0.2 pM. Thereafter, we used site-directed mutagenesis, spectroscopic, ligand-displacement, and Hydrogen/Deuterium Exchange-Mass Spectrometry techniques to map the αT regions relevant for the interaction with full-length haemadin and with the synthetic N- and C-terminal peptides Haem(1-10) and Haem(45-57). Haem(1-10) competitively binds to/inhibits αT active site (KI = 1.9 μM) and its potency was enhanced by 10-fold after Phe3 → β-Naphthylalanine exchange. Conversely to full-length haemadin, haem(45-57) displays intrinsic affinity for exosite-I (KD = 1.6 μM). Hence, we synthesized a peptide in which the sequences 1-9 and 45-57 were joined together through a 3-Glycine spacer to yield haemanorm, a highly potent (KI = 0.8 nM) inhibitor targeting αT active site and exosite-I. Haemanorm can be regarded as a novel class of hirulog-like αT inhibitors with potential pharmacological applications.
Abstract sourced from PubMed (NCBI) for the cited record. See the original publication for the authoritative version.
Zusammenfassung
Synthetic haemadin from Haemadipsa sylvestris (57-aa) inhibits alpha-thrombin (Ki 0.2 pM) at active site + exosite-II, plus engineered haemanorm hybrid peptide (Ki 0.8 nM) targets active site + exosite-I.
Warum dies für die Hirudotherapie relevant ist
This study reports the synthesis and characterization of haemadin, a 57-amino-acid thrombin inhibitor naturally isolated from the land-living leech Haemadipsa sylvestris, and the rational design of a novel bivalent inhibitor termed haemanorm. Haemadin potently inhibits α-thrombin (Ki = 0.2 pM) — matching recombinant hirudin — but uniquely targets exosite-II rather than exosite-I, allowing it to block thrombomodulin-bound thrombin without inhibiting meizothrombin, a property the authors suggest may offer pharmacological advantages over hirudin. The authors further mapped functional domains and engineered haemanorm (Ki = 0.8 nM), joining N- and C-terminal haemadin sequences via a glycine spacer to create a hirulog-like molecule targeting both the active site and exosite-I. This work is directly relevant to ASH's domain, as it characterizes a leech-derived anticoagulant protein and derives a potential new drug candidate from its structure. However, these are biochemical and in-vitro findings; no animal or clinical data are presented, and pharmacological application claims remain preliminary.
Zitation
From haemadin to haemanorm: Synthesis and characterization of full-length haemadin from the leech Haemadipsa sylvestris and of a novel bivalent, highly potent thrombin inhibitor (haemanorm).
Acquasaliente L et al. · Protein science, 2023
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