Two engineered eglin c mutants potently and selectively inhibiting kexin or furin
Basic science published in FEBS Lett (2004)
Abstract
Eglin c with mutants L45R and D42R at the P(1) and P(4) positions has been reported to become a stable inhibitor toward the proprotein convertases (PC), furin and kexin, with a K(i) of 2.3x10(-8) and 1.3x10(-10) M, respectively. The mutant was further engineered at the P(2)'-P(4)' positions to create a more potent and selective inhibitor for each enzyme. The residue Asp at P(1)' which is crucial for stabilizing the conformation of eglin c remained unchanged. The eglin c mutants cloned into the vector pGEX-2T and expressed in Escherichia coli (DH5alpha) were purified to homogeneity, and their inhibitory activities toward the purified recombinant furin and kexin were examined. The results showed that (1) Leu47 at P(2)' replaced with either a positively or negatively charged residue resulted in a decrease in inhibitory activities to both enzymes; (2) the replacement of Arg with Asp at P(3)' was favorable for inhibiting furin with a K(i) of 7.8 x 10(-9) M, but not for inhibiting kexin; (3) the replacement of Tyr with Glu at P(4)' increased the inhibitory activity to kexin with a K(i) of 3 x 10(-11) M, but was almost without any influence on furin inhibition. It was indicated that the inhibitory specificity of eglin c could be changed from inhibiting elastase to inhibiting PCs by site-directed mutation at the P positions, while the inhibitory selectivity to furin or kexin could be optimized by mutation at the P' positions.
Abstract sourced from PubMed (NCBI) for the cited record. See the original publication for the authoritative version.
Summary
Site-directed mutagenesis of eglin C yields potent furin (Ki 7.8x10^-9 M) and kexin (Ki 3x10^-11 M) inhibitors.
Why This Matters for Hirudotherapy
This study examined how site-directed mutations at the P′ positions of eglin c could optimize inhibitory selectivity for the proprotein convertases furin and kexin, reporting engineered mutants with Ki values of 7.8×10⁻⁹ M for furin and 3×10⁻¹¹ M for kexin, and demonstrating that inhibitory specificity could be shifted from elastase toward proprotein convertases. Eglin c is a serine protease inhibitor originally isolated from the medicinal leech Hirudo medicinalis, so this protein-engineering work is directly relevant to ASH's interest in characterizing and therapeutically repurposing bioactive components of the leech secretome. Rational redesign of a leech-derived inhibitor to target specific disease-relevant proteases may inform development of targeted biopharmaceuticals building on hirudotherapy-derived molecules. However, this is an in vitro biochemical study using recombinant purified enzymes and inhibitors expressed in E. coli, not a clinical or in vivo efficacy study, so it does not establish therapeutic utility in leech therapy itself.
Citation
Two engineered eglin c mutants potently and selectively inhibiting kexin or furin.
Liu Z et al. · FEBS letters, 2004
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