Optimization of protease-inhibitor interactions by randomizing adventitious contacts

Polypeptide protease inhibitors are often found to inhibit targets with which they did not coevolve, as in the case of high-affinity inhibition of bacterial subtilisin by the leech inhibitor eglin c. Two kinds of contacts exist in such complexes: (i) reactive site loop-active site contacts and (ii) interactions outside of these that form the broader enzyme-inhibitor interface. We hypothesized that the second class of "adventitious" contacts could be optimized to generate significant increases in affinity for a target enzyme or discrimination of an inhibitor for closely related target proteases. We began with a modified eglin c, Arg-42-Arg-45-eglin, in which the reactive site loop had been optimized for subtilisin-related processing proteases of the Kex2/furin family. We randomized 10 potential adventitious contact residues and screened for inhibition of soluble human furin. Substitutions at one of these sites, Y49, were also screened against yeast Kex2 and human PC7. These screens identified not only variants that exhibited increased affinity (up to 20-fold), but also species that exhibited enhanced selectivity, that is, increased discrimination between the target enzymes (up to 41-fold for furin versus PC7 and 20-fold for PC7 versus furin). One variant, Asp-49-Arg-42-Arg-45-eglin, exhibited a Ki of 310 pM for furin and blocked furin-dependent processing of von Willebrand factor in COS-1 cells when added to the culture medium of the cells. The exploitation of adventitious contact sites may provide a versatile technique for developing potent, selective inhibitors for newly discovered proteases and could in principle be applied to optimize numerous protein-protein interactions.

Abstract sourced from PubMed (NCBI) for the cited record. See the original publication for the authoritative version.

Randomizing ten adventitious contact residues in a modified eglin c inhibitor yielded furin-binding variants with up to 20-fold higher affinity and up to 41-fold greater furin/PC7 selectivity; one variant, with a 310 pM furin Ki, blocked furin-dependent von Willebrand factor processing in COS-1 cells.

Explore how this research connects to clinical practice

More peer-reviewed studies in Salivary Pharmacology.

• Medicinal leeches and hirudotherapy — Gödekmerdan A et al. (2011)
• PEROXIDATION OF SALIVA LIPIDS IN PATIENTS WITH POSTCOVID SYNDROME DURING HIRUDOTHERAPY — Serikbayeva S et al. (2024)
• Medicinal leech therapy in pain syndromes: a narrative review — Koeppen D, Aurich M, Rampp T (2014)
• Immunomodulatory effects of medicinal leech saliva extract on in vitro activated macrophages — Ayhan H et al. (2024)
• Proteinase inhibitors from the medicinal leech Hirudo medicinalis — Baskova I, Zavalova L (2001)
• Polyfunctionality of destabilase, a lysozyme from a medicinal leech — Baskova I, Zavalova L (2008)
• Make it double: identification and characterization of a Tandem-Hirudin from the Asian medicinal leech Hirudinaria manillensis — Lukas P et al. (2022)
• Use of the medicinal leech in the treatment of ear diseases — Seleznev K et al. (1992)
• Recombinant Destabilase from Hirudo medicinalis Is Able to Dissolve Human Blood Clots In Vitro — Bobrovsky P et al. (2021)
• Effects of Carbon Nanotube Environmental Dispersion on an Aquatic Invertebrate, Hirudo medicinalis — Girardello R et al. (2015)

Browse all ASH-analysed research →