Optimization of protease-inhibitor interactions by randomizing adventitious contacts
Structural pharmacology published in PNAS (2003)
Abstract
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.
Resumen
Demonstrates protein-engineering approach to optimize eglin-based protease-inhibitor specificity by randomizing surface contacts, enabling novel inhibitor specificities.
Por qué esto importa para la hirudoterapia
This study examined how optimizing "adventitious" contacts outside the reactive site loop of the leech-derived inhibitor eglin c could enhance its affinity and selectivity for target proteases like furin. By randomizing specific residues, the researchers generated eglin c variants with significantly increased affinity and selectivity for human furin. For ASH, this highlights how leech-derived protease inhibitors can be bioengineered into highly potent and selective candidates for modulating mammalian protease pathways. However, the study is strictly a preclinical, in vitro biotechnological investigation using modified variants in COS-1 cells, not an evaluation of natural hirudotherapy or clinical outcomes.
Citación
Optimization of protease-inhibitor interactions by randomizing adventitious contacts.
Komiyama T et al. · Proceedings of the National Academy of Sciences, 2003
Contexto clínico relacionado
Explore cómo esta investigación se conecta con la práctica clínica
Añadido a la biblioteca ASH: May 27, 2026 · Última actualización del sitio: June 18, 2026