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Related Experiment Videos

Optimization of protease-inhibitor interactions by randomizing adventitious contacts.

Tomoko Komiyama1, Bryan VanderLugt, Martin Fugère

  • 1Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, MI 48109, USA.

Proceedings of the National Academy of Sciences of the United States of America
|July 2, 2003
PubMed
Summary

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Researchers optimized protein-protein interactions by modifying "adventitious" contacts, creating potent and selective protease inhibitors. This technique enhances inhibitor affinity and discrimination for specific enzyme targets.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Drug Discovery

Background:

  • Protein protease inhibitors can bind targets they did not coevolve with, like leech inhibitor eglin c binding bacterial subtilisin.
  • Enzyme-inhibitor complexes involve reactive site contacts and broader interface interactions, termed "adventitious" contacts.

Purpose of the Study:

  • To investigate if optimizing adventitious contacts can increase inhibitor affinity and selectivity for target enzymes.
  • To develop potent and selective protease inhibitors for newly discovered proteases.

Main Methods:

  • Modified eglin c (Arg-42-Arg-45-eglin) was used, with its reactive site optimized for Kex2/furin family proteases.
  • Randomization of 10 potential adventitious contact residues and screening for inhibition of soluble human furin.

Related Experiment Videos

  • Substitutions at Y49 were screened against yeast Kex2 and human PC7 for affinity and selectivity.

    • Main Results:

    • Identified variants with up to 20-fold increased affinity for target enzymes.
    • Developed variants exhibiting enhanced selectivity, with up to 41-fold discrimination between furin and PC7.
    • A variant, Asp-49-Arg-42-Arg-45-eglin, showed a 310 pM Ki for furin and blocked furin-dependent processing in cells.

    Conclusions:

    • Optimizing adventitious contact sites is a versatile strategy for developing potent and selective protease inhibitors.
    • This approach can be applied to enhance numerous protein-protein interactions, aiding in the development of novel therapeutics.