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Engineering of a macromolecular scaffold to develop specific protease inhibitors

Nature Biotechnology volume 21, pages 1063–1068 (2003)Cite this article

Abstract

The specific inhibition of serine proteases, which are crucial switches in many physiologically important processes, is of value both for basic research and for therapeutic applications. Ecotin, a potent macromolecular inhibitor of serine proteases of the S1A family, presents an attractive scaffold to engineer specific protease inhibitors because of its large inhibitor-protease interface. Using synthetic shuffling in combination with a restricted tetranomial diversity, we created ecotin libraries that are mutated at all 20 amino acid residues in the binding interface. The efficacy of these libraries was demonstrated against the serine protease plasma kallikrein (Pkal). Competitive phage display selection yielded a Pkal inhibitor with an apparent dissociation equilibrium constant (Ki*) of 11 pM, whereas Ki* values for related proteases (such as Factor Xa (FXa), Factor XIa (FXIa), urokinase-type plasminogen activator (uPA), thrombin, and membrane-type serine protease 1 (MT-SP1)) were four to seven orders of magnitude higher. The adaptability of the scaffold was demonstrated by the isolation of inhibitors to two additional serine proteases, MT-SP1/matriptase and Factor XIIa.

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Figure 1: Positioning of ecotin surface loops in crystal structure and during synthetic shuffling.
Figure 2: Analysis of the development of ecotin to inhibit Pkal preferentially over FXIa for either pools of variants during sequential rounds of phage display selection or individual variants after seven rounds of selection.
Figure 3: Graphic representation of the Ki* values determined of ecotin wt and ecotin-Pkal for ten proteases.
Figure 4: Graphic representation of the Ki* values determined of ecotin wt, MT-6, XII-18 and ecotin-Pkal for four highly related proteases.

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Acknowledgements

We would like to thank C. Eggers for many helpful discussions regarding the data and the manuscript. This work was supported by a TALENT-stipend from the Netherlands Organization for Scientific Research to A.A.S. and grant CA72006 from the National Institutes of Health to C.S.C.

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  1. Department of Pharmaceutical Chemistry, University of California San Francisco, 600 16th Street Suite S512, San Francisco, 94143-2280, California, USA

    A Allart Stoop & Charles S Craik

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Correspondence to Charles S Craik.

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Stoop, A., Craik, C. Engineering of a macromolecular scaffold to develop specific protease inhibitors. Nat Biotechnol 21, 1063–1068 (2003). https://doi.org/10.1038/nbt860

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