Nature Biotechnology21, 1063 - 1068 (2003)
Published online: 17 August 2003; | doi:10.1038/nbt860
Engineering of a macromolecular scaffold to develop specific protease
inhibitors
A Allart Stoop
& Charles S Craik
Department of Pharmaceutical Chemistry, University
of California San Francisco, 600 16th Street Suite S512,
San Francisco, California 94143-2280,
USA.
Correspondence should be addressed to Charles S Craik craik@cgl.ucsf.edu
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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