Irreversible inhibitors of the 3C protease of Coxsackie virus through templated assembly of protein-binding fragments.
Small-molecule fragments binding to biomacromolecules can be starting points for the development of drugs, but are often difficult to detect due to low affinities. Here we present a strategy that identifies protein-binding fragments through their potential to induce the target-guided formation of covalently bound, irreversible enzyme inhibitors. A protein-binding nucleophile reacts reversibly with a bis-electrophilic warhead, thereby positioning the second electrophile in close proximity of the active site of a viral protease, resulting in the covalent de-activation of the enzyme. The concept is implemented for Coxsackie virus B3 3C protease, a pharmacological target against enteroviral infections. Using an aldehyde-epoxide as bis-electrophile, active fragment combinations are validated through measuring the protein inactivation rate and by detecting covalent protein modification in mass spectrometry. The structure of one enzyme-inhibitor complex is determined by X-ray crystallography. The presented warhead activation assay provides potent non-peptidic, broad-spectrum inhibitors of enteroviral proteases.
Institute of Pharmacy, Medicinal Chemistry, Freie Universität Berlin, Königin-Luise-Straße 2+4, 14195 Berlin, Germany.
Daniel
D
Becker
Institute for Research in Biomedicine, Parc Científic de Barcelona, Baldiri Reixac 10-12, 08028 Barcelona, Spain.|Institut de Biologia Molecular de Barcelona (CSIC), Parc Científic de Barcelona, Baldiri Reixac 10-12, 08028 Barcelona, Spain.
Zuzanna
Z
Kaczmarska
Institute of Pharmacy, Medicinal Chemistry, Freie Universität Berlin, Königin-Luise-Straße 2+4, 14195 Berlin, Germany.
Christoph
C
Arkona
Institute of Pharmacy, Medicinal Chemistry, Freie Universität Berlin, Königin-Luise-Straße 2+4, 14195 Berlin, Germany.
Robert
R
Schulz
Institute of Pharmacy, Medicinal Chemistry, Freie Universität Berlin, Königin-Luise-Straße 2+4, 14195 Berlin, Germany.
Carolin
C
Tauber
Institute of Pharmacy, Medicinal Chemistry, Freie Universität Berlin, Königin-Luise-Straße 2+4, 14195 Berlin, Germany.
Gerhard
http://orcid.org/0000-0002-5344-0048
G
Wolber
Institute of Biochemistry, University of Lübeck, Ratzeburger Allee 160, 23562 Lübeck, Germany.|German Center for Infection Research (DZIF), Hamburg-Lübeck-Borstel Site, University of Lübeck, 23562 Lübeck, Germany.
Rolf
R
Hilgenfeld
Institute for Research in Biomedicine, Parc Científic de Barcelona, Baldiri Reixac 10-12, 08028 Barcelona, Spain.|Institut de Biologia Molecular de Barcelona (CSIC), Parc Científic de Barcelona, Baldiri Reixac 10-12, 08028 Barcelona, Spain.
Miquel
M
Coll
Institute of Pharmacy, Medicinal Chemistry, Freie Universität Berlin, Königin-Luise-Straße 2+4, 14195 Berlin, Germany.
Jörg
J
Rademann
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