Re-engineering of human urokinase provides a system for structure-based drug design at high resolution and reveals a novel structural subsite

Vicki Nienaber*, Jieyi Wang, Don Davidson, Jack Henkin

*Corresponding author for this work

    Research output: Contribution to journalArticlepeer-review

    62 Scopus citations

    Abstract

    Inhibition of urokinase has been shown to slow tumor growth and metastasis. To utilize structure-based drug design, human urokinase was re- engineered to provide a more optimal crystal form. The redesigned protein consists of residues Ile16-Lys243 (in the chymotrypsin numbering system; for the urokinase numbering system it is Ile159-Lys404) and two point mutations, C122A and N145Q (C279A and N302Q). The protein yields crystals that diffract to ultra-high resolution at a synchroton source. The native structure has been refined to 1.5 Å resolution. This new crystal form contains an accessible active site that facilitates compound soaking, which was used to determine the co-crystal structures of urokinase in complex with the small molecule inhibitors amiloride, 4-iodo-benzo(b)thiophene-2- carboxamidine and phenyl-guanidine at 2.0-2.2 Å resolution. All three inhibitors bind at the primary binding pocket of urokinase. The structures of amiloride and 4-iodo-benzo(b)thiophene-2-carboxamidine also reveal that each of their halogen atoms are bound at a novel structural subsite adjacent to the primary binding pocket. This site consists of residues Gly218, Ser146, and Cys191-Cys220 and the side chain of Lys143. This pocket could be utilized in future drug design efforts. Crystal structures of these three inhibitors in complex with urokinase reveal strategies for the design of more potent nonpeptide urokinase inhibitors.

    Original languageEnglish (US)
    Pages (from-to)7239-7248
    Number of pages10
    JournalJournal of Biological Chemistry
    Volume275
    Issue number10
    DOIs
    StatePublished - Mar 10 2000

    ASJC Scopus subject areas

    • Biochemistry
    • Molecular Biology
    • Cell Biology

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