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

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 ILe(16)-Lys(243) (in the chymotrypsin numbering system; for the ur okinase numbering system it is ILe(159)-Lys(404)) and two point mutations, C122A and N145Q (C279A and N302Q). The protein yields crystals that diffrac t to ultra-high resolution at a synchrotron source. The native structure ha s been refined to 1.5 Angstrom resolution. This new crystal form contains a n accessible active site that facilitates compound soaking, which was used to determine the co-crystal structures of urokinase in complex with the sma ll molecule inhibitors amiloride, 4-iodo-benzo(b)thiophene-2-carboxamidine and phenylguanidine at 2.0-2.2 Angstrom resolution. All three inhibitors bi nd 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 prim ary binding pocket. This site consists of residues Gly(218), Ser(146), and Cys(191)-Cys(220) and the side chain of Lys(143). This pocket could be util ized in future drug design efforts. Crystal structures of these three inhib itors in complex with urokinase reveal strategies for the design of more po tent nonpeptidic urokinase inhibitors.