Exploiting subsite S1 of trypsin-like serine proteases for selectivity: Potent and selective inhibitors of urokinase-type plasminogen activator

A nonselective inhibitor of trypsin-like serine proteases, 2-(2-hydroxybiph enyl-3-yl)-1H-indole-5-carboxamidine (1) (Verner, E.; Katz, B. A.; Spencer, J.; Allen, D.; Hataye, J.; Hruzewicz, W.; Hui, H. C.; Kolesnikov, A.; Li, Y.; Luong, C.; Martelli, A.; Radika. K.; Rai, R.; She, M.; Shrader, W.; Spr engeler, P. A.; Trapp, S.; Wang, J.; Young, W. B.; Mackman, R. L. J. Med. C hem. 2001, 44, 2753-2771) has been optimized through minor structural chang es on the S1 binding group to afford remarkably selective and potent inhibi tors of urokinase-type plasminogen activator (uPA). The trypsin-like serine proteases' that comprise drug targets can be broadly categorized into two subfamilies, those with Ser190 and those with Ala190. A single-atom modific ation, for example, replacement of hydrogen for chlorine at the 6-position of the 5-amidinoindole P1 group on 1, generated up to 6700-fold selectivity toward the Ser190 enzymes and against the Ala190 enzymes. The larger chlor ine atom displaces a water molecule (H(2)O1(S1)) that binds near residue 19 0 in all the complexes of 1, and related inhibitors, in uPA, thrombin, and trypsin. The water molecule, H(2)O1(S1), in both the Ser190 or Ala190 enzym es, hydrogen bonds with the amidine N1 nitrogen of the inhibitor. When it i s displaced, a reduction in affinity toward the Ala190 enzymes is observed due to the amidine N1 nitrogen of the bound inhibitor being deprived of a k ey hydrogen-bonding partner. In the Ser190 enzymes the affinity is maintain ed since the serine hydroxyl oxygen O gamma (Ser190) compensates for the di splaced water molecule. High-resolution crystallography provided evidence f or the displacement of the water molecule and validated the design rational e. In summation, a novel and powerful method for engineering selectivity to ward Ser190 proteases and against Ala190 proteases without substantially in creasing molecular weight is described.