Selective inhibitors of the serine protease plasmin: probing the S3 and S3' subsites using a combinatorial library

A combinatorial library of 400 serine protease inhibitors with the general structure Cbz-X(aa)-Trp-cyclohexanone-Trp-Y(aa)-OH has been constructed. The library was synthesized on the solid phase using mix-and-split synthesis, where 20 different amino acids were incorporated at both the X(aa) and Y(aa) positions. These two positions correspond to the S3 and S3' subsites of the active site. Iterative deconvolution was used to identify hits from the library. The library was screened against four serine proteases: plasmin, kallikrein, thrombin, and trypsin. Seven inhibitors from the library that showed promising activities were resynthesized using solution-phase methods. Four of these compounds were good inhibitors of plasmin with IC(50) values in the range of 2.7-3.6 microM. The most potent of these inhibitors showed >150-fold selectivity for plasmin when compared to the other three serine proteases.