Design of inhibitors for human tissue kallikrein using non-natural aromatic and basic amino acids.

We explored the unique substrate specificity of the primary S1 subsite of human urinary kallikrein (hK1), which accepts both Phe or Arg synthesizing and assaying peptides derived from Phenylacetyl-Phe-Ser-Arg-EDDnp, a previously described inhibitor with analgesic and anti-inflammatory activities. Phe was substituted by amino acids contg. larger aliph. or arom. side chains as well as by non-natural basic amino acids, which were designed to combine a large hydrophobic and/or arom. group with a pos.-charged group at their side chains. In general, all peptides with basic amino acids represented better inhibitors than those with hydrophobic amino acids. Furthermore, the S1 subsite specificity proved to be much more selective than the mere distinction between Phe and Arg, for minor differences in the side chains of the non-natural amino acids resulted in major differences in the Ki values. Finally, we present a series of peptides that were assayed as competitive inhibitors for human tissue kallikrein that may lead to the development of novel peptides, which are both more potent and selective.