Highly potent inhibitors of human cathepsin L identified by screening combinatorial pentapeptide amide collections

By screening a combinatorial pentapeptide amide collection in an inhibition assay, we systematically evaluated the potential of 19 proteinogenic amino acids and seven nonproteinogenic amino acids to serve as building blocks f or inhibitors of human cathepsin L. Particularly efficient were aromatic, b ulky, hydrophobic amino-acid residues, especially leucine, and positively c harged residues, especially arginine. Building blocks for potential inhibit ory peptides were combined by random selection from their activity pattern. This random approach for the design of inhibitors was introduced to compen sate for the inaccuracy induced by shifted docking of combinatorial compoun d collections at the active center of cathepsin L. Thereby, we obtained str ucturally defined pentapeptide amides which inhibited human cathepsin L at nanomolar concentrations. Among the most potent novel inhibitors, one pepti de, RKLLW-NH2, shares the amphiphilic character of the nonamer fragment VMN GLQNRK of the autoinhibitory, substrate-like, but reverse-binding prosegmen t of human cathepsin L which blocks the active center of the enzyme. Obviou sly, RKLLW-NH2 carries the functions that are important for enzyme-peptide interaction in a condensed form. This hypothesis was confirmed by structure -activity studies using truncated and modified pentapeptides.