Dipeptide vinyl sulfones suitable for intracellular inhibition of dipeptidyl peptidase I.

In granules of hematopoetic cells, dipeptidyl peptidase I (DPPI) processes inactive proenzymes into active enzymes, e.g., lymphocyte progranzyme A. Our goal was to develop irreversible inhibitors of intracellular DPPI. First, we identified inhibitors with aqueous stability. Then we determined which inhibitors were nontoxic, could enter cells and inactivate intracellular DPPI. We screened nine dipeptide vinyl sulfone (VS) inhibitors (kobs/[I]>72 M−1 s−1) and found six that were nontoxic. Four affected intracellular DPPI at <25 μM. These compounds contained only uncharged amino acid residues; the two less reactive compounds contained charged Glu residues. The best one, Leu–Phe–VS–CH3, inactivated DPPI in cells with an ID50 of ∼5 μM. This inhibitor was not the best inhibitor of purified DPPI. Longer aqueous stabilities were important predictors of cellular efficacy. Leu–Phe–VS–CH3 had a half life of 97 min at the pH of the extracellular medium (7.5) and 1302 min at pH 5.5 (the intracellular environment of DPPI). This VS had no direct effect on granzyme activities. In contrast, the diazomethyl ketone inhibitor Gly–Phe–CHN2 inhibited chymase activity. Several good intracellular DPPI VS inhibitors lacked reactivity with cathepsins B, H and L. In conclusion, we have identified DPPI inhibitors suitable for cellular applications.