Design and synthesis of inhibitors for serine and cysteine proteases

A series of peptide (alpha-aminoalkyl)phosphonate diphenyl ester inhibitors was designed for Granzyme H (GrH), both for predicted greater potency and to help elucidate the specificity of the various rat and mouse chymases, most of which have been incompletely studied. Phosphonates are highly efficient irreversible inhibitors that are effective against serine proteases while showing no activity towards cysteine proteases. They are stable under physiological conditions and are unreactive to most nucleophiles. These phosphonates were designed using the known preference of GrH for Phe or Phe analogs at P1, combined with various N-terminal blocking groups to determine what structures were preferred for potency. A series of 14 inhibitors was designed using the sequences Suc-Phe-Leu-AA1P(OPh)2 and 3-phenoxybenzoyl-AA1P(OPh)2 as starting points, where AA1 was Phe, Hphe, or PhGly. The most potent compound of the series was Suc-Phe-Leu-HpheP(OPh)2, which exhibited 65% inhibition against GrH. A close second in potency was Suc-Phe-Leu-PhGly P(OPh)2, which had 55% inhibition against GrH. Both of these compounds exhibit percent inhibitions with GrH equivalent to or better than the best published inhibitors for this enzyme.; Based on the reported optimal tetrapeptide sequences for caspases 3, 6, and 8, a series of alpha-keto amide inhibitors was designed. alpha-Keto amides are competitive reversible transition state inhibitors that often exhibit slow binding inhibition. They have the advantage of being fairly stable and having decent cell permeability, as well as demonstrated biological activity in vivo. The biggest advantage, however, is that alpha-keto amides can be extended in the P' direction, allowing for additional enzyme-inhibitor interactions and generating increased potency and specificity. With that in mind, the 7 inhibitors synthesized utilized an optimal sequence for either Caspase 6 (Z-Val-Glu-(Ile/Val)-Asp-CO-NH-R) or Caspase 8 (Z-Leu-Glu-Thr-Asp-CO-NH-R), as well as a variety of P ' residues such as those where R = CH2Ph, (CH2)2Ph, (CH2)3Ph, and (CH 2)3OCH3. Two of these compounds, Z-Leu-Glu-Thr-Asp-CO-NH-(CH 2)2Ph and Z-Leu-Glu-Thr-Asp-CO-NH-(CH2)3 OCH3, were potent and selective inhibitors of caspase 8. Although slightly lower in potency than the best previously published inhibitors of caspase 8, these alpha-keto amides are much more selective, making them ideal for further studies and optimization.