Inactivation of cysteine proteases by (acyloxy)methyl ketones using S '-P ' interactions

We have synthesized (acyloxy)methyl ketone inactivators of papain, cathepsi n B, and interleukin-1 beta conversion enzyme (ICE) that interact with both the S and S' subsites. The value of k(inact)/K-i for these inactivators is strongly dependent on the leaving group. For example, Z-Phe-Gly-CH2-X is a poor inactivator of papain when X is OCOCH3 (k(inact)/K-i = 2.5 M-1 s(-1)) but becomes a potent inactivator when X is OCO-L-Leu-Z (k(inact)/K-i = 11 000 M-1 s(-1)). Since these leaving groups have similar chemical reactiviti es, the difference in potency must be attributed to interactions with the S ' sites. The potency of the leaving group correlates with the P' specificit y of papain, Similar results are also observed for the inactivation of cath epsin B by these compounds. A series of inactivators with the general struc ture Fmoc-L-Asp-CH2-X were designed to inactivate ICE. No inhibition was ob served when X was OCOCH3. In contrast, ICE is inactivated when X is OCO-D-P ro-Z (k(inact)/K-i = 131 M-1 s(-1)). These results demonstrate that S'-P' i nteractions can be utilized to increase the efficacy and selectivity of (ac yloxy)methyl ketone inactivators.