Modulation of the Inhibitor Properties of (Acyloxy)methyl Ketones Toward the CaaX Proteases
Dipeptidyl (acyloxy)methyl ketones (AOMKs) have been identified as mechanism-based inhibitors of certain cysteine proteases. These compds. are also inhibitors of the integral membrane proteins Rce1p and Ste24p, which are proteases that independently mediate a cleavage step assocd. with the maturation of certain isoprenylated proteins. The enzymic mechanism of Rce1p is ill-defined, whereas Ste24p is a zinc metalloprotease. Rce1p is required for the proper processing of the oncoprotein Ras and is viewed as a potential target for cancer therapy. In this study, we synthesized a small library of dipeptidyl AOMKs to investigate the structural elements that contribute to the inhibitor properties of this class of mols. toward Rce1p and Ste24p. The compds. were evaluated using a fluorescence-based in vitro proteolysis assay. The most potent dipeptidyl AOMKs contained an arginine residue and the identity of the benzoate group strongly influenced potency. A warhead' free AOMK inhibited Rce1p and Ste24p. The data suggest that the dipeptidyl AOMKs are not mechanism-based inhibitors of Rce1p and Ste24p and corroborate the hypothesis that Rce1p is not a cysteine protease.