Inhibition of the CaaX proteases Rce1p and Ste24p by peptidyl (acyloxy)methyl ketones

Abstract

The CaaX proteases Rce1p and Ste24p can independently promote a proteolytic step required for the maturation of certain isoprenylated proteins. Although functionally related, Rce1p and Ste24p are unrelated in primary sequence. They have distinct enzymatic properties, which are reflected in part by their distinct inhibitor profiles. Moreover, Rce1p has an undefined catalytic mechanism, whereas Ste24p is an established zinc-dependent metalloprotease. This study demonstrates that both enzymes are inhibited by peptidyl (acyloxy)methyl ketones (AOMKs), making these compounds the first documented dual specificity inhibitors of the CaaX proteases. Further investigation of AOMK-mediated inhibition reveals that varying the peptidyl moiety can significantly alter the inhibitory properties of AOMKs toward Rce1p and Ste24p and that these enzymes display subtle differences in sensitivity to AOMKs. This observation suggests that this compound class could potentially be engineered to be selective for either of the CaaX proteases. We also demonstrate that the reported sensitivity of Rce1p to TPCK is substrate-dependent, which significantly alters the interpretation of certain reports having used TPCK sensitivity for mechanistic classification of Rce1p. Finally, we show that an AOMK inhibits the isoprenylcysteine carboxyl methyltransferase Ste14p. In sum, our observations raise important considerations regarding the specificity of agents targeting enzymes involved in the maturation of isoprenylated proteins, some of which are being developed as anti-cancer therapeutic agents.

A novel chemical screening strategy in zebrafish identifies common pathways in embryogenesis and rhabdomyosarcoma development

“…TPCK has been shown to be a potent in vivo inhibitor of S6K1, PDK1 and other related kinases with a conserved domain (known as AGC kinases), although an in vitro kinase assay showed that S6K1 is not the direct molecular target of TPCK (Ballif et al, 2001;Grammer and Blenis, 1996). TPCK has also been described as inhibiting the endoprotease responsible for cleaving the C-terminal AAX sequence on RAS (Porter et al, 2007). Alternative mechanisms have been proposed including recent work demonstrating that TPCK blocks specific cysteine residues on IkappaB kinase (IKK) and p65/RelA (Ha et al, 2009).…”

Biogenesis of the Saccharomyces cerevisiae Pheromone a -Factor, from Yeast Mating to Human Disease

“…Other reports have suggested that Rce1 may be a noncanonical zinc metalloprotease (202). Several residues important for activity have been identified by mutational analysis but do not provide further clarification of the protease category to which Rce1 belongs (204), nor do standard protease inhibitor studies (206). Until Rce1 can be purified, it will not be possible to know whether it is solely responsible for endoproteolysis or instead whether it requires a binding partner or stimulates another protease.…”

“…Purified Ste24 has been demonstrated to be a zinc metalloprotease (255), as discussed in detail below. Rce1, on the other hand, has been refractory to purification and lacks a recognizable protease motif (33,199,204,206). Thus, it remains possible that Rce1 may be necessary, but not sufficient, for AAXing, functioning to activate an as-yet-unidentified protease.…”

“…Because the oncoprotein Ras is mutated in many cancers, small-molecule inhibitors of Rce1 are of clinical and therapeutic interest (99,272). Several of these have been identified, including those from a compound library screening effort (77,171,206). Of the latter inhibitors, some are specific for Rce1, while others inhibit both Rce1 and Ste24.…”

Heterologous Expression Studies of Saccharomyces cerevisiae Reveal Two Distinct Trypanosomatid CaaX Protease Activities and Identify Their Potential Targets

“…To date, the most advanced drug discovery efforts have focused on farnesyltransferase inhibitors (FTIs) (9,53). Inhibitors of the CaaX proteases and ICMT are also being developed (1,11,28,37,39,48). Disrupting CaaX protein modifications has therapeutic application to other diseases as well.…”