Specificity of aza-peptide electrophile activity-based probes of caspases

Abstract

Activity-Based Probes (ABPs) are small molecules that form stable covalent bonds with active enzymes thereby allowing detection and quantification of their activities in complex proteomes. A number of ABPs that target proteolytic enzymes have been designed based on well-characterized mechanism-based inhibitors. We describe here the evaluation of a novel series of ABPs based on the aza-aspartate inhibitory scaffold. Previous in vitro kinetic studies showed that this scaffold has a high degree of selectivity for the caspases, clan CD cysteine proteases activated during apoptotic cell death. Aza-aspartate ABPs containing either an epoxide or Michael acceptor reactive group were potent labels of executioner caspases in apoptotic cell extracts. However they were also effective labels of the clan CD protease legumain and showed unexpected crossreactivity with the clan CA protease cathepsin B. Interestingly, related aza peptides containing an acyloxymethyl ketone reactive group were relatively weak but highly selective labels of caspases. Thus azapeptide electrophiles are valuable new ABPs for both detection of a broad range of cysteine protease activities and for selective targeting of caspases. This study also highlights the importance of confirming the specificity of covalent protease inhibitors in crude proteomes using reagents such as the ABPs described here.

An Optimized Activity-Based Probe for the Study of Caspase-6 Activation

“…The active site of this enzyme has a similar overall fold to the caspases but is thought to predominantly cleave protein substrates after aspargine residues (Abe et al, 1993). However, legumain is also able to bind aspartic acid-containing probes at the reduced pH of the lysosome (Kato et al, 2005; Sexton et al, 2007). To monitor selectivity, we used both AB50 and LE22 to label RAW cells, an immortalized mouse macrophage line that expresses high levels of active legumain and cathepsins (Figure 2A).…”

Noninvasive optical imaging of apoptosis by caspase-targeted activity-based probes

“…However the issue of labeling of this off-target protease can potentially be reduced by pre-treatment with inhibitors that show exquisite specificity for legumain. For example, pre-blocking of legumain activity using aza-epoxide containing a P1 asparagine 32 should allow subsequent exclusive labeling of caspases using the AB50 probe series. Alternatively, it may not be necessary to use probes with absolute caspase selectivity for imaging apoptosis in vivo .…”

Small Molecule Active Site Directed Tools for Studying Human Caspases

“…Likewise aza-aspartate ABP with an elongated peptide motif (b-EVazaD-epoxide 471 ) effectively labeled caspase-3/7 and additionally showed labeling of caspase-9, in a good agreement with previously characterized b-EVD-AOMKMe probe 476 . 397 …”

“…Moreover, Sexton et al pointed out differences between kinetic measurements using recombinant enzymes and experiments on cell lysates. 397 …”