Selective targeting of tumor and stromal cells by a nanocarrier system displaying lipidated cathepsin B inhibitor.

Cathepsin B (CtsB) is a lysosomal cysteine proteinase that is specifically translocated to the extracellular milieu during cancer progression. The development of a lipidated CtsB inhibitor incorporated into the envelope of a liposomal nanocarrier (LNC-NS-629) is described. Ex vivo and in vivo studies confirmed selective targeting and internalization of LNC-NS-629 by tumor and stromal cells, thus validating CtsB targeting as a highly promising approach to cancer diagnosis and treatment.

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

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.

Inhibition of papain-like cysteine proteases and legumain by caspase-specific inhibitors: when reaction mechanism is more important than specificity.

We report here that a number of commonly used small peptide caspase inhibitors consisting of a caspase recognition sequence linked to chloromethylketone, fluoromethylketone or aldehyde reactive group efficiently inhibit other cysteine proteases than caspases. The in vitro studies included cathepsins B, H, L, S, K, F, V, X and C, papain and legumain. Z-DEVD-cmk was shown to be the preferred irreversible inhibitor of most of the cathepsins in vitro, followed by Z-DEVD-fmk, Ac-YVAD-cmk, Z-YVAD-fmk and Z-VAD-fmk. Inactivation of legumain by all the inhibitors investigated was moderate, whereas cathepsins H and C were poorly inhibited or not inhibited at all. Inhibition by aldehydes was not very potent. All the three fluoromethylketones efficiently inhibited cathepsins in Jurkat and human embryonic kidney 293 cells at concentrations of 100 microM. Furthermore, they completely inhibited cathepsins B and X activity in tissue extracts at concentrations as low as 1 microM. These results suggest that data based on the use of these inhibitors should be taken with caution and that other proteases may be implicated in the processes previously ascribed solely to caspases.