A cell-permeable inhibitor and activity-based probe for the caspase-like activity of the proteasome.

The ubiquitin-proteasome pathway degrades the majority of proteins in mammalian cells and plays an essential role in the generation of antigenic peptides presented by major histocompatibility class I molecules. Proteasome inhibitors are of great interest as research tools and drug candidates. Most work on proteasome inhibitors has focused on the inhibition of the chymotryptic-like (beta5) sites; little attention has been paid to the inhibition of two other types of active sites, the trypsin-like (beta2) and the caspase-like (beta1). We report here the development of the first cell-permeable and highly selective inhibitors (4 and 5) of the proteasome's caspase-like site. The selectivity of the compounds is directly and unambiguously established by Staudinger-Bertozzi labeling of proteasome subunits covalently modified with azide-functionalized inhibitor 5. This labeling reveals that the caspase-like site of the immunoproteasome (beta1i) is a preferred target of this compound. These compounds can be used as tools to study roles of beta1 and beta1i sites in generation of specific antigenic peptides and their potential role as co-targets of anti-cancer drugs.

A fluorescent broad-spectrum proteasome inhibitor for labeling proteasomes in vitro and in vivo.

The proteasome is an essential evolutionary conserved protease involved in many regulatory systems. Here, we describe the synthesis and characterization of the activity-based, fluorescent, and cell-permeable inhibitor Bodipy TMR-Ahx(3)L(3)VS (MV151), which specifically targets all active subunits of the proteasome and immunoproteasome in living cells, allowing for rapid and sensitive in-gel detection. The inhibition profile of a panel of commonly used proteasome inhibitors could be readily determined by MV151 labeling. Administration of MV151 to mice allowed for in vivo labeling of proteasomes, which correlated with inhibition of proteasomal degradation in the affected tissues. This probe can be used for many applications ranging from clinical profiling of proteasome activity, to biochemical analysis of subunit specificity of inhibitors, and to cell biological analysis of the proteasome function and dynamics in living cells.

Solid-phase synthesis of succinylhydroxamate peptides: functionalized matrix metalloproteinase inhibitors.

[reaction: see text] A novel solid-phase synthesis strategy toward succinylhydroxamate peptides, using an appropriately protected hydroxamate building block, is described. Rapid and efficient access is gained to amine-functionalized peptides, which can be decorated with, for instance, a fluorescent label. In addition, we demonstrate an on-resin synthesis of a biotinylated photoactivatable hydroxamate peptide, which can be used as an activity-based probe for matrix metalloproteinases and ADAMs.

Bioorthogonal organic chemistry in living cells: novel strategies for labeling biomolecules.

The chemical labeling of biomolecules continues to be an important tool for the study of their function and cellular fate. Attention is increasingly focused on labeling of biomolecules in living cells, since cell lysis introduces many artefacts. In addition, with the advances in biocompatible synthetic organic chemistry, a whole new field of opportunity has opened up, affording high diversity in the nature of the label as well as a choice of ligation reactions. In recent years, several different two-step labeling strategies have emerged. These rely on the introduction of a bioorthogonal attachment site into a biomolecule, then ligation of a reporter molecule to this site using bioorthogonal organic chemistry. This Perspective focuses on these techniques, their implications and future directions.

Blocking endothelial adhesion molecules: a potential therapeutic strategy to combat atherogenesis.

PURPOSE OF REVIEW: This review provides a concise update of the involvement of endothelial adhesion molecules in atherogenesis, an overview of current advances in the development of adhesion molecule blocking agents, as well as an insight into the potential of these molecules in cardiovascular therapy. RECENT FINDINGS: As endothelial adhesion molecules are deemed to play an important role in the development and progression of atherosclerotic lesions, they are interesting targets for therapeutic intervention in this process. In particular, P-selectin and vascular cell adhesion molecule 1 are widely considered to hold promise in this regard. Current research efforts centre on the design of agents that directly block the interaction of the receptor with its ligand (e.g. soluble P-selectin glycoprotein ligand 1, blocking antibodies, EWVD-based peptides) or that interfere with their synthesis (e.g. antisense oligonucleotides) or their regulatory control by nuclear factor kappa B or peroxisome proliferator-activated receptor gamma. Furthermore, adhesion molecules have been exploited as a target for the specific delivery of drug carriers (e.g. biodegradable particles with entrapped dexamethasone) or therapeutic compounds (e.g. dexamethasone) to the plaque. All approaches have been shown to be effective in blocking adhesion molecule function in in-vitro studies and in-vivo models for inflammation or atherosclerosis. SUMMARY: Although the field has achieved considerable progress in recent years, leading to the development of a number of interesting leads, final proof of their efficacy in cardiovascular therapy is eagerly awaited.

Activity profiling of papain-like cysteine proteases in plants.

Transcriptomic and proteomic technologies are generating a wealth of data that are frequently used by scientists to predict the function of proteins based on their expression or presence. However, activity of many proteins, such as transcription factors, kinases, and proteases, depends on posttranslational modifications that frequently are not detected by these technologies. Therefore, to monitor activity of proteases rather than their abundance, we introduce protease activity profiling in plants. This technology is based on the use of biotinylated, irreversible protease inhibitors that react with active proteases in a mechanism-based manner. Using a biotinylated derivative of the Cys protease inhibitor E-64, we display simultaneous activities of many papain-like Cys proteases in extracts from various tissues and from different plant species. Labeling is pH dependent, stimulated with reducing agents, and inhibited specifically by Cys protease inhibitors but not by inhibitors of other protease classes. Using one-step affinity capture of biotinylated proteases followed by sequencing mass spectrometry, we identified proteases that include xylem-specific XCP2, desiccation-induced RD21, and cathepsin B- and aleurain-like proteases. Together, these results demonstrate that this technology can identify differentially activated proteases and/or characterize the activity of a particular protease within complex mixtures.

Development of an isotope-coded activity-based probe for the quantitative profiling of cysteine proteases.

Quantification studies of complex protein mixtures have been restricted mainly to whole cell extracts. Here we describe the synthesis of two sets of isotope-coded activity-based probes that allow quantitative functional proteomics experiments on the cathepsins.

Synthesis and elaboration of functionalised carbohydrate-derived spiroketals.

The scope of a stereoselective three-step approach for the synthesis of sugar derived spiroketals is presented. The methodology consists of Grignard addition of vinyl- or allylmagnesium bromide to a carbohydrate lactone, followed by K-10 clay mediated glycosidation with a terminal alkenol and subsequent ring-closing metathesis of the resulting diene. The generality of this procedure is demonstrated by the synthesis of various pyranose- and furanose-derived spiroketals, as well as more advanced tricyclic spiroketal derivatives. It is shown that functionalisation of the double bond in the resulting spiroketals leads to fused polycyclic ethers.

Olefin metathesis in glycobiology: new routes towards diverse neoglycoconjugates.

This report provides a historical overview of the implementation of olefin metathesis in carbohydrate chemistry, with the emphasis on the construction of neoglycoconjugates. The current state-of-the-art in catalyst design allows the preparation of carbohydrate-containing molecules with great structural and functional diversity. Recent examples of complex neoglycoconjugates, including glycopolymers, glycoclusters, glycopeptide and glycolipid analogues are highlighted. Finally, future perspectives in the scope of olefin metathesis mediated neoglycoconjugate synthesis are discussed.