Customising lactacystins: Applications in cancer chemotherapy & as chemical tools to dissect proteasome functions in vivo

The 20S proteasome is at the core of the normal cycling of proteins in eukaryotic cells. It is responsible for the hydrolytic fragmentation of ubiquitinated proteins and is implicated in the processing of proteins involved in cell-growth regulation, gene transcription, and metabolism (Biochem. Soc. Trans. 2007, 12). As well as their use as important chemical tools to study proteasome regulation of biochemical networks, selective proteasome inhibitors also have great therapeutic potential for the treatment of allergies, asthma and cancer. Over the past 10-15 years, several beta-lactone derived natural products (PS-519, Salinosporamides A-B, Cinnabaramides A-C) and the beta-hydroxyl CysNAc thioester derivatives (lactacystin and cinnabaramide-F) have been identified, with similar modes of activity, as extremely potent covalent inhibitors of the 20S proteasome. Of these, the most intensively studied has been lactacystin (a beta-hydroxy CysNAc thioester-derived metabolite of Streptomyces sp. OM-6519). Its anti-proteolytic activity is mainly due to its lactonic derivative clasto-lactacystin beta-lactone (omuralide). Lactacystin activity is attributed to the spontaneous in vivo formation of this cell-permeable beta-lactone (J. Biol. Chem. 1997, 182), which is a highly specific covalent inhibitor of the 20S proteasome subunit, by O-acylation of its N-terminal threonine sidechain. Once inside the cell, Omuralide undergoes rapid, but reversible, conjugation with glutathione to give a glutathione-thioester derivative of lactacystin. This serves as an intracellular reservoir of Omuralide and minimises its irreversible inactivation by hydrolysis to the beta-hydroxy acid. Reaction kinetics for each of these individual pathways have only been analysed for lactacystin. Cinnabaramides A-C have not yet been studied. The interplay between these competing pathways evidently influences the steady state concentration of the biologically active lactone (ie. immediate potency) and its controlled release (ie. duration of activity) in vivo. A clear understanding of substituent effects on the kinetics of these reaction pathways will ultimately aid the design of proteasome inhibitors whose potency and duration of action can be tailored for optimum effect. Resistance to reversible thiolysis by GSH would elevate the steady-state concentration of the biologically active beta-lactone form of these proteasome inhibitors. Proteasome inhibitors are also used to target the nuclear transcription factor NF-kappaB, which plays a central role in inflammation, proliferation and anti-apoptotic pathways. Therefore, new and improved derivatives of Lactacystins, Salinosporamides & Cinnabaramides can also serve as chemical tools for cell biologists to help unravel the functions of NF-kappaB signalling pathways in vivo. It is noteworthy that Salinosporamide-A is already in clinical trial for the treatment of multiple myeloma. However the difficulties faced in the synthesis of such molecules limits the accessibility to improved analogues of Salinosporamide-A, which itself is currently best prepared by whole cell synthesis. We have developed a remarkably short and flexible synthetic approach to this group of compounds, which can be readily modified and extended to provide access to the salinosporamides & cinnabaramides, and which circumvents the limitations of previous (and more laborious) preparations. OBJECTIVES 1. Synthesis of lactacystin, Omuralide, Cinnabaramides and a range of structural derivatives. 2. Mechanistic studies of these compounds in terms of their susceptibility to inactivation (by hydrolysis) and intracellular trapping by reaction with glutathione, and their potency as proteasome inhibitors 3. Whole cell studies of inhibitor effects on cytotoxicity, cell proliferation, NF-kappaB activation and duration of activity in cancer cell lines.