Introduction

The degradation of intracellular proteins into their component amino acids is a highly complex and closely regulated process that leads to downstream effects on a wide array of essential cellular activities. This degradative process fulfills two main functions: (1) It eliminates defective proteins that could potentially harm the cell and (2) it ensures proper

From: Cancer Drug Discovery and Development: The Oncogenomics Handbook Edited by: W. J. LaRochelle and R. A. Shimkets © Humana Press Inc., Totowa, NJ

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Fig. 1. Ubiquitin-proteasome pathway. Ubiquination of a proteins substrate starts with the linkage of ubiquitin with E1 through a thiolester bond (ATP-dependent process). This "activated" ubiquitin is transferred to E2. With the aid of ubiquitin ligase (E3), ubiquitin is covalently linked to the lysine residue of the protein substrate. This now "marked" protein substrate is recognized by the proteas-ome, deubiquination occurs, and the protein enters the proteolytic core for degradation. (Courtesy of Millenium Pharmaceuticals, Inc., Cambridge, MA. Used with permission.)

regulation of cellular metabolism by maintaining adequate levels of enzymes and regulatory proteins. Interestingly, prior to the discovery of the "ubitiquitin-proteasome pathway," intracellular protein degradation was believed to be an unregulated process conducted principally through nonselective lysosomal protein degradation.

A substrate protein begins its degradative pathway by being "marked" through covalent linkage with ubiquitin. Ubiquitin is a highly conserved 76-amino-acid protein that attaches to a target protein through a three-step process involving several enzymes. First, ubiquitin is activated by ubiquitin-activating enzyme (E1) in an ATP-requiring reaction. Activated ubiquitin is then transferred to ubiquitin-conjugating enzyme (E2), which, in turn, presents it to ubiquitin protein ligase (E3). Finally, E3 facilitates covalent linkage of the activated ubiquitin to a target protein. Multiple ubitiquin molecules could tandemly bind to a target protein, resulting in polyubiquination. This ubiquitin-marked protein is then recognized by the proteasome and it is degraded within its proteolytically active 20S chamber, thus the term "ubiquitin-proteasome pathway" (UPP) (1-4) (see Fig. 1).

The 26S proteasome is a 2.5-kDa multiprotein complex that consists of a 20S core particle, flanked by one or two 19S regulatory proteins. It is expressed in all eukaryotic cells, both in the nucleus and cytoplasm, and its main function is the degradation of a large number of intracellular proteins. These include proteins involved in cell cycle regulation (5), apoptosis (6), angiogenesis, transcription factors (7,8), growth factor receptors (9), and signal transduction molecules. Interfering with the normal cycling of these proteins through proteasome inhibition could lead to derangement of key processes, such as cell mitosis,

Fig. 2. Chemical structure ofbortezomib. (Courtesy of Millenium Pharmaceuticals, Inc., Cambridge, MA. Used with permission.)

Fig. 3. Structure of the proteasome. (Left) The 26S proteasome has a proteolytically active 20S core, usually flanked by one or two 19S regulatory subunits, which act as "gate-keepers," recognizing and controlling access of ubiquinated proteins into the core; (right) cross-sectional view of the proteolytic core. Bortezomib binds to the proteolytically active P-subunit, thus inhibiting its chymotrypsin-like activity. (Courtesy of Millenium Pharmaceuticals, Inc., Cambridge, MA. Used with permission.)

Fig. 3. Structure of the proteasome. (Left) The 26S proteasome has a proteolytically active 20S core, usually flanked by one or two 19S regulatory subunits, which act as "gate-keepers," recognizing and controlling access of ubiquinated proteins into the core; (right) cross-sectional view of the proteolytic core. Bortezomib binds to the proteolytically active P-subunit, thus inhibiting its chymotrypsin-like activity. (Courtesy of Millenium Pharmaceuticals, Inc., Cambridge, MA. Used with permission.)

cell adhesion, neoplastic growth, and metastasis, and cause cell apoptosis. Consequently, proteasome inhibition has drawn considerable attention as a potential novel approach for anticancer therapy.

A large number of molecules that interfere with proteasome function, both naturally occurring and synthetic, have been described. Many bind either irreversibly or reversibly to the proteolytically active sites within the 20S core particle, thus inhibiting its function. Among them, boronic acid peptides have shown great promise from the clinical standpoint because of their high potency, selectivity for the proteasome, and stability under physiologic conditions.

Bortezomib (VELCADE; formerly PS-341) is a boronic acid dipeptide derivative and is the first proteasome inhibitor to have progressed to clinical trials (see Fig. 2). It inhibits the proteasome in a highly selective manner through the stability of the boron-Thr'Ogdative bond that forms at the active site of the proteosome, inhibiting its chymotrypsinlike activity (see Fig. 3). Its highly selective property allows other common proteases to be unaffected by this molecule. Bortezomib has shown activity in both preclinical and clinical studies in both solid tumors and hematological malignancies. Its clinical activity in multiple myeloma was found to be especially impressive in phase II trials, leading to its accelerated approval by the Federal Drug Administration (FDA) for the treatment of multiple myeloma in heavily pretreated patients (10). Most recently, a phase III trial in multiple myeloma comparing bortezomib to high-dose dexamethasone in refractory patients was terminated early because of significant advantage of the bortezomib arm.

This chapter will present the molecular rationale, key preclinical data, as well as current clinical trials data evaluating proteasome inhibitors, namely bortezomib, in the management of solid tumors.

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