In October 2004, the Royal Swedish Academy of Sciences awarded the Nobel Prize in Chemistry to Aaron Ciechanover, Avram Hersko, and Irwin Rose "for the discovery of ubiquitin-mediated protein degradation." Their seminal work in the 1970s and 1980s opened the door for the next generation of "ubiquitinologists," including Alexander Varshavsky, Keith Wilkinson, and Arthur Haas, who discovered that ubiquitin (8 kDa), a highly conserved protein, becomes covalently attached to lysine residues of target proteins (ubiquitination). The researchers subsequently showed that this attachment occurs through a three-step process involving ubiquitin-activating (E1), ubiquitin-conjugating (E2), and ubiquitin-ligating (E3) enzymes.
The work has led to a new class of therapeutic targets associated with the ubiquitin-proteasome pathway that offers the potential for treatment of cachexia (also known as muscle atrophy) and other refractory diseases. This pathway closely regulates the selective degradation of cellular proteins and is therefore...
MULTIPLE FUNCTIONS
The ubiquitin pathway degrades a multitude of cellular proteins, including cell cycle regulators, growth- and differentiation-controlling factors, transcriptional activators, cell-surface receptors and ion channels, endoplasmic reticulum proteins, antigenic proteins destined for presentation on class I MHC molecules, and abnormal and misfolded proteins.567 Potential therapeutic indications for effectors of ubiquitin-pathway components are numerous, including cancer, diabetes, cardiovascular disease, metabolic disease, inflammation, and neurodegeneration. While most of the ubiquitin-based research has been related to protein degradation, other ubiquitin functions and applications are equally exciting, such as the discovery of the N-end rule (the identity of the N-terminal amino acid governs the half-life of a protein) by Alexander Varshavsky and functions of ubiquitination not related to proteasomal degradation.89 Both discoveries have therapeutic implications.
For example, ubiquitination controls membrane receptor trafficking to endosomes and thus receptor activity. Therapeutic implications include cancer and inflammation. In the case of Varshavsky's N-end rule, engineering specific N-terminal amino acids of a protein therapeutic can enhance the in vivo half-life of the protein. This discovery could potentially have an impact on the entire protein therapeutic market; nevertheless, to date, the authors are unaware if the N-end rule has been utilized to optimize the half-life of a protein therapeutic. An obvious reason could be the arduous task of manufacturing biologically active proteins.
Velcade, the only approved therapeutic agent that targets the ubiquitin-proteasome pathway, achieved $143 million in net product sales with quarter-over-quarter growth in 2004, according to Millennium's annual report. Currently, it is marketed only for refractory multiple myeloma, but trials for other indications are underway and there is considerable off-label use. Velcade may soon face direct competition from Nereus Pharmaceuticals' NPI-0052 candidate proteasome inhibitor, which is active not only against the chymotryptic component of the proteasome (as is Velcade) but also against the tryptic component (which is not affected by Velcade). Nereus hopes to file an investigational new drug application for NPI-0052 by the end of 2005.
Apart from Velcade, no drug candidate that targets the ubiquitin-proteasome pathway has yet reached the clinic. This does not indicate that research activity has lessened in intensity (see Table). Ubiquitin-related research continues to increase in the pharmaceutical industry and, even more impressively, in academia, where hundreds of papers on the subject have been published. This reflects the greater freedom to pursue interesting questions in academic research than in pharmaceutical research, which is restricted by the business model.
A cell with various ubiquitin and ubiquitin-like (Ubl) pathway-related functions. While protein degradation in the proteasomal machinery is the first discovered and best known effect of ubiquitin pathway enzymes, recent studies have revealed additional physiological consequences of protein ubiquitination and de-ubiquitination.
In addition to Millennium and Nereus, the list of pharmaceutical industry ubiquitin pioneers includes Celgene, Progenra, Regeneron, and Rigel. After Millennium and Nereus, the company with a compound closest to the clinic is Celgene, whose inhibitors of E2 ligases are in advanced pre-clinical development. Both Regeneron and Rigel are interested in E3 ligases but have not yet announced a clinical candidate. Regeneron is focusing on muscle atrophy and Rigel on cancer, inflammatory diseases, and virology. Rigel collaborated with Merck last November to investigate E3 ligases, and references to Genentech, Novartis, and Roche are appearing more in the literature. Among the large pharmas, Roche may be the most advanced, based on its recent publication in
E3S AND DUBS
Of the potential targets, which include E1, E2s (approx. 50), E3s (~500 to 700), and DUBs (~65 to 100), E3s were initially considered the most attractive due to their quantity, diversity, and selectivity. Each unique E3 is believed to ubiquitinate only a few substrates. Despite several years of focus on E3s, an inhibitor has yet to reach the clinic. While E2s and DUBs may be less selective than E3s, the number of DUBs can, in theory, afford a good deal of selectivity. DUBs received comparatively less attention than E3 ligases as therapeutic targets early on, but Progenra recently filed patents for DUB assays, indicating that they are now being evaluated as therapeutic targets. The rationale is similar to that for E3-based drug discovery efforts: hastening or delaying the natural ubiquitin-assisted degradation of critical proteins can be therapeutic. Since a protein needs a polyubiquitin tag (four or more ubiquitins) to enter the proteasome for degradation, DUB catalyzed removal of a single ubiquitin would preserve a tagged protein. In this respect, DUBs perform an even more critical function as "editors" of the ubiquitin pathway.
Biochemical and genetic evidence implicates several ubiquitin E3 ligases and DUBs in various pathologies, and several comprehensive reviews linking ubiquitin-pathway components to disease are available.512 In addition to MuRF1, examples of E3 ligases are pVHL, mutated in certain cancers and behaving as a tumor suppressor,13 and parkin, mutated in juvenile parkinsonism.14 A DUB called UCHL1 (Pgp9.5) has been overexpressed in certain refractory cancers and associated with poor prognoses.1516 By developing and validating therapeutic hypotheses based on this emerging body of knowledge, it should be possible to discover inhibitors or activators of ubiquitin-pathway targets that will become part of the next generation of molecularly targeted drugs for a variety of refractory and devastating illnesses.
John Hall (
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