The Ubiquitin System and Drug Discovery

By John Mayer, Professor of Molecular Cell Biology, School of Biomedical Sciences, University of Nottingham Medical School, Queen's Medical Centre, Nottingham, UK.

The successful launch of the proteasome inhibitor, bortezomib (Velcade) by Millennium in 2003, for the treatment of multiple myeloma, has brought significant interest in the ubiquitin-proteasome system (UPS) as a potential area to target for the development of new therapies.  Many have been surprised by the specificity and effectiveness of this reversible inhibitor of proteasomal catalytic activity, since proteasomes are found in all cell types. Several companies are now actively working to develop inhibitors of different targets within the UPS, including Novartis, Hybrigenics, Rigel Pharmaceuticals, Proteolix, Millennium and Nereus Pharmaceuticals.

Ubiquitin and disease
One of the reasons that the UPS has sparked so much interest so rapidly in the drug discovery sector is its involvement in nearly every process in the cell, and hence in many of the world’s major diseases.  This includes, for example, chronic neurodegenerative diseases [1], myeloma [2], lymphoma [3], renal cell carcinoma [4], immune and inflammatory disorders [5] metabolic diseases such as diabetes [6], conditions involving obesity [7] and muscle wasting [8].

The importance of the original discovery of ubiquitin and the enzymes involved in activating and conjugating ubiquitin (E1, E2 and E3 proteins) and then removing ubiquitin from protein targets (deubiquitylating enzymes - DUBs) was recently recognised by the founders of the field (Avram Hershko, Aaron Ciechanover and Irwin Rose) receiving the Nobel Prize for Chemistry in 2004. One reason for this award was that the complexity and sophistication of the ubiquitin system rivals and compliments phosphorylation and dephosphorylation in the cell, demonstrating its importance in numerous cellular processes during development and homeostasis, and ultimately, disease.

Role of ubiquitin
Ubiquitin was discovered as a small 76 amino acid protein that could be covalently conjugated to target proteins for intracellular protein degradation. The roots of the ubiquitin conjugation reactions were found to be in prokaryotic biosynthetic pathways for thiamine and molybdopterin [9-11].

As might be anticipated, there are many downstream receptors for ubiquitylated proteins in the cell including proteins that contain ubiquitin-binding/-interacting domains, e.g. UIM, UBA, CUE, GAT, UEV, NZF, UBM and UBZ domains [12]. 

The fact that ubiquitin has so many regulatory roles in the cell raises the question of how many genes exist that code for the enzymes of the UPS.  In humans, in addition to the nine genes coding for the E1s (the ubiquitin activating enzymes), there appear to be 34 genes for the E2s (the ubiquitin conjugating enzymes), and 531 genes for the E3s (the ubiquitin-protein ligases).  There are also a further 88 genes for the DUBs.  As a basis for comparison, there are 518 kinase genes in the “kinome” [13].

Ubiquitin is not only conjugated to lysine residues in target proteins via isopeptide bonds, but can be conjugated to itself by isopeptide bonds to form polyubiquitin chains on target proteins.  Furthermore, different lysine residues in ubiquitin can be used to form chains and analyses in yeast indicate that all seven lysines in ubiquitin can be used to form such chains [14].  Differentially tagged proteins may be recognised by yet more downstream signalling proteins and other molecules in the cell to control diverse cellular functions, and a wide range of ubiquitin binding sites in proteins has already been discovered [12].

Next frontiers
For progress in research surrounding the UPS to move towards the development of major new therapies on the market for diseases such as cancers and chronic neurodegenerative diseases, the substrates of many of the E3s and DUBs need to be identified. Modern proteomics coupled with sophisticated mass spectrometic analyses is ideally suited to the task of detecting post-translational modifications of regulatory and housekeeping proteins by ubiquitin [14].  However, the approaches will need to be quantitative and involve time-related analyses of the patterns and types of ubiquitylation of proteins in response to the stimulation of specific signal transduction pathways, for example, the resolution of the protein-ubiquitin landscape in response to cellular growth factors or apoptotic stimuli.

Elucidation of the combination of E3s and DUBs as specific protein targets in key regulatory pathways would lead to screens for compounds that inhibit either an E3 or DUB respectively to increase or decrease the activity of some signal transduction pathway as a prelude to therapeutic intervention in diseases controlled by different signal transduction pathways in the cell.  Once verified at the cultured cellular level, these approaches could be extended to the analyses of patterns of ubiquitylation/deubiquitylation in animal models of disease.  Transgenic and gene targeted mice could be used to focus on specific changes in the landscape of ubiquitylation/deubiquitylation in specific tissues in relation to the expression of disease-causing genes (transgenes) or ablation of expression of the genes (gene targeting).

Conclusions
The proteasomal catalytic inhibitor, bortezomib (Millennium) has led the way as an effective drug for the treatment of multiple myeloma. Additionally, Millennium is now in Phase 2 clinical trials with bortezomib for other haematological malignancies and solid tumours. There is growing interest in the development of compounds that inhibit DUBs and particular interest in the search for inhibitors of E3s to cause disruption of the cell cycle and therefore be effective as new anti-tumour agents. Clearly, the current proteasome inhibitors and attempts to control E3s and DUBs represent just the tip of the iceberg in terms of the potential for novel therapeutic interventions based on the manipulation of ubiquitin-related pathways in the cell.

References
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