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1.  Strategies for the Identification of Ubiquitin Ligase Inhibitors 
Biochemical Society transactions  2010;38(Pt 1):132-136.
Dysregulation of the ubiquitin-proteasome system (UPS) has been implicated in a wide range of pathologies including cancer, neurodegeneration, and viral infection. Inhibiting the proteasome has been shown to be an effective therapeutic strategy in humans; yet toxicity with this target remains high. Ubiquitin Ligases (E3s) represent an alternative attractive therapeutic target in the UPS. Here we will discuss current platforms that report on E3 ligase activity and can detect E3 inhibitors, while underlining the advantages and disadvantages of each approach.
PMCID: PMC3060714  PMID: 20074047
Ubiquitin; ligase; E3; RING; HECT; high-throughput screening
2.  Selective Dual Inhibitors of the Cancer-Related Deubiquitylating Proteases USP7 and USP47 
ACS Medicinal Chemistry Letters  2012;3(10):789-792.
Inhibitors of the cancer-related cysteine isopeptidase human ubiquitin-specific proteases 7 (USP7) and 47 (USP47) are considered to have potential as cancer therapeutics, owing to their ability to stabilize the tumor suppressor p53 and to decrease DNA polymerase β (Polβ), both of which are potential anticancer effects. A new class of dual small molecule inhibitors of these enzymes has been discovered. Compound 1, a selective inhibitor of USP7 and USP47 with moderate potency, demonstrates inhibition of USP7 in cells and induces elevated p53 and apoptosis in cancer cell lines. Compound 1 has been shown to demonstrate modest activity in human xenograft multiple myeloma and B-cell leukemia in vivo models. This activity may be the result of dual inhibition of USP7 and USP47. To address issues regarding potency and developability, analogues of compound 1 have been synthesized and tested, leading to improvements in potency, solubility, and metabolic reactivity profile. Further optimization is expected to yield preclinical candidates and, ultimately, clinical candidates for the treatment of multiple myeloma, prostate cancer, and other cancers.
PMCID: PMC4025646  PMID: 24900381
Ubiquitin isopeptidase; deubiquitylase; p53; Polβ
3.  Analysis of SUMOylated proteins using SUMO-traps 
Scientific Reports  2013;3:1690.
SUMO-modified proteins are recognized by SUMO interacting motifs (SIMs), thus triggering diverse cellular responses. Here SIMs were used to develop SUMO-traps to capture endogenous SUMOylated proteins. Our results show that these small peptides are transferable motifs that maintain their SUMO binding capacity when fused to the heterologous carrier protein GST. The tandem disposition of SIMs increases the binding capacity of SUMO-traps to specifically interact with polySUMO but not poly-Ubiquitin chains. We demonstrate that this SUMO capturing system purifies SUMOylated proteins such as IκBα, PTEN, PML or p53 in vitro and in vivo. These properties can be used to explore the many critical functions regulated by protein SUMOylation.
PMCID: PMC3631770  PMID: 23604351
4.  Characterization of Selective Ubiquitin and Ubiquitin-Like Protease Inhibitors Using a Fluorescence-Based Multiplex Assay Format 
The reversible conjugation of ubiquitin and ubiquitin-like (UbL) proteins to protein substrates plays a critical role in the regulation of many cellular pathways. The removal of ubiquitin from target proteins is performed by ubiquitin proteases also known as deubiquitylases (DUBs). Owing to their substrate specificity and the central role ubiquitylation plays in cell signaling pathways, DUB are attractive targets for therapeutic development. The development of DUB inhibitors requires assays that are amenable to high-throughput screening and provide rapid assessment of inhibitor selectivity. Determination of inhibitor selectivity at an early stage of drug discovery will reduce drug failure in the clinic as well as reduce overall drug development costs. We have developed two novel assays, UbL-Enterokinase light chain and UbL-Granzyme B, for quantifying ubiquitin and UbL protease activity. In our quest to discover and characterize novel chemical entities, we have combined these assays with a previously developed assay in a multiplex format. This multiplex format allows for the detection of three distinct protease activities simultaneously, in a single well. We have demonstrated that the multiplex format is able to distinguish between selective and nonselective protease inhibitors. Specifically, we have used this assay format to characterize P022077, a selective ubiquitin-specific protease 7 inhibitor discovered at Progenra.
PMCID: PMC3065724  PMID: 21133675
5.  The Ubiquitin-Specific Protease USP34 Regulates Axin Stability and Wnt/β-Catenin Signaling▿ 
Molecular and Cellular Biology  2011;31(10):2053-2065.
Wnt proteins control multiple cell behaviors during development and tissue homeostasis. However, pathological activation of Wnt signaling is the underlying cause of various human diseases. The ubiquitin-proteasome system plays important regulatory functions within the Wnt pathway by regulating the activity of several of its core components. Hence, multiple E3 ubiquitin ligases have been implicated in its regulation. Less is known, however, about the role of ubiquitin-specific proteases in Wnt signaling. Analysis of purified axin-containing protein complexes by liquid chromatography-tandem mass spectrometry revealed the presence of the ubiquitin protease USP34. Our results indicate that USP34 functions downstream of the β-catenin destruction complex to control the stability of axin and opposes its tankyrase-dependent ubiquitination. Reflecting on the requirement for tight control of axin homeostasis during Wnt signaling, interfering with USP34 function by RNA interference leads to the degradation of axin and to the inhibition of β-catenin-mediated transcription. Given the numerous human diseases exhibiting spurious Wnt pathway activation, the development of USP34 inhibitors may offer a novel therapeutic opportunity.
PMCID: PMC3133363  PMID: 21383061
6.  Strategies for the Identification of novel inhibitors of deubiquitinating enzymes 
Biochemical Society transactions  2008;36(Pt 5):828-832.
Dysregulation of the ubiquitin-proteasome system (UPS) has been implicated in a wide range of pathologies including cancer, neurodegeneration, and viral infection. Inhibiting the proteasome has been shown to be an effective therapeutic strategy in humans; yet toxicity with this target remains high. Deubiquitinating enzymes (DUBs) represent an alternative target in the UPS with low predicted toxicity. Currently, there are no DUB inhibitors that have entered the clinic. To address this situation, Progenra has developed a novel assay to measure the proteolytic cleavage of ubiquitin or UBL (ubiquitin like protein) conjugates such as SUMO, NEDD8 or ISG15 by isopeptidases. Here we will discuss current platforms for detecting DUB inhibitors and underline the advantages and disadvantages of the approaches.
PMCID: PMC3061546  PMID: 18793145
Ubiquitin; deubiquitylase; deSUMOylase; deISGylase; deNEDDylase; isopeptidase; high-throughput screening

Results 1-6 (6)