Expanding the deubiquitinase biochemical toolbox with assays, activity-based probes, and small molecule inhibitors
Date
2018
Authors
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Publisher
University of Delaware
Abstract
Ubiquitination is an important, reversible post-translational modification (PTM) that plays a crucial role in many eukaryotic cellular processes. Dysregulation of ubiquitination and deubiquitination of cellular pathways is often associated with various human diseases ranging from neurological disorders and cancer, to viral infection. Thus, this PTM is thought to be suitable for therapeutic intervention, and have garnered tremendous interest into the enzymes involved. The deubiquitinases, or DUBs, are a class of enzymes that oppose the ubiquitination cascade and have been associated with various human diseases. ☐ Chapter 1: Drug discovery campaigns against DUBs require enzymatic deubiquitination assays amenable for high-throughput screening (HTS). Although several DUB substrates and assays have been developed in recent years, they are limited to recombinantly purified DUBs. Many DUBs are large multi-domain proteins that are difficult to obtain recombinantly in sufficient quantities for HTS. Therefore, an assay that obviates the need of purified recombinant protein, and also recapitulates a physiologically relevant environment, is highly desirable. Such an assay will open doors for drug discovery against many therapeutically relevant, but currently inaccessible DUBs. To address this need, I developed a cell lysate DUB assay based on AlphaLISA (amplified luminescent proximity homogenous assay linked immunosorbent assay) technology for HTS. This assay platform uses a biotin-tagged ubiquitin probe and a HA-tagged DUB expressed in human cells. I validated and adapted this assay to a 1536-well format, which enabled screening against UCHL1 as proof of principle using a library of approximately 15,000 compounds. Initial hits were validated as true inhibitors of USP15 utilizing a combination of counter-screens, orthogonal, reversibility, and redox cycling assays. I expect that the new platform can be readily adapted to other DUBs to allow the identification of more potent and selective small molecule inhibitors and chemical probes. ☐ Chapter 2: Close to 100 DUBs have been identified in humans, and as previously discussed, are implicated in human diseases including different types of cancer, neurodegenerative diseases, viral infections, and inflammatory diseases. Consequently, there has been a surge in efforts to develop DUB inhibitors. To my knowledge, DUB inhibitors have yet to enter late stage clinical trials. However, many DUB inhibitors show promise for the treatment of human diseases in preclinical models. Recently, ubiquitin specific protease 15 (USP15) has received increased attention as a promising drug target. USP15 has been linked to aggressive and invasive cancers and neurodegeneration due to its involvement in the TGF-β/SMAD signaling pathway and mitophagy, respectively. Therefore, I embarked to identify a potent and selective inhibitor of USP15. I performed a pilot quantitative High-Throughput Screening (qHTS) of 24,000 compounds against USP15. 165 compounds were chosen for follow-up testing in orthogonal diUb cleavage assays. Additionally, lead compounds were assessed for redox cycling capability, reversibility, and selectivity for USP15 against closely related DUBs. One small molecule inhibitor from the HTS campaign, NCGC00013130-04, was found to be of particular interest and is currently being evaluated further in cellular systems. Furthermore, NCGC00013130-04 and other lead molecule were utilized to initiate a quantitative structure-activity relationship (QSAR) though a machine learning approach to identify additional active chemotypes. ☐ Chapter 3: Advances in the knowledge of DUB activity and biological functions have largely been hindered due to the lack of biochemical tools available capable of accommodating physiologically relevant settings. However, activity-based probes (ABPs), which form a covalent bond between probe and enzyme, have been developed targeting the cysteine protease subfamilies of DUBs. Not only can DUB ABPs be used to study purified recombinant proteins, they also enable the analysis of DUBs in a more physiologically relevant setting, cell lysates. DUB ABPs have fueled the explosive development in the field, identifying new DUBs, shedding light on DUB specificity and catalyzing inhibitor development. Nevertheless, most DUB ABPs are not cell permeable, limiting their utility to purified proteins and cell lysates. Labeling of DUBs in their native cellular environment is highly desirable. Therefore in Chapter 3, I sought to fulfill this need through the development of a broad-spectrum, cell-penetrating Ub-based DUB ABP. To do so I employed a strategy of delivering DUB ABPs to cells through the usage of cell-penetrating peptides (CPPs). I found that coupling CPPs (TAT and cR10) to my Ub-based probes fostered the generation of a new class of cell permeable Ub-based DUB ABPs. Of which, enabled the profiling of DUBs in a cellular setting, such as HeLa cells, which was investigated using immunoblotting and mass spectrometry-based proteomics. Cell permeability of the probes was confirmed using live-cell fluorescent confocal microscopy. Additionally, a pan-DUB inhibitor, PR-619, was chosen to assess the potential usage of cell permeable Ub-based DUB ABPs in drug discovery. I expect that the new DUB probe can be readily adapted to various cell-based investigations of DUB cellular functions and provide much needed insight into the roles of DUBs in human diseases.
Description
Keywords
Activity-based probes, Drug discovery campaign, Inhibitors, Ubiquitination