The ubiquitin-like protein NEDD8 functions to regulate the activation of cullin-RING ubiquitin ligases (CRLs). Although CRLs have well-established roles in many fundamental aspects of cellular physiology, recent pre- clinical studies have found that cancer cells are more reliant on NEDD8 for proliferation and survival than normal cells. These observations have invigorated interest in devising strategies to pharmacologically inhibit NEDD8 to treat cancer and other diseases. The NEDD8-activating enzyme (NAE) represents the most attractive target of the NEDD8 system as it possess a catalytic pocket suitable for binding small molecules and undergoes structural rearrangements important for its activities. MLN4924, a mechanism-based inhibitor that generates a NEDD8-adenylate mimetic in the NAE catalytic pocket, represents the only NEDD8 system inhibitor and is currently being evaluated on cancer patients. We and other have found that cancer cells and experimental tumors rapidly lose sensitivity to MLN4924 through treatment-emergent mutations in NAE that impact inhibitor binding by altering the enzyme's biochemical properties. Although the implications of these on MLN4924 therapy are currently unknown, they suggest cancer cells tolerate considerable variability in NAE function. These mutations render cells broadly resistant to molecules targeting the enzyme's catalytic pocket. This project, submitted in response to PAR-13-364 Development of Assays for High-Throughput Screening for Use in Probe and Pre-therapeutic Discovery, seeks to develop highly innovative biophysical and cell-based assays focused on the NEDD8 system and to use these in pilot screens to identify proof-of-concept probes. Through our preliminary studies, we have devised a homogeneous biophysical assay that measures ligand- dependent changes in NAE thermal stability. We hypothesize that this assay will allow for different classes of equilibrium-binding reversible inhibitors to be identifie by screening chemically diverse small molecule libraries. To test this hypothesis and achieve the overall objective of the research, three Specific Aims are proposed: 1) to establish and validate biophysical assays that measure NAE thermal stability and supporting specificity assays; 2) to develop cell-based assays that measure biological effects of NAE inhibition; and 3) to conduct focused high-throughput screens with selected libraries, verify hits, and determine hit potency and selectivity. IMPACT: This work is highly innovative with translational relevance based on the extraordinary opportunity it provides to develop new research tools and therapeutic approaches to inhibit NAE and the NEDD8 system for the treatment of cancers and other diseases. Through our efforts focused on generating novel assays and chemical probes, these studies are expected to provide important new insight into the NEDD8 system and other ubiquitin and ubiquitin-like protein modification systems to improve human health.
In the quest for new and innovative cancer treatments, targeting functions of the ubiquitin-like protein NEDD8 has emerged as a highly promising therapeutic strategy. Pre-clinical data have clearly demonstrated that cancers are more reliant on NEDD8 for proliferation and survival than normal cells, suggesting that pharmacological inhibition of this post-translational modification system could be broadly applicable to treat the disease. In the work proposed here, we aim to develop novel biophysical and cell-based assays to devise and test new strategies focused on identifying small molecule inhibitors of the NEDD8 system.
| Wei, Yang; Toth, Julia I; Blanco, Gabrielle A et al. (2018) Adapted ATPase domain communication overcomes the cytotoxicity of p97 inhibitors. J Biol Chem 293:20169-20180 |
| Her, Nam-Gu; Toth, Julia I; Ma, Chen-Ting et al. (2016) p97 Composition Changes Caused by Allosteric Inhibition Are Suppressed by an On-Target Mechanism that Increases the Enzyme's ATPase Activity. Cell Chem Biol 23:517-28 |
