View Abstract

A2986 - Further Development of a Small Molecule Inhibitor of the Deubiquitinase STAMBP
Author Block: J. S. Bednash1, N. M. Weathington1, B. Chen1, R. K. Mallampalli2; 1Acute Lung Injury Center of Excellence, Division of Pulmonary, Allergy and Critical Care Medicine, University of Pittsburgh, Pittsburgh, PA, United States, 2Division of Pulmonary, Allergy and Critical Care Medicine, University of Pittsburgh, Medical Specialty Service Line, Veterans Affairs Pittsburgh Healthcare System, Pittsburgh, PA, United States.
INTRODUCTION: Innate immune signaling by the inflammasome contributes to the pathogenesis of the acute respiratory distress syndrome (ARDS). Inflammasomes are multi-protein complexes that critically regulate the early immune response by facilitating the maturation and release of the potent pro-inflammatory cytokines IL-1β and IL-18. Increased circulating IL-1β and IL-18 are associated with increased mortality in ARDS. Pharmacologic targeting of the deubiquitinase (DUB) STAMBP reduces inflammation in pre-clinical models by reducing inflammasome activity and secretion of IL-1β.
RATIONALE: Targeting of the ubiquitin-proteasome system in cancer has led to the development of multiple small molecule proteasome inhibitors. To date, there exist no FDA-approved DUB inhibitors for any indication. Development of a small molecule inhibitor of STAMBP deubiquitinase activity provided proof of concept for targeting DUBs as anti-inflammatory targets. Further optimization of a lead compound targeting STAMBP is necessary prior to use in animal and human models.
METHODS: Cell culture and treatment, ELISA, Western blotting, co-immunoprecipitation, qPCR, in silico modeling and drug design, and in vitro DUB and caspase activity assays.
RESULTS: In a fluorescence-based DUB enzyme assay, our lead compound STAMBP antagonist decreased the initial velocity of STAMBP DUB activity as well as the maximum velocity of this enzymatic reaction. We then tested nine additional compounds with a similar backbone structure to the lead compound with substituted functional groups on the periphery of the lead compound molecular structure. Results of these studies were used to further modify the lead compound, which will subsequently be used for additional testing in our enzymatic assay.
CONCLUSIONS: Our small molecule inhibitor establishes STAMBP as a potential therapeutic target to reduce the effects of injurious inflammasome-driven pro-inflammatory stress. Prior development of a lead compound targeting STAMBP provided proof of concept, but further development is necessary prior to further testing in pre-clinical models. Using a fluorescence-based enzymatic assay, we identified functional group modifications that decrease the mean inhibitory concentration of this compound in cell-based systems. Further efforts in DUB inhibitor optimization may lead to use in clinical studies and eventual development of a novel class of anti-inflammatory agents for clinical use.