A High Throughput Cell-Based Deubiquitinase Screening Assay

Abstract ID 129141 Poster Board 206 One of the functions of ubiquitination, the cellular process of ligating ubiquitin to proteins, is to mark proteins for degradation. Ubiquitination is carried out by E1, E2, and E3 enzyme cascades. Deubiquitinases (DUBs) are a family of enzymes responsible for hydrolyzing and removing ubiquitin from marked proteins. An imbalance in the addition and removal of ubiquitin is a hallmark of many neurodegenerative diseases. A DUB, USP15, has been found to counteract the ubiquitinating efforts of E3-ligase Parkin and in turn prevent the degradation of dysfunctional mitochondria. The buildup of faulty mitochondria is a characteristic of Parkinson's disease. The role USP15 plays in antagonizing Parkin and restricting mitophagy makes it an important target in drug discovery. Our goal was to discover inhibitors of USP15 through the development of a high throughput cell-based small molecule screening assay. Inhibitors found in traditional biochemical assays tend to fail in cell-based experiments. The cell-based assay subjects' inhibitors to physiologically accurate environment, helping to identify molecules capable of inhibiting the intended target under true cellular conditions. The biotin-cR₁₀-Ub-PA probe was first expressed as an Avi-C-Ub-Intein construct in E. coli overexpressing a biotin ligase. Upon chitin incubation and cleavage from the resin, the PA warhead was added. The probe includes an N-terminal cysteine residue for conjugation of a cell-permeable peptide, cyclic polyarginine (cR10), that allows the biotin-cR₁₀-Ub-PA probe to enter the cell and covalently bind to DUBs. Stable HeLa cells expressing HA-tagged USP15 were generated by a PiggyBac transposon system. AlphaLISA technology was utilized to measure the binding of the biotinylated Ub-probe to the HA-tagged USP15 via streptavidin donor beads and Anti-HA acceptor beads respectively. Small molecules that prevent binding of the Ub-probe to USP15 cause a decrease in AlphaLISA signal and indicate a potential USP15 inhibitor. To miniaturize and automate the assay for higher throughput, optimization of cell density, assay volumes, detergent concentrations, and automated dispensing methods were conducted. As a counter screen we established AlphaLISA TruHits and HA-double-tagged Ub probe (biotin-Ub-HA)- based assays. An enzymatic assay was conducted with recombinant USP15 and Ub-Rho110 substrate to use as a comparison to the cell-based screening assay. As proof of concept, we screened a library of 872 compounds at a single concentration. A total of 46 compounds showed an inhibition efficiency of greater than 50%. A follow up screen was conducted with the 46 compounds in 11-point dose response in the same cell-based assay. AlphaLISA TruHits and a Biotin-Ub-HA probe counter screens were used to test for false positives that may interfere with the AlphaLISA technology or tag-bead interaction. Cytotoxicity of the compounds was measured in a CellTiter-Glo assay. After removal of false positive and cytotoxic compounds 24 hits were identified, and 14 of these were novel compounds not detected in the biochemical assay. We developed and optimized a high throughput amenable cell-based assay which identified small molecule inhibitors of USP15. Using this assay, we detected new DUB inhibitor molecules that were not observed in the biochemical assay. Cellular thermal shift assays and microscale thermophoresis target engagement experiments were conducted to confirm hits from cell-based DUB screening.