Small-Molecule Suppressors of Cytokine-Induced Beta-Cell Apoptosis

Abstract

Type-1 diabetes is caused by the autoimmune destruction of insulin-producing beta cells in the pancreas. Beta-cell apoptosis involves a complex set of signaling cascades initiated by \(interleukin-1\beta (IL-1\beta)\), \(interferon-\gamma (IFN-\gamma)\), and \(tumor necrosis factor-\alpha (TNF-\alpha)\). \(IL-1\beta\) and \(TNF-\alpha\) induce \(NF\kappa B\) expression, while \(IFN-\gamma\) induces STAT1 activation. These cytokines lead to a decrease of beta-cell function. The goal of this thesis is to identify small-molecule suppressors of cytokine-induced beta-cell apoptosis using high-throughput screening approach. Using the rat INS-1E beta-cell line, I developed an assay to measure cellular viability after 48 hours of cytokine treatment. I screened 29,760 compounds for their ability to suppress the negative effects of the cytokines. I identified several compounds to be suppressors of beta-cell apoptosis. These efforts led to the discovery of \(GSK-3\beta\) and HDAC3 as novel targets for suppressing beta-cell apoptosis. I also followed up on BRD0608, a novel suppressor that increased ATP levels and decreased caspase activity in the presence of cytokines. To follow up this compound, 35 analogs related to BRD0476 were synthesized using solid-phase synthesis and tested for their protective effects in the presence of cytokines. A structurally related analog, BRD0476, was found to be more potent and active in human islets, decreasing caspase activation and increasing insulin secretion after a 6-day treatment. I performed gene-expression profiling of INS-1E cells treated with the cytokine cocktail in the absence or presence of \(10\mu M\) BRD0476. Gene-set enrichment analysis revealed that the gene sets most significantly changed by BRD0476 involved cellular responses to \(IFN-\gamma\). I therefore assessed the effects of BRD0476 on STAT1 transcriptional activity. Cytokine treatment increased the reporter-gene luciferase activity, while co-treatment with BRD0476 reduced this activity significantly. To identify the intracellular target(s) of BRD0476, I collaborated with the Proteomics Platform in Broad Institute using SILAC (stable isotope labeling by amino acids in cell culture). SILAC is a mass spectrometry-based method to identify proteins that bind a small molecule attached to a bead. Deubiquitinase USP9X was pulled down by BRD0476. Knock-down of USP9X by siRNA phenocopied the protective effects of BRD0476. Binding assays were performed to identify interactions between BRD0476 and USP9X.