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Kinase-Independent Small-Molecule Inhibition of JAK-STAT Signaling

By Danny Hung-Chieh Chou, Amedeo Vetere, Amit Choudhary, Stephen S. Scully, Monica Schenone, Alicia Tang, Rachel Gomez, Sean M. Burns, Morten Lundh, Tamara Vital, Eamon Comer, Patrick W. Faloon, Vlado Dančík, Christie Ciarlo, Joshiawa Paulk, Mingji Dai, Clark Reddy, Hanshi Sun, Matthew Young, Nicholas Donato, Jacob Jaffe, Paul A. Clemons, Michelle Palmer, Steven A. Carr, Stuart L. Schreiber and Bridget K. Wagner


Phenotypic cell-based screening is a powerful approach to small-molecule discovery, but a major challenge of this strategy lies in determining the intracellular target and mechanism of action (MoA) for validated hits. Here, we show that the small-molecule BRD0476, a novel suppressor of pancreatic β-cell apoptosis, inhibits interferon-gamma (IFN-γ)-induced Janus kinase 2 (JAK2) and signal transducer and activation of transcription 1 (STAT1) signaling to promote β-cell survival. However, unlike common JAK-STAT pathway inhibitors, BRD0476 inhibits JAK-STAT signaling without suppressing the kinase activity of any JAK. Rather, we identified the deubiquitinase ubiquitin-specific peptidase 9X (USP9X) as an intracellular target, using a quantitative proteomic analysis in rat β cells. RNAi-mediated and CRISPR/Cas9 knockdown mimicked the effects of BRD0476, and reverse chemical genetics using a known inhibitor of USP9X blocked JAK-STAT signaling without suppressing JAK activity. Site-directed mutagenesis of a putative ubiquitination site on JAK2 mitigated BRD0476 activity, suggesting a competition between phosphorylation and ubiquitination to explain small-molecule MoA. These results demonstrate that phenotypic screening, followed by comprehensive MoA efforts, can provide novel mechanistic insights into ostensibly well-understood cell signaling pathways. Furthermore, these results uncover USP9X as a potential target for regulating JAK2 activity in cellular inflammation

Topics: Chemistry (all), Catalysis, Biochemistry, Colloid and Surface Chemistry
Year: 2015
DOI identifier: 10.1021/jacs.5b04284
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