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

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• 作者：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 Dan膷铆k ; 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 ; Bridget K. Wagner
• 刊名：Journal of the American Chemical Society
• 出版年：2015
• 出版时间：June 24, 2015
• 年：2015
• 卷：137
• 期：24
• 页码：7929-7934
• 全文大小：424K
• ISSN：1520-5126

文摘

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 尾-cell apoptosis, inhibits interferon-gamma (IFN-纬)-induced Janus kinase 2 (JAK2) and signal transducer and activation of transcription 1 (STAT1) signaling to promote 尾-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 尾 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.