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J Am Chem Soc DOI:10.1021/jacs.5b04284

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

Publication TypeJournal Article
Year of Publication2015
AuthorsChou, DHung-Chieh, Vetere, A, Choudhary, A, Scully, SS, Schenone, M, Tang, A, Gomez, R, Burns, SM, Lundh, M, Vital, T, Comer, E, Faloon, PW, Dančík, V, Ciarlo, C, Paulk, J, Dai, M, Reddy, C, Sun, H, Young, M, Donato, N, Jaffe, J, Clemons, PA, Palmer, M, Carr, SA, Schreiber, SL, Wagner, BK
JournalJ Am Chem Soc
Date Published2015 Jun 24
KeywordsAnimals, Apoptosis, Cell Line, Cell Survival, Humans, Insulin-Secreting Cells, Interferon-gamma, Janus Kinase 2, Phosphorylation, Protective Agents, Rats, Signal Transduction, STAT1 Transcription Factor, Ubiquitin Thiolesterase, Ubiquitination

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.


Alternate JournalJ. Am. Chem. Soc.
PubMed ID26042473
PubMed Central IDPMC5003570
Grant ListUL1RR024924 / RR / NCRR NIH HHS / United States
T34 GM008751 / GM / NIGMS NIH HHS / United States
RL1HG004671 / HG / NHGRI NIH HHS / United States
RL1CA133834 / CA / NCI NIH HHS / United States
RL1GM084437 / GM / NIGMS NIH HHS / United States
DP2 DK083048 / DK / NIDDK NIH HHS / United States
UL1 RR024924 / RR / NCRR NIH HHS / United States
/ / Howard Hughes Medical Institute / United States
RL1 GM084437 / GM / NIGMS NIH HHS / United States
DP2-DK083048 / DK / NIDDK NIH HHS / United States
5T34GM008751 / GM / NIGMS NIH HHS / United States
RL1 HG004671 / HG / NHGRI NIH HHS / United States
RL1 CA133834 / CA / NCI NIH HHS / United States