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Kinase-Independent Small-Molecule Inhibition of JAK-STAT Signaling.
Chou, Danny Hung-Chieh; Vetere, Amedeo; Choudhary, Amit; Scully, Stephen S; Schenone, Monica; Tang, Alicia; Gomez, Rachel; Burns, Sean M; Lundh, Morten; Vital, Tamara; Comer, Eamon; Faloon, Patrick W; Dancík, Vlado; Ciarlo, Christie; Paulk, Joshiawa; Dai, Mingji; Reddy, Clark; Sun, Hanshi; Young, Matthew; Donato, Nicholas; Jaffe, Jacob; Clemons, Paul A; Palmer, Michelle; Carr, Steven A; Schreiber, Stuart L; Wagner, Bridget K.
Afiliação
  • Chou DH; ‡Department of Chemistry and Chemical Biology, Harvard University, 12 Oxford Street, Cambridge, Massachusetts 02138, United States.
  • Choudhary A; §Society of Fellows, Harvard University, 78 Mount Auburn Street, Cambridge, Massachusetts 02138, United States.
  • Sun H; #Department of Internal Medicine, University of Michigan Comprehensive Cancer Center, 1500 E. Medical Center Drive, Ann Arbor, Michigan 48103, United States.
  • Young M; ∇Department of Pharmacology, University of Michigan Medical School, 1150 W. Medical Center Drive, Ann Arbor, Michigan 48109, United States.
  • Donato N; #Department of Internal Medicine, University of Michigan Comprehensive Cancer Center, 1500 E. Medical Center Drive, Ann Arbor, Michigan 48103, United States.
  • Schreiber SL; ‡Department of Chemistry and Chemical Biology, Harvard University, 12 Oxford Street, Cambridge, Massachusetts 02138, United States.
J Am Chem Soc ; 137(24): 7929-34, 2015 Jun 24.
Article em En | MEDLINE | ID: mdl-26042473
ABSTRACT
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 ß-cell apoptosis, inhibits interferon-gamma (IFN-γ)-induced Janus kinase 2 (JAK2) and signal transducer and activation of transcription 1 (STAT1) signaling to promote ß-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 ß 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.
Assuntos

Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Interferon gama / Substâncias Protetoras / Células Secretoras de Insulina / Fator de Transcrição STAT1 / Janus Quinase 2 Tipo de estudo: Prognostic_studies Limite: Animals / Humans Idioma: En Revista: J Am Chem Soc Ano de publicação: 2015 Tipo de documento: Article País de afiliação: Estados Unidos

Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Interferon gama / Substâncias Protetoras / Células Secretoras de Insulina / Fator de Transcrição STAT1 / Janus Quinase 2 Tipo de estudo: Prognostic_studies Limite: Animals / Humans Idioma: En Revista: J Am Chem Soc Ano de publicação: 2015 Tipo de documento: Article País de afiliação: Estados Unidos