title: Kinase-Independent Small-Molecule Inhibition of JAK-STAT Signaling.
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 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.
authors.affiliation: ‡Department of Chemistry and Chemical Biology, Harvard University, 12 Oxford Street, Cambridge, Massachusetts 02138, United States.
authors.forename: Danny Hung-Chieh
authors.initials: DH
authors.lastname: Chou
authors.forename: Amedeo
authors.initials: A
authors.lastname: Vetere
authors.affiliation: §Society of Fellows, Harvard University, 78 Mount Auburn Street, Cambridge, Massachusetts 02138, United States.
authors.forename: Amit
authors.initials: A
authors.lastname: Choudhary
authors.forename: Stephen S
authors.initials: SS
authors.lastname: Scully
authors.forename: Monica
authors.initials: M
authors.lastname: Schenone
authors.forename: Alicia
authors.initials: A
authors.lastname: Tang
authors.forename: Rachel
authors.initials: R
authors.lastname: Gomez
authors.forename: Sean M
authors.initials: SM
authors.lastname: Burns
authors.forename: Morten
authors.initials: M
authors.lastname: Lundh
authors.forename: Tamara
authors.initials: T
authors.lastname: Vital
authors.forename: Eamon
authors.initials: E
authors.lastname: Comer
authors.forename: Patrick W
authors.initials: PW
authors.lastname: Faloon
authors.forename: Vlado
authors.initials: V
authors.lastname: Dančík
authors.forename: Christie
authors.initials: C
authors.lastname: Ciarlo
authors.forename: Joshiawa
authors.initials: J
authors.lastname: Paulk
authors.forename: Mingji
authors.initials: M
authors.lastname: Dai
authors.forename: Clark
authors.initials: C
authors.lastname: Reddy
authors.affiliation: #Department of Internal Medicine, University of Michigan Comprehensive Cancer Center, 1500 E. Medical Center Drive, Ann Arbor, Michigan 48103, United States.
authors.forename: Hanshi
authors.initials: H
authors.lastname: Sun
authors.affiliation: ∇Department of Pharmacology, University of Michigan Medical School, 1150 W. Medical Center Drive, Ann Arbor, Michigan 48109, United States.
authors.forename: Matthew
authors.initials: M
authors.lastname: Young
authors.affiliation: #Department of Internal Medicine, University of Michigan Comprehensive Cancer Center, 1500 E. Medical Center Drive, Ann Arbor, Michigan 48103, United States.
authors.forename: Nicholas
authors.initials: N
authors.lastname: Donato
authors.forename: Jacob
authors.initials: J
authors.lastname: Jaffe
authors.forename: Paul A
authors.initials: PA
authors.lastname: Clemons
authors.forename: Michelle
authors.initials: M
authors.lastname: Palmer
authors.forename: Steven A
authors.initials: SA
authors.lastname: Carr
authors.affiliation: ‡Department of Chemistry and Chemical Biology, Harvard University, 12 Oxford Street, Cambridge, Massachusetts 02138, United States.
authors.forename: Stuart L
authors.initials: SL
authors.lastname: Schreiber
authors.forename: Bridget K
authors.initials: BK
authors.lastname: Wagner
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D006801:Humans; D050417:Insulin-Secreting Cells / Q000166:cytology / Q000187:drug effects* / Q000276:immunology; D007371:Interferon-gamma / Q000276:immunology*; D053614:Janus Kinase 2 / Q000276:immunology*; D010766:Phosphorylation / Q000187:drug effects; D020011:Protective Agents / Q000737:chemistry* / Q000494:pharmacology*; D051381:Rats; D050794:STAT1 Transcription Factor / Q000276:immunology*; D015398:Signal Transduction / Q000187:drug effects; D043222:Ubiquitin Thiolesterase / Q000276:immunology; D054875:Ubiquitination / Q000187:drug effects
chemical_list: D020011:Protective Agents; D050794:STAT1 Transcription Factor; C506234:USP9X protein, human; D007371:Interferon-gamma; D053614:Janus Kinase 2; D043222:Ubiquitin Thiolesterase