title: HCS campaign to identify selective inhibitors of IL-6-induced STAT3 pathway activation in head and neck cancer cell lines.
abstract: Signal transducer and activator of transcription factor 3 (STAT3) is hyperactivated in head and neck squamous cell carcinomas (HNSCC). Cumulative evidence indicates that IL-6 production by HNSCC cells and/or stromal cells in the tumor microenvironment activates STAT3 and contributes to tumor progression and drug resistance. A library of 94,491 compounds from the Molecular Library Screening Center Network (MLSCN) was screened for the ability to inhibit interleukin-6 (IL-6)-induced pSTAT3 activation. For contractual reasons, the primary high-content screening (HCS) campaign was conducted over several months in 3 distinct phases; 1,068 (1.1%) primary HCS actives remained after cytotoxic or fluorescent outliers were eliminated. One thousand one hundred eighty-seven compounds were cherry-picked for confirmation; actives identified in the primary HCS and compounds selected by a structural similarity search of the remaining MLSCN library using hits identified in phases I and II of the screen. Actives were confirmed in pSTAT3 IC50 assays, and an IFNγ-induced pSTAT1 activation assay was used to prioritize selective inhibitors of STAT3 activation that would not inhibit STAT1 tumor suppressor functions. Two hundred three concentration-dependent inhibitors of IL-6-induced pSTAT3 activation were identified and 89 of these also produced IC50s against IFN-γ-induced pSTAT1 activation. Forty-nine compounds met our hit criteria: they reproducibly inhibited IL-6-induced pSTAT3 activation by ≥70% at 20 μM; their pSTAT3 activation IC50s were ≤25 μM; they were ≥2-fold selective for pSTAT3 inhibition over pSTAT1 inhibition; a cross target query of PubChem indicated that they were not biologically promiscuous; and they were ≥90% pure. Twenty-six chemically tractable hits that passed filters for nuisance compounds and had acceptable drug-like and ADME-Tox properties by computational evaluation were purchased for characterization. The hit structures were distributed among 5 clusters and 8 singletons. Twenty-four compounds inhibited IL-6-induced pSTAT3 activation with IC50s ≤20 μM and 13 were ≥3-fold selective versus inhibition of pSTAT1 activation. Eighteen hits inhibited the growth of HNSCC cell lines with average IC50s ≤ 20 μM. Four chemical series were progressed into lead optimization: the guanidinoquinazolines, the triazolothiadiazines, the amino alcohols, and an oxazole-piperazine singleton.
authors: 1 Department of Pharmaceutical Sciences, School of Pharmacy, University of Pittsburgh , Pittsburgh, Pennsylvania.|2 University of Pittsburgh Cancer Institute , Pittsburgh, Pennsylvania.|3 Pittsburgh Specialized Application Center, University of Pittsburgh Drug Discovery Institute , Pittsburgh, Pennsylvania.
authors: Paul A
authors: PA
authors: Johnston
authors: 4 Department of Otolaryngology, University of Pittsburgh , Pittsburgh, Pennsylvania.
authors: Malabika
authors: M
authors: Sen
authors: 1 Department of Pharmaceutical Sciences, School of Pharmacy, University of Pittsburgh , Pittsburgh, Pennsylvania.
authors: Yun
authors: Y
authors: Hua
authors: 1 Department of Pharmaceutical Sciences, School of Pharmacy, University of Pittsburgh , Pittsburgh, Pennsylvania.
authors: Daniel P
authors: DP
authors: Camarco
authors: 3 Pittsburgh Specialized Application Center, University of Pittsburgh Drug Discovery Institute , Pittsburgh, Pennsylvania.
authors: Tong Ying
authors: TY
authors: Shun
authors: 5 Departments of Pharmacology and Chemistry, University of Virginia , Charlottesville, Virginia.
authors: John S
authors: JS
authors: Lazo
authors: 6 University of Pittsburgh Chemical Diversity Center , Pittsburgh, Pennsylvania.|7 Department of Health Services and Health Administration, College of Nursing and Health Professions, University of Southern Indiana , Evansville, Indiana.
authors: Gabriela Mustata
authors: GM
authors: Wilson
authors: 6 University of Pittsburgh Chemical Diversity Center , Pittsburgh, Pennsylvania.
authors: Lynn O
authors: LO
authors: Resnick
authors: 6 University of Pittsburgh Chemical Diversity Center , Pittsburgh, Pennsylvania.
authors: Matthew G
authors: MG
authors: LaPorte
authors: 1 Department of Pharmaceutical Sciences, School of Pharmacy, University of Pittsburgh , Pittsburgh, Pennsylvania.|2 University of Pittsburgh Cancer Institute , Pittsburgh, Pennsylvania.|6 University of Pittsburgh Chemical Diversity Center , Pittsburgh, Pennsylvania.|8 Department of Chemistry, University of Pittsburgh , Pittsburgh, Pennsylvania.
authors: Peter
authors: P
authors: Wipf
authors: 1 Department of Pharmaceutical Sciences, School of Pharmacy, University of Pittsburgh , Pittsburgh, Pennsylvania.|6 University of Pittsburgh Chemical Diversity Center , Pittsburgh, Pennsylvania.
authors: Donna M
authors: DM
authors: Huryn
authors: 9 Clinical and Translational Science Institute, Otolaryngology - Head and Neck Surgery, University of California , San Francisco, California.
authors: Jennifer R
authors: JR
authors: Grandis
mesh_terms: D002294:Carcinoma, Squamous Cell / Q000188:drug therapy*; D045744:Cell Line, Tumor; D006258:Head and Neck Neoplasms / Q000188:drug therapy*; D057166:High-Throughput Screening Assays; D006801:Humans; D007371:Interferon-gamma / Q000494:pharmacology; D015850:Interleukin-6 / Q000502:physiology; D050794:STAT1 Transcription Factor / Q000502:physiology; D050796:STAT3 Transcription Factor / Q000037:antagonists & inhibitors* / Q000502:physiology; D015398:Signal Transduction / Q000187:drug effects*; D000077195:Squamous Cell Carcinoma of Head and Neck; D000093242:Interleukin-6 Inhibitors
chemical_list: D007371:Interferon-gamma; D015850:Interleukin-6; D050794:STAT1 Transcription Factor; D050796:STAT3 Transcription Factor; D000093242:Interleukin-6 Inhibitors; C508600:IL6 protein, human; C494079:STAT1 protein, human; C494086:STAT3 protein, human