Novel potent azetidine-based compounds irreversibly inhibit Stat3 activation and induce antitumor response against human breast tumor growth in vivo

• Published in: Cancer letters Vol. 534; p. 215613
• Main Authors: Yue, Peibin, Zhu, Yinsong, Brotherton-Pleiss, Christine, Fu, Wenzhen, Verma, Nagendra, Chen, Jasmine, Nakamura, Kayo, Chen, Weiliang, Chen, Yue, Alonso-Valenteen, Felix, Mikhael, Simoun, Medina-Kauwe, Lali, Kershaw, Kathleen M., Celeridad, Maria, Pan, Songqin, Limpert, Allison S., Sheffler, Douglas J., Cosford, Nicholas D.P., Shiao, Stephen L., Tius, Marcus A., Lopez-Tapia, Francisco, Turkson, James
• Format: Journal Article
• Language: English
• Published: Ireland Elsevier B.V 28.05.2022; Elsevier Limited
• Subjects: Animals; Antitumor activity; Antitumor cell effects; Apoptosis; Azetidines - pharmacology; Breast cancer; Cancer therapies; Cell death; Cell Line, Tumor; Covalent modification; Epidermal growth factor; Fibroblasts; Gene expression; Humans; Hydrochloric acid; Janus kinase; Kinases; Laboratory animals; Mass spectroscopy; Mice; Peptides; Phosphorylation; Proteins; Scientific imaging; Signal transducer and activator of transcription; Signal Transduction; Site-directed mutagenesis; Small-molecule inhibitors; Stat1 protein; Stat3 protein; STAT3 Transcription Factor - metabolism; Stat5 protein; Therapeutic targets; Transcription; Triple Negative Breast Neoplasms - drug therapy; Triple Negative Breast Neoplasms - genetics; Tumor growth inhibition; Xenografts; Signal transducer and activator of transcription; Antitumor cell effects; Small-molecule inhibitors; Covalent modification; Tumor growth inhibition
• ISSN: 0304-3835; 1872-7980
• DOI: 10.1016/j.canlet.2022.215613

Signal transducer and activator of transcription (Stat)3 is a valid anticancer therapeutic target. We have discovered a highly potent chemotype that amplifies the Stat3-inhibitory activity of lead compounds to levels previously unseen. The azetidine-based compounds, including H172 (9f) and H182, irreversibly bind to Stat3 and selectively inhibit Stat3 activity (IC50 0.38–0.98 μM) over Stat1 or Stat5 (IC50 > 15.8 μM) in vitro. Mass spectrometry detected the Stat3 cysteine peptides covalently bound to the azetidine compounds, and the key residues, Cys426 and Cys468, essential for the high potency inhibition, were confirmed by site-directed mutagenesis. In triple-negative breast cancer (TNBC) models, treatment with the azetidine compounds inhibited constitutive and ligand-induced Stat3 signaling, and induced loss of viable cells and tumor cell death, compared to no effect on the induction of Janus kinase (JAK)2, Src, epidermal growth factor receptor (EGFR), and other proteins, or weak effects on cells that do not harbor aberrantly-active Stat3. H120 (8e) and H182 as a single agent inhibited growth of TNBC xenografts, and H278 (hydrochloric acid salt of H182) in combination with radiation completely blocked mouse TNBC growth and improved survival in syngeneic models. We identify potent azetidine-based, selective, irreversible Stat3 inhibitors that inhibit TNBC growth in vivo. •Azetidine-based compounds, exemplified by H182 inhibit Stat3 activity with nanomolar-sub-micromolar potency•Azetidine-based compounds irreversibly bind to Stat3•Mass spectrometry detected the covalent binding to the key residues, Cys328, Cys426, Cys468, and Cys542•H182, H120 and other azetidine compounds inhibit constitutive and ligand-induced Stat3 signaling in triple-negative breast cancer cells•H120 or H182 as a single agent inhibited growth of triple-negative breast tumor xenografts•H278 (hydrochloric acid salt of H182) in combination with radiation completely blocked mouse triple-negative breast cancer growth and improved survival in syngeneic models