Novel high-throughput screening system for identifying STAT3–SH2 antagonists Yutaka Uehara a , Masato Mochizuki a , Kenji Matsuno a , Takeharu Haino b , Akira Asai a, * a Center for Drug Discovery, Graduate School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526, Japan b Department of Chemistry, Graduate School of Science, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima 739-8526, Japan article info Article history: Received 23 January 2009 Available online 27 January 2009 Keywords: STAT3 SH2 Transcription Anticancer Screening Porphyrin abstract Constitutive activation of the oncogenic transcription factor STAT3 frequently occurs in various human malignancies. STAT3 activation involves dimerization via intermolecular pTyr–SH2 interaction. Thus, antagonizing this interaction is a feasible approach to inhibit STAT3 activation for cancer therapy. In order to identify selective STAT3 inhibitors, we developed a biochemical HTS system based on Alpha- Screen technology, which measures the abilities of test compounds to antagonize pTyr–SH2 interactions. We screened our chemical libraries using this system and identified 5,15-diphenylporphyrin (5,15-DPP) as a selective STAT3–SH2 antagonist. Selective inhibition of STAT3 nuclear translocation and DNA biding activity was observed in cells treated with 5,15-DPP. IL-6-dependent dimerization of STAT3, c-myc pro- moter binding and c-myc protein expression were all suppressed by 5,15-DPP, whereas no decrement in either expression or phosphorylation level of STAT3 was observed. Thus, the HTS assay system repre- sented herein may be useful for identifying novel STAT3–SH2 antagonists. Ó 2009 Elsevier Inc. All rights reserved. Signal Transducer and Activator of Transcription (STAT) pro- teins were originally discovered as latent cytoplasmic transcription factors that mediate cytokine and growth factor-related extracellu- lar signals. These signaling pathways involve the activation of receptor tyrosine kinases, such as epidermal growth factor recep- tor and platelet-derived growth factor receptor, and Janus kinases (JAKs). Following phosphorylation at tyrosine residue 705 (Tyr705), two STAT monomers dimerize through reciprocal phos- photyrosine and Src homology 2 (SH2) domain interactions. A dimeric form of STAT translocates into the nucleus and binds to STAT-specific DNA-response elements of target genes for transcrip- tion activation. The STAT family comprises seven members, namely, STAT1–STAT4, STAT6, and the closely related STAT5a and STAT5b proteins. The members play a role in diverse biological functions, including cell proliferation, cell survival, angiogenesis, apoptosis, and inflammation. Several lines of evidence have impli- cated some of the STAT family members in malignant transforma- tion and tumor cell survival. In particular, STAT3 is constitutively activated in many types of hematopoietic and solid tumors, such as leukemia and breast and prostate cancers. In light of these find- ings, STAT3 has been considered an attractive target for the devel- opment of new anticancer drugs [1–5]. Because of its central role just downstream of protein tyrosine kinases, aberrant STAT3 activ- ity is often associated with transforming mechanisms induced by oncogenic tyrosine kinases. In addition, recent reports showed that STAT3 was constitutively activated both in tumor cells and in immune cells confined in tumor microenvironments, and that con- stitutively activated STAT3 inhibited the expression of mediators necessary for immune responses against the tumor cells [6–7]. Although a large number of small molecules have been reported to inhibit STAT3 signaling, the vast majority of them act on STAT3 by inhibiting the upstream tyrosine kinases responsible for STAT3 activation. In particular, small-molecule inhibitors of JAK2 abol- ished STAT3 activity and induced tumor cell apoptosis [2,3,5,8]. In another study, peptide-based STAT3 inhibitors designed to tar- get the STAT3–SH2 domain were effective in suppressing the cellu- lar functions of STAT3 [3,5,9]. In addition, several small molecules such as STA-21, Stattic, and S3I-201 have been reported to inhibit STAT3 by targeting to the SH2 domain [10–12]. S3I-201 exhibited antitumor efficacy in a mouse xenografted with human breast tu- mor harboring constitutively active STAT3, and thus presenting a proof-of-concept for the potential clinical use of such small mole- cules [12]. In order to identify a novel structural class of STAT3 inhibi- tors, we developed a biochemical assay using an AlphaScreen technology-based HTS system. Using the HTS assay, we identified 5,15-diphenylporphyrin (5,15-DPP) as a selective STAT3–SH2 antagonist. Selective inhibition of STAT3 translocation and DNA biding activity was observed in cells treated with 5,15-DPP. IL- 6-dependent dimerization, c-myc promoter binding and c-myc protein expression were all suppressed by 5,15-DPP in a dose- dependent manner, while no decrement in either expression or phosphorylation level of STAT3 was observed. Herein, we report the development of the HTS system and biological activities of 5,15-DPP. 0006-291X/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2009.01.137 * Corresponding author. Fax: +81 54 264 5231. E-mail address: aasai@u-shizuoka-ken.ac.jp (A. Asai). Biochemical and Biophysical Research Communications 380 (2009) 627–631 Contents lists available at ScienceDirect Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc