Small Molecule Inhibitors of Signal Transducer and Activator of Transcription 3 (Stat3) Protein Bikash Debnath, † Shili Xu, † and Nouri Neamati* Department of Pharmacology and Pharmaceutical Sciences, School of Pharmacy, University of Southern California, 1985 Zonal Avenue, Los Angeles, California 90089, United States 1. INTRODUCTION Signal transducers and activators of transcription (STATs) are 79-113 kDa proteins playing dual roles as signal transducers and transcription factors. At least seven members have been identified in this family, including Stat1, Stat2, Stat3, Stat4, Stat5A, Stat5B, and Stat6. Although the Stat proteins are structurally related, they participate in different cellular processes. 1,2 Among the members in the STAT family, Stat3 has received particular attention and is the most studied member primarily because of its role in cancer progression, inflammation, cardiomyogenesis, ischemia/reperfusion injury, and stem cell self-renewal. Stat3 was independently discovered and studied by two research groups and described in 1994. Akira et al. purified and cloned Stat3 from mouse liver nuclear extracts, named it as acute-phase response factor (APRF), and also identified Stat3 as a DNA-binding factor that selectively binds to the IL-6- responsive element within the acute-phase gene promoter. 3 Zhong et al. discovered Stat3 as a DNA-binding protein in response to epidermal growth factor. 4 Since then, multiple Stat3 isoforms have been identified, including the long form Stat3α, the truncated forms Stat3β and Stat3γ, and a putative novel form Stat3δ, 5 all derived from a single gene located within chromosome 17q21 via alternative splicing of the transcript’s3′ end. 5 Stat3α (p92), a 770 amino acid protein, is the predominantly expressed form of Stat3 in most cell types. 6 Stat3β (p83) is an alternatively spliced RNA form of Stat3α, in which the 55 C-terminal amino acids of the transactivation domain are replaced by seven distinct amino acids. Stat3β was generally regarded as a dominant negative Stat3 isoform 7 until recent in vivo experimental evidence showed that Stat3β rescued the embryonic lethality of a Stat3-null mutation and was capable by itself of activating the expression of Stat3 target genes. 8 Compared with Stat3α and Stat3β, the physiologic roles of Stat3γ and Stat3δ are less clear. Stat3γ (p72) is another C- terminal truncated form of Stat3α derived post-translationally through limited proteolysis. Stat3γ is primarily activated in terminally differentiated neutrophils. 9 Stat3δ exists at low levels and decreases with cell differentiation. 5 In this review, we summarize the signaling pathways of Stat3, its role in different diseases as well as in stem cell maintenance, and the progress in the design, discovery, and development of Stat3 inhibitors since 2006. 2. STAT3 PROTEIN STRUCTURE The Stat3β structure consists of a coiled coil, a DNA binding, a linker, as well as an SH2 domain and lacks the N-terminal cooperative and C-terminal transactivation domains (Figure 1a). Currently, two crystal structures of mouse Stat3β (1BG1 and 3CWG) are available in the Protein Data Bank. 10,11 1BG1 consists of a Stat3β homodimer bound to its DNA target sequence with a 2.25 Å resolution (Figure 1b). 10 The phosphorylated Tyr705 along with neighboring residues (702-709) in each monomer (amino acid residues 136-716) is bound to the Src homology 2 (SH2) domain in the other monomer. The SH2 domain comprises three subpockets that can be targeted by small-molecule inhibitors. Residues Lys591, Arg609, Ser611, and Ser613 from the SH2 domain are involved in polar interactions with phospho-Tyr705 (Figure 1c). Leu706 of the phosphopeptide is bound to a hydrophobic pocket of the SH2 domain. Four loops, three from the DNA binding domain and one from the linker domain, form interactions with both DNA strands. 10 Figure 1d shows that the unphosphorylated Stat3 core fragment comprising amino acid residues 136-688 is loosely bound through the SH2 domain (PDB code 3CWG). 11 In the 3CWG crystal structure, Stat3 does not bind DNA because the DNA binding domains are away from each other and are in reverse orientation compared to the 1BG1 structure. The root-mean-square deviation (rmsd) value for C α atoms of these two crystal structures (core fragment of monomers) is only 0.9 Å, suggesting minor conformational changes between the phosphorylated and unphosphorylated forms. 11 3. STAT3 SIGNALING PATHWAY As part of the Janus kinase (JAK) Stat pathway, Stat3 signaling can be activated by both receptor and nonreceptor tyrosine kinases via the tyrosine phosphorylation cascade (Figure 2). The growth factor receptors that are known to cause the activation of Stat3 include epidermal growth factor receptors (EGFRs), human epidermal growth factor receptor (HER2, also known as NEU), fibroblast growth factor receptors (FGFRs), insulin-like growth factor receptors (IGFRs), hepatocyte growth factor receptors (HGFRs, also known as MET), platelet-derived growth factor receptors (PDGFRs), and vascular endothelial growth factor receptors (VEGFRs). After autocrine or paracrine cytokines/growth factors binding to their respective receptors, they undergo homo- or heterodimeriza- tion leading to the subsequent activation of intrinsic receptor tyrosine kinases that culminate in the phosphorylation of Stat3 at Tyr705. For receptors lacking intrinsic tyrosine-kinase activity, ligand engagement induces recruitment and activation of receptor-associated tyrosine kinases, such as JAK and SRC. Subsequently, JAK and SRC proteins phosphorylate certain tyrosine residues in the intracellular domain of the receptor, creating docking sites for cytosolic Stat3 via its SH2 domain. Received: February 15, 2012 Published: May 31, 2012 Perspective pubs.acs.org/jmc © 2012 American Chemical Society 6645 dx.doi.org/10.1021/jm300207s | J. Med. Chem. 2012, 55, 6645-6668