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Discovery of Chromone‐Based Inhibitors of the Transcription Factor STAT5

Molecular signals originating at the cell surface are conveyed by a complex system of interconnected signaling pathways to the nucleus. They converge at transcription factors, which in turn regulate the transcription of sets of genes which ultimately determine the cellular phenotype. Whereas enzymes involved in signaling pathways, that is, intracellular kinases and phosphatases and receptor tyrosine kinases, have been recognized and exploited as intervention points for modulating cellular properties with small organic molecules, transcription factors are often considered “nondruggable” because of their lack of enzymatic activities. However, as many transcription factors require interactions with themselves or other proteins, cell-permeable inhibitors of protein–protein interactions could provide an approach towards the inhibition of this important class of proteins, and would thereby allow for the analysis of transcription factor functions and for therapeutic intervention of diseased states. 3] Initially regarded as unfeasible, a growing body of evidence indicates that the inhibition of protein–protein interactions can be potently and selectively achieved by drug-like molecules, some of which are even undergoing clinical trials. STATs (signal transducers and activators and transcription) are a family of transcription factors, which require their Src-homology 2 (SH2) domain at two steps of the signaling process to be active. Firstly, STATs need to bind via their SH2 domain to activated receptors and nonreceptor tyrosine kinases (NRTKs), and can subsequently be phosphorylated at a conserved tyrosine residue C-terminal of their SH2 domain (Scheme 1). Secondly, upon tyrosine phosphorylation, STATs dissociate from the respective receptor or NRTK, and form dimers via reciprocal interactions between their SH2 domains and the sequences surrounding the phosphorylated tyrosine residue. Therefore, a small molecule which inhibits the protein–protein interactions mediated by the SH2 domain of STATs could inhibit STAT functions efficiently (Scheme 1). Direct inhibition of STATs is less likely to result in unintentional inhibition of additional signaling pathways than the targeting of upstream kinases. Two STAT family members, STAT3 and STAT5, have been recognized as therapeutic targets for many human tumors. 16] We have recently identified a small-molecule inhibitor of STAT3, which acts by selectively inhibiting the function of the STAT3 SH2 domain, thus validating the outlined approach toward STAT inhibition. Two isoforms of STAT5 exist, dubbed STAT5a and STAT5b, which are 93% identical at the amino acid level. STAT5 is overactive in several kinds of leukemias, and also in breast cancer, uterine cancer, prostate cancer, and squamous cell carcinoma of the head and neck (SCCHN). As the inhibition of signaling by STAT5 has been shown to inhibit tumor growth and to induce apoptosis of tumor cells, direct inhibition of the STAT5 protein would be desirable to help dissect and counteract the role of STAT5 in cancer. Smallmolecule inhibitors of STAT5 could furthermore be useful tools to clarify the relevance of STAT5 for various cellular processes in genetically unmodified systems. Despite the significant interest in small-molecule inhibitors of STAT5, to the best of our knowledge, nonpeptidic molecules which inhibit the function of the STAT5 SH2 domain have not been published to date. To identify organic molecules which can inhibit the function of the SH2 domain of STAT5, we used a homogeneous assay based on fluorescence polarization which monitors binding of the peptide 5-carboxyfluorescein-GYACHTUNGTRENNUNG(PO3H2)LVLDKW, which is derived from the erythropoietin (EPO) receptor, to the SH2 domain of STAT5b. Screening of diverse chemical libraries consisting of a total of 17298 molecules for compounds which disrupt the interaction between STAT5b and its binding peptide led to the identification of the chromone-derived acyl hydrazone 1 (Table 1, apparent IC50=47 17 mm). The functions of the SH2 domains of STAT3, STAT1, and of the tyrosine kinase Lck were inhibited to a lesser extent (Table 1, Figure 1). Scheme 1. Simplified model of STAT signaling induced by activated cytokine receptors. The signaling steps indicated by the dashed arrows (phosphorylation and dimerization) could be inhibited by an inhibitor of the SH2 domain of STAT family members.