3-D Pharmacophore Identification of Second Generation Small Molecule Inhibitors of Stat3 SH2-Phosphotyrosine Binding That Induce Apoptosis in Acute Myeloid Leukemia Cells.

Abstract 1727

Poster Board I-753

Signal Transducer and Activator of Transcription 3 (Stat3) is a critical signaling intermediate in hematopoietic cells that is activated by recruitment to tyrosine-phosphorylated receptor complexes, including c-kit, IL-6 receptor, and G-CSF receptor. Stat3 proteins interact via their Src homology 2 (SH2) domain with specific phosphotyrosine (pY) motifs on activated receptors, leading to phosphorylation of Stat3 on tyrosine 705 (pY705). Reciprocal interactions between the SH2 domains and pY705 residues within Stat3 proteins promote tail-to-tail dimerization, which results in nuclear accumulation. Nuclear Stat3 dimers bind to specific DNA sequences and regulate the expression of genes involved in cell survival, proliferation, and angiogenesis. Stat3 is aberrantly activated in many types of cancers, including acute myeloid leukemia (AML), and this finding is associated with poor prognosis. Dominant negative, antisense, and RNAi strategies have repeatedly shown that inhibition of Stat3 activity in cancer cells induces apoptosis and impairs tumor growth, but these approaches are clinically problematic. Candidates for small molecule Stat3 inhibitors have been described, but none has yet entered into clinical use. We used a combination of virtual ligand screening, surface plasmon resonance (SPR) screening, and functional assays to identify 3 novel lead compounds that competitively inhibited Stat3 binding to its pY-peptide ligand, thereby blocking recruitment / phosphorylation and dimerization of Stat3 proteins. Each was selective for Stat3 v. Stat1, inhibited Stat3 nuclear accumulation and induced apoptosis preferentially in breast cancer cell lines with constitutively activated Stat3. The most active of these, Compound 188 (C188), however, was only weakly active in inducing apoptosis of AML cell lines. Two-dimensional fingerprint screening for similarity using the scaffold of C188 and a LifeChemicals compound library identified 202 compounds (C188-1 to C188-202). Each was overlayed onto C188 using a 3-D pharmacophore program (LigandScout) and ranked. The top ranking 39 compounds were tested by SPR for inhibition of Stat3 binding to pY-peptide ligand; 33 of 39 had measurable IC₅₀s with C188-9 having one of the lowest at 2.5 μM. AML cell lines were pre-treated for 1 hour with increasing concentrations of C188-9, then stimulated with G-CSF (100 ng/ml), and pY-Stat3 was quantified by FACS. The IC₅₀s for 4 out of 5 cell lines tested were approximately 4 μM. Immunoblot analyses confirmed that pre-treatment with C188-9 inhibited Stat3 phosphorylation without affecting the level of total Stat3. The IC₅₀s for inhibition of IFN-γ-induced Stat1 phosphorylation were greater than 7 μM in 4 out of 5 cell lines, indicating that C188-9 was relatively selective for Stat3. We also tested apoptosis induction by C188-9 in 7 AML cell lines representing a variety of FAB and cytogenetic subtypes, including GDM-1, HL-60, Kasumi-1, KG-1, K562, NB-4, and THP-1. Cells were exposed to increasing concentrations of C188-9 for 24 hours and then labeled with annexin V-FITC. Apoptotic cells were quantified by FACS. C188-9 induced apoptosis in all cell lines tested, with EC₅₀s ranging from 7.4 μM (GDM-1) to 44.4 μM (K562). The range of EC₅₀s was similar to that found with JSI-124, a commercially available small molecule reported to inhibit Stat3 activity by an unknown mechanism. Similar to C188-9, JSI-124 induced apoptosis in these AML cell lines with EC₅₀s ranging from 1.2 μM (Kasumi-1 and THP-1) to 44.9 μM (K562). Thus, scaffold similarity and 3-D pharmacophore analysis identified second generation chemical probes that target Stat3 at two steps in its activation. One of the compounds, C188-9, induced apoptosis in AML cells at low micromolar concentrations, and therefore merits further study as a promising new targeted therapy for AML and other malignancies.