Novel inhibitors of signal transducer and activator of transcription 3 (STAT3) show potent activity in cell cultures and tumor xenografts.

The transcription factor (TF) STAT3 is an attractive target for development of anticancer drugs. STAT3 is over-expressed and activated in many human malignancies and has an important role in multiple oncogenic signaling pathways affecting proliferation, survival and metabolic adaptation of cancer cells. It has been difficult, however, to develop effective inhibitors of STAT3. In this study, we performed an in-depth analysis of the mechanism of action of two compounds, OPB-31121 and OPB-51602, which are currently undergoing clinical testing. We combined computational docking (CD), molecular dynamic simulation (MDS) and in vitro binding assays to study the compounds interaction with STAT3. CD predicted that OPB-31121 and OPB-51602 could bind to a common pocket in the STAT3 SH2 domain, which was not shared with other STAT3 inhibitors. MDS and in silico mutational analysis allowed refinement of the binding site predictions and an estimate of the relative binding affinities. Isotermal titration calorimetry (ITC) studies confirmed that OPB-31121 and OPB-51602 bound with high affinity (Kd, 5-10 nM) to recombinant STAT3 SH2 domain. Binding of the two compounds was disrupted by mutations of aminoacid residues in the predicted binding pocket and was mutually exclusive in competition assays. In contrast, OPB-31121 and OPB-51602 did not compete for binding with other STAT3 inhibitors, confirming that they occupied distinct pockets in the SH2 domain. In cell culture assays, OPB-31121 and OPB-51602 interfered with both Tyr705 and Ser727 phosphorylation, which are required for full transcriptional activity of STAT3. Proliferation of cancer cells was strongly affected in vitro by OPB-51602 and OPB-31121 in anchorage-dependent growth and soft-agar assays. In these assays OPB-51602 and OPB-31121 were active at low nanomolar concentrations (IC50, 5-10 nM). Interestingly, the compounds were more effective in metabolic stress conditions (e.g., nutrient and glucose depletion), suggesting that STAT3 inhibition interfered with relevant metabolic functions in cancer cells. In vivo treatment with OPB-51602 (PO, 20-40 mg/kg, 3-5 days) reduced Tyr705 and Ser727 phosphorylation in tumor xenografts. Growth of DU145 prostate tumor xenografts was almost completely arrested by daily treatment with OPB-51602 (PO, 20-40 mg/kg, 2 weeks). Interestingly, tumor growth did not resume after discontinuation of the 2-weeks treatment, indicating a persistent impairment of tumor-initiating capability. Altogether, this study demonstrates that STAT3 is the relevant intracellular target of OPB-51602 and OPB-31121. The two compounds bound with high affinity to a distinct pocket in the SH2 domain of STAT3 and interfered with STAT3 functions both in cells and tumor xenografts. These features resulted in distinctive biological activity and pharmacological properties of these novel compounds.