Nitro Group-Bearing Small Molecules: Highly Potent STAT3 Inhibitors Targeting Glioblastoma Stem Cells

Introduction. Signal Transducer and Activator of Transcription 3 (STAT3) signaling is disregulated in cancer, including glioblastoma (GBM), and often correlates with a worse prognosis. STAT3 activity contributes to tumor growth, anti-apoptosis, cancer stem cell maintenance, and immune evasion. Although several STAT3 inhibitors are being investigated in pre-clinical and clinical trials, no direct STAT3 inhibitor has been FDA-approved. GBM is a lethal brain tumor with limited treatment options therefore, new therapeutics are urgently needed. In drug discovery, the nitro functional group has strong electron-withdrawing capabilities, and nitro-containing compounds are being explored as anti-cancer compounds. Methods. 41 nitro-containing chalcones and cyclic C5-curcumin analogs were synthesized and tested for cytotoxicity against 3 GBM stem cell (GSC) lines. GSCs were treated with 0.1-10 μM and viability was examined 72 hours later by MTS assay. Neurosphere assays were performed to investigate effect on stem cell self-renewal pathways. Western blot analysis was performed to explore effects on cell signaling pathways. SwissDock was used to predict STAT3 binding. Results. Of 41 compounds tested, over half had an average GSC IC50 value of ≤10 μM, and 4 compounds had an IC50 value in the lower nanomolar range. One of the most effective compounds is a nitro-containing cyclic C5-curcumin analog, termed Compound 31, with an average GSC IC50 value of 0.76 μM, significantly lower than the STAT3 signaling inhibitor WP1066 (IC50 value = 2.12 μM) currently being investigated in clinical trials for GBM. Compound 31 significantly reduced GSC neurosphere formation and STAT3 phosphorylation and expression of downstream genes as determined by western blot analysis. Molecular docking experiments demonstrate that compound 31 binds to the SH2 domain of STAT3, an area critical for STAT3 phosphorylation and activation. Conclusion. The nitro-group is a potent and versatile functional group known to induce cancer cell death by several mechanisms. We identified several promising nitro-containing lead compounds with potent anti-GSC activity. Specifically, we identified a novel STAT3 inhibitor that directly targets STAT3 activity via SH2 domain binding. Given its role in tumor progression, cancer stem cell maintenance, and immune modulation, the STAT3 pathway is a promising therapeutic GBM target.