Next-generation ERbeta agonists with superior brain uptake suppress glioblastoma and enhance temozolomide response

Background: Glioblastoma (GBM) is the most common and aggressive primary brain malignancy in adults, with a dismal 5-year overall survival rate. Epidemiologic studies suggest that estrogen receptor beta (ERbeta) exerts tumor-suppressive effects in the brain, supporting its potential as a therapeutic target. However, the lack of potent, selective, and brain-penetrant ERbeta ligands has hindered translational progress. We recently identified indanone- and tetralone-keto/hydroxyloxime derivatives (CIDD-0149897) as promising ERbeta agonist. Here, we report the structure-guided design, synthesis, and preclinical validation of next-generation ERbeta agonists with markedly improved potency, selectivity, safety, and blood-brain barrier permeability for GBM therapy. Methods: To optimize the CIDD-0149897 scaffold and define key pharmacophores, we designed and synthesized 177 analogues using structure-based drug design incorporating ERbeta-LBD X-ray data across multiple chemotypes. ERbeta versus ERalpha selectivity was quantified using dual-luciferase reporter assays. Cytotoxicity and apoptosis were assessed in patient-derived and mouse glioblastoma stem cells (GSCs) via CellTiter-Glo and Annexin-V assays. Mechanistic studies employed Western blotting, RT-qPCR, comet assays, RNA-seq, and mass spectrometry. In vitro ADME and PK were performed through the UTSA Center for Innovative Drug Discovery. Therapeutic efficacy of lead ERbeta agonists was evaluated in orthotopic syngeneic and patient-derived xenograft GBM models. Results: Five optimized ERbeta agonists displayed substantially improved potency and selectivity. ADME analyses demonstrated 20-50-fold higher aqueous solubility and excellent plasma stability relative to CIDD-0149897. Two analogues CIDD-0169124 and CIDD-0166596 showed ≥10-fold higher brain exposure than CIDD-0149897, with a peak brain/plasma ratio of 3.12 ± 0.84 at 15 minutes, indicating rapid brain uptake. Both lead molecules reduced human and mouse GSC viability with low-micromolar IC₅₀ values and significantly enhanced temozolomide (TMZ) response, increasing DNA damage and suppressing neurosphere formation. Comet assays revealed marked increases in olive tail moment with combination treatment compared to monotherapy. In vivo, CIDD-0169124 and CIDD-0166596 significantly reduced tumor burden and prolonged survival in orthotopic xenograft GBM models. Combination with TMZ further extended survival. Immunohistochemistry of tumor sections showed decreased Ki-67 and increased cleaved caspase-3 in combination-treated tumors. Conclusion: Two improved ERbeta agonists outperformed CIDD-0149897 in potency, selectivity, safety, and brain penetration. These compounds show strong anti-GBM action and enhance TMZ efficacy, supporting their development as next-generation ERbeta-targeted therapeutics for GBM. Supported by NIH RO1 CA269866.