EACR26-1905

Selective JAK1 Targeting by Novel Biotinylated Isoxazoles Drives Potent Antitumor Activity in 2D and 3D Spheroid Models

M. Ergüven¹, H. Çatalak Yılmaz², D. Lengerli³, R. Acar¹, S. Bagheralmoosavi⁴, S. Banerjee², G. Yeşilöz⁴, Ö. Konu¹, E. Banoğlu³, O. Çizmecioğlu¹

¹İhsan Doğramacı Bilkent University, Molecular Biology and Genetics, Ankara, Turkey
²Middle East Technical Universityty, Biological Sciences, Ankara, Turkey
³Gazi University, Department of Pharmaceutical Chemistry, Ankara, Turkey
⁴Bilkent University, National Nanotechnology Research Center (UNAM), Ankara, Turkey

Introduction:

Isoxazole derivatives represent a promising scaffold in anticancer drug discovery, yet their therapeutic capacity remains to be fully elucidated. In this study, we describe the synthesis and biological characterization of two biotinylated isoxazole derivatives, C160 and C161, which are structurally derived from the parent compound EB38.

Material and method:

Breast (T47D and MDA-MB-231), liver (SNU475 and Mahlavu), and prostate (C4-2, DU145, and PC3) cancer cell lines were utilized in this study to characterize biotinylated derivatives. Anti-proliferative capacities of biotinylated derivatives were determined by conducting cell viability assays. To investigate cellular targets of biotinylated derivatives, biochemical characterizations with western blot, CETSA, and DARTS experiments were followed. 3D growth assay was performed utilizing the liquid-overlay method to investigate anti-growth effects of biotinylated derivatives in spheroid models.

Result and discussion:

Across breast, prostate, and liver cancer cell lines, both biotinylated derivatives demonstrated markedly enhanced antiproliferative and pro-apoptotic effects compared to EB38, with consistently reflected on reduced IC50 values in 2D cultures and enhanced anti-growth capacity in tumor spheroids. Mechanistically, C160 and C161 remarkably suppressed the phosphorylation of JAK1 and STAT1, inhibiting JAK/STAT pathway and triggering apoptotic cleavage of PARP, caspase-3, and caspase-7. Direct target engagement was rigorously confirmed through CETSA and DARTS assays, which revealed thermal and proteolytic stabilization of endogenous JAK1 in the presence of C160. In silico molecular docking and HADDOCK simulations supported JAK1 as a direct cellular target with high-affinity binding comparable to FDA-approved Ruxolitinib. Importantly, embryotoxicity tests in in vivo models establish that biotinylated derivatives do not show evidence of general toxicity in zebrafish embryos, even at concentrations exceeding in vitro effective doses.

Conclusion:

Taken together, our findings identified biotinylated isoxazole derivatives as potent, selective JAK1 inhibitors with pronounced antitumorigenic capacity, highlighting their promise for further development in targeted cancer therapies.