In silico screening, synthesis, and biological evaluation of pyrazolopyrimidine-derived mTOR inhibitors for...

Cellular senescence is a stress-induced state characterized by irreversible cell cycle arrest. Senescent cells accumulate during aging and contribute to age-related diseases, including neurodegeneration, cancer, and type 2 diabetes mellitus. The mTOR signaling pathway plays a critical role in maintaining and regulating senescence-associated features.

We employed virtual high-throughput screening and fragment-based design to identify novel small-molecule competitive mTOR kinase inhibitors with favorable physicochemical properties. Six lead compounds (1-6) were selected, and torkinib (7) was synthesized and used as a reference.

Biochemical and cell-based assays revealed that torkinib and compounds 5 and 6 inhibited mTORC1-mediated phosphorylation of p70 S6K. Compound 5 exhibited cytostatic effects in both non-transformed human cells and glioma cancer cells, with greater sensitivity observed in the latter. Unlike the rapalog temsirolimus, both torkinib and compound 5 suppressed migration in multiple glioblastoma cell lines. Notably, compound 5 induced a transient autophagy flux distinct from that elicited by other tested mTOR inhibitors. Furthermore, compound 5 reduced radiation-induced expression of senescence-associated secretory phenotype (SASP) markers, including IL-1α, IL-6, and IL-8. Additional senomorphic effects included decreased cell size and reduced senescence-associated β-galactosidase activity. In vivo, compound 5 showed slightly higher toxicity than torkinib, likely due to improved solubility.

Compound 5 demonstrates distinct biological effects compared to torkinib and represents a promising candidate for further development as an mTOR inhibitor targeting both cancer and senescent cells.