Dual targeted Ugi-bisamides induce caspase-mediated apoptosis and inhibit PI3K/mTOR signaling in NSCLC

Abstract

Non-small cell lung cancer (NSCLC) continues to be a major leading cause of cancer-related deaths globally. Its poor prognosis and resistance to conventional therapies necessitate further investigation into NSCLC's molecular mechanisms to develop novel treatments. Aberrant activation of the PI3K/AKT/mTOR signaling pathway coupled with dysregulation of the apoptotic caspase cascade can severely impair apoptotic processes in NSCLC that result in tumor progression and resistance to therapy. This study unveiled novel bisamide-Ugi adducts designed to trigger caspase-dependent apoptosis and suppress the PI3K/mTOR signaling pathway in NSCLC. The targeted adducts were synthesized using an optimized one-pot Ugi four-component reaction (Ugi-4CR). The anti-proliferative activity of the test compounds was assessed in lung A549 cancer cells and compared to normal fibroblasts (WI-38) using the MTT assay. Most of the screened derivatives surpassed the cytotoxic activity of sorafenib (IC₅₀ = 4.78 μM); in particular, the Ugi adduct 8 stood out as the most cytotoxic and safest hit (IC₅₀ = 1.39 μM; SI = 15.23). Flow cytometric analysis revealed that 8 induced apoptotic cell death in the A549 cancer cells by 21-fold with total apoptosis = 25.03%, and arrested the cell cycle at the G1-phase. Mechanistically, it upregulated the activity levels of caspases-3, 7, 8, and 9 in A549 cells and showed notable selectivity for caspase-3 and -8. Additionally, qRT-PCR gene expression analyses for apoptotic markers revealed that 8 induced p53 expression by approximately 8.38 and upregulated the Bax/Bcl-2 ratio by 22 times relative to the control. Conversely, it downregulated key oncogenic genes involved in the EGFR/PI3K/mTOR pathway. At the protein level, Ugi adduct 8 significantly suppressed PI3K and mTOR kinase activity with IC₅₀ values of 1.21 μM, and 0.26 μM, respectively. Docking simulations elucidated the potential binding modes of hit 8 within the ATP binding pockets of PI3K, and mTOR, aiding in the identification of its structural activity determinants. Lastly, in silico ADMET profiling confirmed its favorable drug-like properties.