A quinazoline scaffold designed to be structurally similar to traditional EGFR inhibitors, called TTT-008, has demonstrated significant anticancer activity in SW48 colorectal cancer cells (IC₅₀ = 1.432 µM). Additionally, TTT-008 exhibited a 173-fold selectivity for SW48 malignant colon cells over CCD-841 non-malignant colon cells, which implies strong potential therapeutic benefits. Despite TTT-008 computationally possessing structural similarity to other kinase inhibitors, we have ruled out this mechanism of action via a K192 panel as well as ADP-Glo and EGFR assays. Alternatively, thorough in vitro analysis of TTT-008 indicates a disruption of cellular redox metabolism and iron homeostasis, classic indicators of ferroptosis. In vitro mechanistic studies elucidated a statistically significant (p <0.0001) depletion of NADPH levels, a 10X reduction in NADPH compared to nontreated colorectal cancer cells. A parallel significant decrease of intracellular glutathione (GSH) compared to control untreated cells (p <0.0001) is consistent with diminished antioxidant capacity of the cells. These results correlate with the findings that cellular lipid peroxides increased with treatment of TTT-008 (p = 0.0001). Additionally, a dose dependent (1.8-30 µM) increase of intracellular iron compared was observed with statistical significance achieved within 24 hours at 1.8 µM TTT-008 treated cells compared to ferroptosis inhibited cells (p =0.0216). Thus, we report a small molecule ferroptosis inducer that exploits the inherent ROS abundance of malignant cells, with selective µM potency in colorectal cancer cells.

Tesfay Tesfatsion

Colorado Chromatography Labs LLC

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