KRA-533, a potent KRAS agonist, directly targets the GTP/GDP binding pocket within the KRAS protein, inhibiting GTP cleavage. This inhibition leads to the buildup of constitutively active GTP-bound KRAS, which subsequently activates both apoptotic and autophagic cell death pathways specifically in cancer cells.

KRA-533, at a concentration of 10 μM and after 48 hours of treatment, significantly increases KRAS activity in HCC827 cells. In H157 cells, a dosage range of 0 to 15 μM over 48 hours elevates KRAS activity proportionally, correlating with higher levels of pERK, an increased ratio of active caspase 3 to procaspase 3, and PARP cleavage, culminating in apoptosis. Furthermore, a 10-day exposure of H292 cells to 10 μM of KRA-533 suppresses cell growth, especially in cells with KRAS mutations. KRA-533 also demonstrates the ability to bind directly to wild-type and G12C, G12D, and G13D mutant KRAS proteins, activating wild-type KRAS and intensifying the activity of mutant KRAS in a dose-responsive manner. Western Blot analysis on HCC827 cells and apoptosis analysis on H157 cells, both conducted at varying concentrations and durations, confirm the compound's dual effects of enhancing KRAS activity and inducing apoptotic cell death.

KRA-533, administered intraperitoneally (I.p.) at dosages ranging from 0 to 30 mg/kg over 28 days, exhibits dose-dependent suppression of tumor growth in lung cancer mutant KRAS xenografts in Nu/Nu nude mice. It also induces apoptosis and autophagy within tumor tissues in a similar dose-dependent fashion. The therapeutic efficacy of KRA-533 is determined to be optimal at dosages between 7.5 mg/kg and 30 mg/kg.