Figure 6.

Rapamycin bound to W-5FA and L-5FA inhibits mTORC1 more potently than the untargeted drug. BT474 cells were treated with rapamycin at various concentrations and times. Upon obtaining cell lysates, mTORC1 inhibition was assessed by western blot of rpS6 phosphorylation (upper bands) using GAPDH as a control (lower bands). The relative phosphorylation was estimated (eq 1). (A,B) After treatment at concentrations between 0 and 1000 nM for 120 min at 37 °C, free rapamycin was effective at concentrations above 10 nM. In contrast, untargeted 5FA and W and L-5FA-rapamycin were maximally effective between 1 and 10 nM, an order of magnitude better. P6 and P13–5FA-rapamycin were the least effective, with complete inhibition above 100 nM. (C) Log AUC was quantified for each curve and compared against by ANOVA. Statistical significance was detected between free rapamycin and every other carrier formulation. (D,E) Cells were next treated at 1 nM and lysates were obtained after indicated incubations. W and L-5FA-rapamycin inhibited mTOR almost completely by 120 min. (F) To quantify this difference, the AUC under the relative expression versus time curve was compared, which showed that only W-5FA and L-5FA significantly decreased the AUC compared to free rapamycin (n = 3 per group, **p < 0.005). Mean ± SD.