A similar number of compounds had Δ Fn = 50-100% and were defined as iron uptake inhibitors. About 10 of these inhibitors blocked the in vitro quenching of calcein by iron and were therefore presumably iron chelators. An additional 80 structural analogs of the hydrazone class of facilitators obtained from TimTec were subsequently assessed with 16 more facilitators identified. The ability to facilitate iron uptake was verified using a dose response curve from 0.1 – 100 μM of a putative facilitator with the same calcein quenching

assay as well as by measuring the effect of the presumed facilitators on 55Fe uptake into K562 cells. Additionally, we arbitrarily chose as the lead compound LS081, the first compound to be verified by a dose-response curve (Figure Cytoskeletal Signaling inhibitor 1). The ability to facilitate iron uptake was confirmed by dose response curves in 14 of the 16 facilitators identified on the initial screen. The EC50 for LS081 was 1.22 ± 0.48 μM with a range of EC50 of 0.5-2 μM for the remainder of the iron facilitators. Within the range of concentrations used over the length of the screening neither cell number nor cell viability was affected;

in addition, the chemicals did not affect the in vitro quenching of buy I-BET151 calcein by iron (data not shown). Figure 1 Dose response curve of LS081 on 55 Fe uptake in K562 cells. 55Fe uptake was measured as described in the Methods. Briefly, 3 × 105 K562 cells were incubated with LS081 for 30 min at concentrations of 0.1-100 μM prior to the addition of 1 μM 55Fe-1 mM AA with subsequent determination of intracellular 55Fe radioactivity. Results were expressed as fold increase in 55Fe radioactivity relative to cells treated with 0.1% DMSO alone. Shown are the means ± SEM of 3 separate experiments with triplicates for each experiment. The insert

shows Cediranib (AZD2171) the chemical structure of LS081. Caco2 cells grown in bicameral chambers for 2-3 weeks to reach the desired trans-epithelial electrical resistance were used as a model for AZD3965 intestinal iron absorption. Under these conditions the Caco2 cells differentiate to form a confluent, polarized monolayer with the brush border membrane of the apical surface in contact with the buffer of the top chamber which then mimics the intestinal lumen and the basal layer in contact with the bottom chamber which represents the systemic circulation. This model allows assaying in the presence of LS081 the transport of 55Fe from the apical chamber into the cells and then into the bottom chamber. In this model over 2 hours, LS081 increased 55Fe uptake into the Caco2 cells and into the basal chamber by 4.0 ± 0.66 and 3.71 ± 0.29 fold, respectively, compared to the DMSO-treated control (mean fold change ± SEM of 3 experiments) with P < 0.001 for both uptake and transport into the basal chamber.