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. 2019 Dec 5;8:e51913. doi: 10.7554/eLife.51913

Figure 1. Chemical structure of compounds and their inhibition of human DMT1.

(A) Chemical structures of compounds used in this work. Their synthesis is described in detail in Appendix 1. Abbreviations are indicated. (B) IC50 values determined by measuring radioactive 55Fe2+ transport (at 1 μM) into HEK293 cells stably expressing hDMT1. Data from brominated compounds are colored in lilac and data from compounds with modified isothiourea groups in violet. Values show averages of 6–8 biological replicates, errors are s.d.

Figure 1.

Figure 1—figure supplement 1. Characterization of compounds.

Figure 1—figure supplement 1.

(A) pH titration of TMBIT and Br-BIT with NaOH (right) or with HCl (left). The compounds were dissolved in H2O to a final concertation of 1 mM and titrated with the respective solutions. Data points for H2O are shown for comparison. (B) Isothermal titration of MnCl2 to solutions of TMBIT and Br-BIT. Control experiments were performed using buffer or EDTA solutions in the sample cell. For each titration, the absorbed heats upon addition of MnCl2 (top) and the integrated heats (bottom) are shown. Addition of MnCl2 to TMBIT and Br-BIT did not result in enthalpic contributions beyond what is observed in titrations of buffer solution. Titration of EDTA, shown as control, resulted in large exothermic signals. The integrated data was fitted to a model assuming one set of binding sites. The binding isotherm (solid line) depicts an affinity in the low nanomolar range.
Figure 1—figure supplement 2. Inhibition of hDMT1.

Figure 1—figure supplement 2.

Dose-dependent inhibition of hDMT1 mediated transport assayed at 1 μM Fe2+ of HEK293 cells stably expressing the protein. Values show averages of 6–8 biological replicates, errors are s.d. Solid lines show a fit to a single binding site isotherm with IC50 values displayed in Figure 1B. Compound 1, Br-DBFIT (A), compound 2, TMBIT (B), compound 3, TEBIT (C), compound 4, Br-BIT (D), compound 5, oBr-BIT (E), compound 6 (F) and compound 7 (G).
Figure 1—figure supplement 3. Transport kinetics of hDMT1 at different inhibitor concentrations.

Figure 1—figure supplement 3.

Compound 1, Br-DBFIT (A), compound 2, TMBIT (B), compound 3, TEBIT (C) and compound 4, Br-BIT (D). Experiments where performed with HEK293 cells stably expressing hDMT1. The solid lines are fits to the Michaelis-Menten equation. Values show averages of 3 biological replicates, errors are s.d.