Restored iron transport by a small molecule promotes absorption and hemoglobinization in animals

Abstract

Multiple human diseases ensue from a hereditary or acquired deficiency of iron-transporting protein function that diminishes transmembrane iron flux in distinct sites and directions. Because other iron-transport proteins remain active, labile iron gradients build up across the corresponding protein-deficient membranes. Here we report that a small-molecule natural product, hinokitiol, can harness such gradients to restore iron transport into, within, and/or out of cells. The same compound promotes gut iron absorption in DMT1-deficient rats and ferroportin-deficient mice, as well as hemoglobinization in DMT1- and mitoferrin-deficient zebrafish. These findings illuminate a general mechanistic framework for small molecule-mediated site- and direction-selective restoration of iron transport. They also suggest that small molecules that partially mimic the function of missing protein transporters of iron, and possibly other ions, may have potential in treating human diseases.

Fig. 1. Restoring physiology to iron transporter-deficient organisms

(A) A small molecule that autonomously performs transmembrane iron transport is hypothesized to harness local ion gradients of the labile iron pool that selectively accumulate in the setting of missing protein iron transporters. Brown spheres represent labile iron, which includes both ionic iron and iron weakly bound to small molecules such as citrate. (B) Structures of hinokitiol (Hino) and the transport-inactive derivative C2-deoxy hinokitiol (C2deOHino). (C) Disc diffusion with hinokitiol of fet3Δftr1Δ cells streaked on a low iron SD-agar plate containing 10 μM FeCl₃ restored yeast cell growth at intermediate concentrations of small molecule. (D) In the absence of hinokitiol, reduced fet3Δftr1Δ yeast cell growth was observed on low iron SD-agar plates containing 10 μM FeCl₃ by serial 10-fold dilution plating (from OD₆₀₀ = 1.0). Under identical conditions, restored cell growth was observed on the same low iron SD-agar plates containing 10 μM hinokitiol. (E) Yeast cell growth in liquid SD media containing 10 μM FeCl₃ in the absence or presence of 10 μM hinokitiol. N = 3. (F) Hinokitiol restored growth of fet3Δftr1Δ yeast while C2deOHino did not. N = 3. (G) Hinokitiol increases ⁵⁵Fe influx into fet3Δftr1Δ yeast while C2deOHino does not. N = 3. (E-G) NS, not significant; **** P ≤ 0.0001; Graphs depict means ± SEM.

Fig. 2. Physical characteristics of hinokitiol binding and transport

(A) Opposite to water soluble chelators, such as deferiprone, the hinokitiol-iron complex partitions into non-polar solvents. (B) UV-Vis titration study of hinokitiol with increasing FeCl₃ indicates hinokitiol binds iron. Arrows indicate changes in UV spectrum with increasing iron from 0:1 Fe:Hino to 6:1 Fe:Hino. (C and D) In contrast to water soluble iron chelators and C2deOHino, hinokitiol autonomously promotes the efflux of (C) ferrous and (D) ferric iron from model POPC liposomes. N = 3. (E) X-ray crystal structure of a C1-symmetric Fe(Hino)₃ complex. (F) Cyclic voltammogram of the iron-hinokitiol complex in 0.1 M Tris buffer in 1:1 MeOH:H₂O at pH=7.2 using 500 μM Hino and 100 μM Fe(NO₃)₃ with a 100 mV/s scan rate. (C, D) Graphs depict representative runs of three independent experiments. (F) Graph depicts a representative run of four independent experiments.

Fig. 3. Hinokitiol restores mammalian cell physiology

(A) ⁵⁵Fe uptake into DMT1-deficient Caco-2 monolayers and (B) transepithelial transport (apical to basolateral) indicated hinokitiol (500 nM) restored normal iron absorption. N = 3. (C) Hinokitiol-promoted ⁵⁵Fe transport occurs on times commensurate with dwell times in the gut. N = 3. (D) Cell pellets from shControl and hinokitiol-treated (1 μM) DMT1-deficient MEL cells appear pink, characteristic of hemoglobin, while DMT1-deficient cell pellets do not. (E) ImageJ quantification of MEL cells stained brown with o-dianisidine. Dotted line represents shControl levels. N = 6-48. (F) ⁵⁵Fe incorporation into heme in hinokitiol-rescued DMT1-deficient MEL cells. Dotted line represents shControl levels. N = 3-23. (G) Hinokitiol increases the number of o-dianisidine stained Mfrn1-deficient MEL cells. Dotted line represents DS19 levels. N = 21-48. (H) Hinokitiol (1 μM) restores ⁵⁵Fe transepithelial transport across FPN1-deficient Caco-2 monolayers (I) without affecting iron uptake. N = 12. (J) Hinokitiol (5 μM) promotes the release of ⁵⁵Fe from hepcidin-treated FPN1-deficient J774 macrophages (t = 2 hours). N = 6-20. (K) Time-dependent release of ⁵⁵Fe from wild type and FPN1-deficient J774 macrophages treated with or without hinokitiol and C2deOHino. N = 6-20. (A, B, E-J) NS, not significant; ** P ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.0001; Graphs depict means ± SEM.

Fig. 4. Hinokitiol leverages built-up iron gradients

(A) Representative fluorescence images of differentiated shControl and DMT1-deficient MEL cells in the absence or presence of hinokitiol (1 μM) using oxyburst green, calcein green, and RPA to detect relative endosomal, cytosolic, and mitochondrial iron levels, respectively. A build-up of labile iron was observed in endosomes of DMT1-deficient cells, which was released after hinokitiol treatment. (B and C) A build-up of intracellular labile iron is observed in FPN1-deficient J774 macrophages treated with 200 μM FeSO₄ by quenching of calcein green fluorescence. N = 3. (D) Iron (III) uptake into J774 macrophages with 50 μM FeCl₃ similarly revealed a build-up of total intracellular iron in FPN1-deficient cells after 4 hours using ⁵⁵Fe as a radiotracer. N = 8. (E) Increased extracellular iron (III) levels increased rates of iron uptake into J774 macrophages when treated with hinokitiol (1 μM) using ⁵⁵Fe as a radiotracer. N = 3. (F and G) Increased intraliposomal (F) ferrous iron and (G) ferric iron leads to increased rates of iron efflux in the presence of hinokitiol (10 μM). No efflux was observed in the absence of hinokitiol. N = 3. (H) Fluorescence imaging of cytosolic iron with calcein green using artificially created iron gradients in J774 macrophages in opposite directions. Cells were loaded with FeSO₄ (200 μM), rinsed, then hinokitiol (100 μM) was added at t = 5 min. An increase in fluorescence was observed, consistent with decreased intracellular labile iron. The gradient was then reversed in these same cells by addition of 100 μM FeCl₃ to the media at t = 12 min. Fluorescence quenching was observed, consistent with iron uptake. (I) Representative ImageJ quantification of calcein green fluorescence in iron-loaded J774 cells with addition of DMSO, hinokitiol, or C2deOHino at t = 5 min and FeCl₃ at t = 12 minutes. Scale bar = 10 μm (A), 20 μm (B, H). (C-E) ** P ≤ 0.01; **** P ≤ 0.0001; Graphs depict means ± SEM. (F, G) Graphs depict means of three independent experiments. (I) Representative graph from six independent experiments.

Fig. 5. The endogenous network is involved in hinokitiol-mediated Caco-2 transport

(A) Representative western blot images of proteins involved in iron absorption and regulation indicate an anemic state is observed in shDMT1 Caco-2 monolayers to promote maximal iron absorption. (B) Unidirectional hinokitiol-mediated transport in shDMT1 Caco-2 monolayers by apical or basolateral addition of hinokitiol (500 nM) and ⁵⁵Fe radiotracer. N = 3. (C) Determination of ⁵⁵Fe levels in immunoprecipitated ferritin in Caco-2 monolayers. N = 3. (D) Knockdown of FPN1 in shDMT1 Caco-2 monolayers with quercetin abrogates hinokitiol-mediated transport. N = 3. (E) Rates of Caco-2 transport with varying concentrations of iron treated with DMSO or hinokitiol (500 nM) after 4 hours. The rates of transport level off with increasing iron concentrations. N = 3. (F) Increased doses of hinokitiol increase uptake into shDMT1 Caco-2 monolayers apically treated with 25 μM FeCl₃; however, a bimodal effect is observed in transepithelial iron transport at 5 μM hinokitiol. N = 3. (G) Representative western blot images of proteins involved in iron absorption and regulation after treatment with increasing hinokitiol and 25 μM FeCl₃. Bimodal effects were similarly observed in protein levels involved in iron absorption and regulation. (H) Intermediate concentrations of hinokitiol lead to significant calcein green quenching in shDMT1 monolayers treated with 25 μM FeCl₃ after 1 hour, consistent with increased labile iron. This effect was reversed at high doses of hinokitiol. (I and J) ImageJ quantification of calcein green fluorescence in these monolayers. N = 3-6. Scale bar = 20 μm (H). (B-F, I, J) NS, not significant; **** P ≤ 0.0001; Graphs depict means ± SEM.

Fig. 6. Hinokitiol restores physiology in iron transporter-deficient animals

(A and B) Oral gavage of 1.5 mg/kg hinokitiol promotes the gut absorption of ⁵⁹Fe into (A) DMT1-deficient Belgrade (b/b) rats and (B) FPN1-deficient Flatiron (ffe/+) mice after 1 hour. N = 4-7. (C) Hinokitiol treatment (1 μM) to the water to embryos at 24 hpf and incubation for an additional forty-eight hours increases the number of GFP-positive erythroids by FACS analysis in DMT1-deficient morphant zebrafish using a transgenic fish containing GFP-tagged erythroids. N = 7-17. (D) Hinokitiol decreases the number of anemic fish from a heterozygous cross of +/cdy fish as determined by o-dianisidine staining, while C2deOHino does not. (E) Hinokitiol (1 μM) increases the number of GFP-positive erythrocytes in Mfrn1-deficient morphant zebrafish. N = 3-13. (F) Hinokitiol increases the number of non-anemic embryos from a heterozygous cross of +/frs fish. (G) Embryos from a heterozygous cross of +/frs fish were genotyped by restriction enzyme digestion with BsrI. Lanes 4 and 5 correspond to frs/frs fish treated with hinokitiol for forty-eight hours. (H) Hinokitiol-treated frs/frs fish stain brown with o-dianisidine while anemic frs/frs fish do not, indicating increased hemoglobin levels after hinokitiol treatment. (A-F) NS, not significant; * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; Graphs depict (A-C, E) means ± SEM or (D, F) weighted means ± SEM.