|Abstract:||In the classic paradigm of pharmacology, small molecules bind to and modulate dysfunctional or overactive proteins. However, in cases where a protein is missing, this strategy is no longer viable. Disorders of iron metabolism are the most prevalent genetic diseases worldwide and currently, there are more than 25 different human hereditary diseases associated with loss of iron transporter function and therefore aberrant iron transport, homeostasis and/or metabolism. Given all of the advantageous features that make small molecules effective therapeutics, we questioned whether small molecules could autonomously replicate the functions of deficient iron-transporting proteins and thereby restore physiology in human disease relevant models. As the remaining endogenous networks of transporters and regulators in the cell remain active, we hypothesized that a build-up of labile iron would occur upstream of membranes missing a protein transporter. We therefore hypothesized that a small molecule mimic may be able to restore physiology by facilitating the movement of iron in a site- and direction-selective manner down the established gradient. In this vein, we discovered that a small molecule natural product from the bark of Cypress trees, hinokitiol, could leverage built-up iron gradients to restore the movement of iron into, within, and/or out of cells, and restore physiology to the system. Hinokitiol promotes the movement of iron in cells deficient in Fet3Ftr1, DMT1, Mfrn1, or FPN1 to restore growth in Fet3Ftr1 yeast, transepithelial iron transport across DMT1-deficient gut epithelia, hemoglobinization in DMT1- and Mfrn1-deficient erythroid progenitors, and iron release from FPN1-deficient gut epithelia and reticuloendothelial macrophages. To test the hypothesis that deficiencies of iron transporters lead to a build-up of labile iron upstream of protein-deficient membranes, we chose to study DMT1-deficient mouse erythroleukemia (MEL) cells. We observed increased endosomal iron and reduced cytosolic and mitochondrial iron in DMT1-deficient MEL cells, using spatiotemporal imaging with organelle-specific, iron-sensitive fluorescent dyes. When treated with hinokitiol, these endosomal iron gradients were released and a subsequent increase in cytosolic and mitochondrial iron levels was observed. We were able to artificially manipulate the direction of hinokitiol mediated iron transport by first loading J774 Mouse Macrophages with iron, adding hinokitiol and watching release of iron, followed by addition of a large excess of iron to the extracellular media. Upon addition of the extracellular iron, the direction reversed from exporting iron out of the cell to importing iron into the cell, further demonstrating hinokitiol’s ability to facilitate the movement of iron down an established gradient. We provide evidence for collaboration between hinokitiol and endogenous IRE- and Hif2α-mediated regulatory networks, with levels of ferritin, IRP2, TfR2, and FPN1 responding to changes in the dynamic iron status upon hinokitiol treatment. Administration of hinokitiol via oral gavage promotes gut iron absorption in DMT1-deficient Belgrade (b/b) rats and FPN1- deficient flatiron (ffe/+) mice. Chronic injection of hinokitiol decreases liver non-heme iron and increases hematocrit in flatiron mice and hinokitiol treatment also restores hemoglobinization and reverses anemia in DMT1- and Mfrn1-deficient zebrafish embryos. I was further able to demonstrate that hinokitiol at concentrations orders of magnitude higher than efficacious doses mainly cause loss of efficacy by sequestering intracellular iron. Thus, a small molecule restores site- and direction-selective iron transport in cells deficient in three distinct iron-transport proteins, and promotes gut iron absorption and/or peripheral hemoglobinization in corresponding animal models. Mechanistic studies support the role of ion gradients that build up in cases of missing iron transporters, enabling hinokitiol to restore site- and direction-selective transmembrane iron transport. Furthermore, endogenous protein-based homeostatic mechanisms interface with this small molecule to promote iron-related physiological processes without disrupting other cellular processes. Collectively, these results suggest that small molecules that partially mimic the function of missing protein transporters of iron, and possibly other ions, may have untapped potential in the treatment of diseases resulting from a deficiency of ion transporting proteins.