Restored iron transport by a small molecule promotes absorption and hemoglobinization: discovery, development, and mechanistic studies

Grillo, Anthony Steven

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https://hdl.handle.net/2142/99210 Copy

Description

Title

Restored iron transport by a small molecule promotes absorption and hemoglobinization: discovery, development, and mechanistic studies

Author(s)

Grillo, Anthony Steven

Issue Date

2017-11-28

Director of Research (if dissertation) or Advisor (if thesis)

Burke, Martin D.

Doctoral Committee Chair(s)

Burke, Martin D.

Committee Member(s)

• Denmark, Scott E.
• Mitchell, Douglas A.
• Zimmerman, Steven C.

Department of Study

Chemistry

Discipline

Chemistry

Degree Granting Institution

University of Illinois at Urbana-Champaign

Degree Name

Ph.D.

Degree Level

Dissertation

Keyword(s)

• Hinokitiol
• Iron transport
• Divalent metal transporter 1 (DMT1)
• Ferroportin-1 (FPN1)
• Mitoferrin 1 (MFRN1)
• Small molecule iron transporter
• Iron transporter
• Molecular prosthetic
• Molecular prosthetics
• Iron biology
• Hemoglobinization
• Iron homeostasis

Abstract

Traditional approaches in the pharmaceutical industry center around the development of small molecule therapeutics that bind to and inhibit overactive protein function. This has been transformational in the treatment of diseases resulting from an excess of protein function, however, thousands of diseases are alternatively caused by a deficiency of protein function and thus remain incurable. A major subset of these are characterized by mutations in critical ion channels and transporters, such as microcytic anemias, cystic fibrosis, and cardiovascular diseases. Deficiencies of these ion transporting proteins diminish ion flux in distinct sites and directions. Noting the many features that make small molecules advantageous as drugs, we asked can small molecules that imperfectly replicate the function of these missing proteins be sufficient to restore physiology in protein-deficient organisms. Since the networks of active and passive ion transporting proteins remain active, we hypothesized an imperfect small molecule ion transporter could restore site- and direction-selective transport by leveraging ion gradients that selectively buildup in ion-transport protein deficiencies, thereby restoring physiology to the protein deficient organism. Iron is a critical cofactor in all forms of life, yet its excess is paradoxically toxic. Organisms have thus developed sophisticated homeostatic networks of iron-transport proteins and their regulators to maintain iron at levels sufficient for normal iron-dependent physiological processes without causing ferritoxicity. Acquired or congenital deficiencies of proteins involved in iron transport, homeostasis, or metabolism often impede the movement of iron into, within, and/or out of cells and are associated with more than 25 Mendelian diseases. We therefore asked whether a small molecule iron transporter could leverage transmembrane gradients of the labile iron pool that selectively build up in such situations to restore the movement of iron into, within, and/or out of cells and thereby enable its use in endogenous iron-dependent physiological processes. We first used iron-deficient yeast as a discovery platform to find a small molecule candidate. In a modified functional complementation assay, the small molecule natural product, hinokitiol, restored growth to yeast missing the iron transporting complex Fet3Ftr1. Hinokitiol promoted iron uptake, and concomitantly restored cell growth back to wild-type levels. In contrast to water soluble iron chelators, hinokitiol:iron complexes are lipid soluble, and readily diffuse through lipid membranes. Extensive biophysical studies suggest this growth restoration can be attributed to the capacity for hinokitiol to promote the transmembrane transport of iron. Encouraged by these results, we next tested the capacity for hinokitiol to promote gut iron absorption and/or hemoglobinization in cells and animals missing three different iron-transport proteins by promoting iron mobilization and utilization. Hinokitiol restored the uptake and transepithelial transport of iron into and across Caco-2 gut epithelia missing the apical iron importer DMT1. It also promoted the efflux of iron out of endosomes into the cytosol in DMT1-deficient erythroblasts, thereby restoring proper differentiation and hemoglobinization. Hinokitiol restored hemoglobinization in mitoferrin-deficient erythroblasts by mobilizing iron into the mitochondrial matrix from the intermembrane space. Treatment with this natural product also enabled the movement of iron out of J774 macrophages deficient in ferroportin. 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. Quantification of labile iron levels using iron-sensitive fluorophores demonstrated a site-selective build-up of iron gradients are selectively formed in iron-transport protein deficiencies upstream of the membrane that normally hosts the missing protein. This enables a metallophore to leverage these gradients to promote the site- and direction-selective movement of iron into, within, and/or out of cells. Further, hinokitiol is functionally integrated into the endogenous homeostatic network of protein transporters, storage proteins, and their regulators to allow for the absorption of iron while preventing iron overload. Collectively, these results 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. This approach may be effective in treating the millions of patients possessing acquired or genetic deficiencies of ferroportin, such as is the case with anemia of inflammation associated with rheumatoid arthritis, inflammatory bowel disease, ulcerative colitis, and other autoimmune disorders.

Graduation Semester

2017-12

Type of Resource

text

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http://hdl.handle.net/2142/99210

Copyright and License Information

Copyright 2017 Anthony S. Grillo

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