The role of frataxin in mitochondrial iron and haem metabolism and the development of iron chelators as potential therapeutic agents for the treatment of Friedreich's ataxia

Becker, Erika Michelle. (2003). The role of frataxin in mitochondrial iron and haem metabolism and the development of iron chelators as potential therapeutic agents for the treatment of Friedreich's ataxia PhD Thesis, School of Medicine, The University of Queensland.

       
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Author Becker, Erika Michelle.
Thesis Title The role of frataxin in mitochondrial iron and haem metabolism and the development of iron chelators as potential therapeutic agents for the treatment of Friedreich's ataxia
School, Centre or Institute School of Medicine
Institution The University of Queensland
Publication date 2003
Thesis type PhD Thesis
Supervisor Professor Des Richardson
Dr Judith Greer
Total pages 212
Language eng
Subjects 1101 Medical Biochemistry and Metabolomics
1108 Medical Microbiology
Formatted abstract Friedreich's ataxia (FA) is a severe neurodegenerative condition with an incidence of 1:50,000 in the European population. In 97% of patients this disease is due to an intronic GAA triplet repeat expansion in the FRDA gene resulting in a marked decrease in its expression. The protein encoded by this gene is known as frataxin which is found within the mitochondrion. Upon deletion of the homologous gene (YFH1) in the yeast, there was an accumulation of iron (Fe) within the mitochondrion. When the YFH1 gene was reintroduced back into the yeast cell, Fe was exported out of the mitochondrion and into the cytosol. Evidence that human frataxin is also involved in mitochondrial Fe overload comes from studies in FA patients that have shown an accumulation of Fe within the heart. While the precise role of human frataxin remains to be determined, the molecule appears to be involved in indirectly regulating the export and/or import of mitochondrial Fe.

We examined the function of frataxin in Fe metabolism by using a well-characterised model of erythroid differentiation, namely. Friend cells induced by treatment with DMSO. We characterised the changes in frataxin expression compared to molecules that play key roles in Fe metabolism (Nramp2 and TfR) and hemoglobinization (β-globin). DMSO-induction of hemoglobinization results in a marked decrease in frataxin gene (Frda) expression and protein levels. To a lesser extent, Nramp2 mRNA levels were decreased upon erythroid differentiation, while transferrin receptor (TfR) and β-globin mRNA levels increased. Intracellular Fe depletion using desferrioxamine (DFO) or pyridoxal isonicotinoyl hydrazone (PIH) which chelate cytoplasmic or cytoplasmic and mitochondrial Fe pools respectively, have no effect on frataxin expression. Further, cytoplasmic or mitochondrial Fe-loading of induced Friend cells with ferric ammonium citrate, or the haem synthesis inhibitor, succinylacetone, respectively, also had no effect on frataxin expression. Significantly, protoporphyrin IX down-regulates frataxin protein levels, suggesting a regulatory role of frataxin in Fe and/or haem metabolism. Since decreased frataxin expression leads to mitochondrial Fe-loading in FA, our data suggest that reduced frataxin expression during erythroid differentiation results in mitochondrial Fe sequestration for haem biosynthesis. These findings of mitochondrial Fe overload suggest that the use of specific Fe chelators which can permeate the mitochondrion may have potential in the treatment of this disease.

Previous studies have demonstrated that aroylhydrazone iron (Fe) chelators of the pyridoxal isonicotinoyl hydrazone (PIH) class have high Fe chelation efficacy both in vitro and in vivo. Depending upon their design, these drugs may have potential as agents for the treatment of Fe overload disease or cancer. Considering the high potential of this class of ligands, we have synthesized 7 novel aroylhydrazones in an attempt to identify Fe chelators more efficient than desferrioxamine (DFO) and more soluble than those of the PIH class. These compounds belong to a new series of tridentate chelators known as the 2-pyridylcarboxaldehyde isonicotinoyl hydrazones (PCIH). We have examined the Fe chelation efficacy and anti-proliferative activity of these chelators including their effects on the expression of genes (WAFl and GADD45) known to be important in mediating cell cycle arrest at G1/S. From 7 chelators synthesized, 3 ligands, namely 2- pyridylcarboxaldehyde benzoyl hydrazone (PCBH), 2-pyridylcarboxaldehyde mbromobenzoyl hydrazone (PCBBH), and 2-pyridylcarboxaldehyde 2-thiophenecarboxyl hydrazone (PCTH), showed greater Fe chelation activity than DFO and comparable or greater efficiency than PIH. These ligands were highly effective at both mobilizing 59Fe from cells and preventing 59Fe uptake from 59Fe-transferrin, and caused a marked increase in the RNA-binding activity of the iron-regulatory proteins (IRPs). Our studies have also demonstrated that in comparison to the cytotoxic Fe chelator, 2-hydroxy-lnaphthylaldehyde isonicotinoyl hydrazone (311; Richardson and Milnes (1997) Blood 89:3025), these ligands have far less effect on cellular growth and 3H-thymidine, 3Hleucine, or 3H-uridine incorporation. In addition, in contrast to 311 that markedly increased WAFl and GADD45 mRNA expression, PCBH and PCTH did not have any effect, while PCBBH increased the expression of GADD45 mRNA. Collectively, the present results demonstrate the potential of several of these ligands as agents for the treatment of Fe overload disease.

In order to develop effective (Fe) chelators for clinical use for Fe overload, significantly FA, we have synthesized and characterised three classes of structurally-related aroylhydrazone analogues based on: (1) 2-pyridylcarboxaldehyde isonicotinoyl hydrazone (PCIH), (2) 2-quinoline carboxaldehyde isonicotinoyl hydrazone (QCIH), and (3) di-2-pyridylketone isonicotinoyl hydrazone (PKIH). These chelators were prepared in order to further understand the structure-activity relationships of these ligands. Despite structural similarities amongst these chelators, marked differences in biological activity were found. Of the seven PCIH ligands synthesized, three were found to have high Fe chelation efficacy and low anti-proliferative activity. Furthermore, in terms of their ability to inhibit Fe uptake from transferrin and increase Fe mobilization from cells, these three ligands were far more effective than DFO. Therefore, these ligands have high potential for the treatment of Fe overload disease, such as FA. Considering that high lipophilicity of the PCIH analogues was correlated to marked Fe chelation efficacy, the 2-pyridylcarboxaldehyde moiety was replaced with the more lipophilic 2-quinolinecarboxaldehyde to generate the QCIH series. Despite the high lipophilicity of the QCIH ligands, none showed appreciable Fe chelation activity or anti-proliferative effects, even though they contained the same Fe-binding site as the PCIH group. The low activity of the QCIH series may be related to the delocalization of electron density into the quinoline ring, resulting in lower Fe-binding affinity. In contrast, the PKIH analogues showed both marked anti-proliferative activity and Fe chelation efficacy. Indeed, the anti-proliferative activity of these ligands was equal to or exceeded that found for one of the most cytotoxic chelators yet identified i.e., 2- hydroxy-1-naphthylaldehyde isonicotinoyl hydrazone (311). The PKIH group of compounds markedly increase the mRNA levels of molecules involved in cell cycle arrest and show potential as anti-cancer agents. Consequently, this study identifies not only structural features useful in the logical design of chelators for the treatment of Fe overload but also for cancer.
Keyword Friedreich's ataxia
Ataxia

 
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