Copper (Cu) is a necessary trace element in humans and animals required for the activity and stability of several Cu-containing enzymes – known as cuproenzymes. These enzymes are involved in a diverse array of physiological processes, including mitochondrial energy generation, iron homeostasis, and collagen cross-linking. Human genetic mutations that prevent Cu transport across cellular membranes or Cu delivery to these enzymes result in lethal disorders such as Menkes disease.
Recently, research led by Dr. Vishal M. Gohil, Associate Professor of Biochemistry & Biophysics at Texas A&M University, and Dr. Mohammad Zulkifli, Assistant Research Scientist from Dr. Gohil’s laboratory, showed that elesclomol, an experimental anticancer drug, could have potential to also treat Cu-deficiency diseases, including Menkes disease. Although this Cu-transporting compound has been shown to relieve Cu deficiency in a variety of animal models, the mechanism by which it delivers Cu to where it is needed inside cells has been unknown.
A prior study had shown that elesclomol acts as a new substrate for a mitochondrial enzyme known as ferredoxin 1 (FDX1). Based on this study, Drs. Gohil and Zulkifli hypothesized that FDX1 might catalyze the release of Cu from elesclomol by transforming elesclomol-bound Cu(II) to Cu(I) by transferring an electron. In this new study, Dr. Gohil and his collaborators used a variety of techniques, including X-ray fluorescence microscopy (XFM) performed on beamlines 9-ID-B and 2-ID-D at the APS, to test this mechanism. Their findings revealed both FDX1-dependent and FDX1-independent pathways for copper release.
The researchers worked with heart cell lines with cuproenzyme deficiencies resulting from a lack of Cu importer. These mutant cells multiplied at a significantly lower rate than healthy cells and had lower mitochondrial oxygen consumption rates, characteristics that could be “rescued” by giving the cells very low amounts of elesclomol. However, when the researchers used a gene editing technique to remove the gene that produces the FDX1 protein, elesclomol no longer had any rescuing effect. Together, these experiments suggested that FDX1 is necessary to release Cu from elesclomol in mitochondria.
Additional experiments confirmed the FDX1-dependent reducing of Cu(II) bound to elesclomol to Cu(I) facilitated the release of Cu from this drug. To directly visualize Cu inside cells, the researchers used XFM, a technique that prompts metals to fluoresce inside cells, a glow that can be seen through a microscope. When the researchers administered elesclomol to cells lacking Cu importer that were also missing the gene for FDX1, XFM showed that Cu still collected inside, although in lower amounts than in cells carrying the FDX1 gene. Further research showed that this Cu made its way to SOD1, the major cuproenzyme found in the cytoplasm, even in cells missing FDX1. Collectively, these experiments suggest that another mechanism beyond targeting FDX1 in mitochondria exists to distribute Cu from elesclomol to cuproenzymes elsewhere in cells.
Finally, the researchers tested whether other Cu-transporting drugs – compounds already tested in clinical trials for amyotrophic lateral sclerosis (ALS, also known as Lou Gehrig’s disease), cancer, and Menkes disease – also target FDX1. Although these drugs did appear to rely somewhat on FDX1 to supply Cu to cuproenzymes, they did so to a far lesser extent than elesclomol. For example, the researchers needed to use Cu-histidine, a drug tested to treat Menkes disease, at a concentration about 1,000 times higher than elesclomol to rescue defects in copper-depleted cells, suggesting that elesclomol is far superior in delivering Cu into cells than Cu-histidine.
Together, these findings show that elesclomol targets FDX1 to deliver Cu to cuproenzymes in mitochondria, but an unknown mechanism also exists to deliver Cu to cuproenzymes elsewhere in cells. These findings could aid researchers in repurposing this experimental anticancer drug for Cu deficiency disorders. It may also lead to further research to better understand how Cu is distributed beyond mitochondria in cells, work that could lead to treatments for a variety of disorders that hinge on Cu over- or under-abundance. An in-progress upgrade to the APS will dramatically transform studies of this type, by greatly improving both the spatial resolution for locating the Cu and the speed that these measurements can be taken. – Christy Brownlee
See: M. Zulkifli1,, A.N. Spelbring1, Y. Zhang2, S. Soma1, S. Chen3, L. Li3, T. Le1, V. Shanbhag4, M.J. Petris4, T-Y Chen2, M. Ralle5, D.P. Barondeau1, V.M. Gohil1, “FDX1-Dependent and Independent Mechanisms of Elesclomol-Mediated Intracellular Copper Delivery,” PNAS 120 (10) Feb. 2023 https://doi.org/10.1073/pnas.2216722120
Author affiliations: 1Texas A&M University; 2University of Houston; 3Argonne National Laboratory; 4University of Missouri; 5Oregon Health and Sciences University
Research reported in this publication was supported by the National Institute of General Medical Sciences of the NIH awards R01GM143630 and R01GM111672 to V.M.G., R21GM129592 to M.R., R35GM133505 to T.-Y.C., R01GM096100 to D.P.B. and National Institute of Diabetes and Digestive and Kidney Diseases award DK131190 and National Cancer Institute award CA262664 to M.J.P. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH. Use of the Advanced Photon Source part of the Argonne National Laboratory is supported by the Department of Energy, Office of Basic Energy Sciences, under contract no. DEAC02-06CH11357. The authors have research support to disclose: The corresponding author’s university (Texas A&M) has entered into a licensing agreement with Engrail Therapeutics for the development of elesclomol:copper as a therapeutic agent for the disorders of copper metabolism.
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