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A Selective and Cell-Permeable Mitochondrial Calcium Uniporter (MCU) Inhibitor Preserves Mitochondrial Bioenergetics after Hypoxia/Reoxygenation Injury
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  • Joshua J. Woods
    Joshua J. Woods
    Robert F. Smith School for Chemical and Biomolecular Engineering, Cornell University, Ithaca, New York 14853, United States
    Department of Chemistry and Chemical Biology, Cornell University, Ithaca, New York 14853, United States
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    Orcidhttp://orcid.org/0000-0002-6213-4093
  • Neeharika Nemani
    Neeharika Nemani
    Department of Medical Genetics and Molecular Biochemistry, Lewis Katz School of Medicine at Temple University, Philadelphia, Pennsylvania 19140, United States
    Center for Translational Medicine, Lewis Katz School of Medicine at Temple University, Philadelphia, Pennsylvania 19140, United States
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  • Santhanam Shanmughapriya
    Santhanam Shanmughapriya
    Department of Medical Genetics and Molecular Biochemistry, Lewis Katz School of Medicine at Temple University, Philadelphia, Pennsylvania 19140, United States
    Center for Translational Medicine, Lewis Katz School of Medicine at Temple University, Philadelphia, Pennsylvania 19140, United States
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  • Akshay Kumar
    Akshay Kumar
    Department of Medical Genetics and Molecular Biochemistry, Lewis Katz School of Medicine at Temple University, Philadelphia, Pennsylvania 19140, United States
    Center for Translational Medicine, Lewis Katz School of Medicine at Temple University, Philadelphia, Pennsylvania 19140, United States
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  • MengQi Zhang
    MengQi Zhang
    Department of Physiology and Pharmacology, Western University, London, Ontario N6A 5C1, Canada
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  • Sarah R. Nathan
    Sarah R. Nathan
    Department of Chemistry and Chemical Biology, Cornell University, Ithaca, New York 14853, United States
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  • Manfred Thomas
    Manfred Thomas
    Department of Medical Genetics and Molecular Biochemistry, Lewis Katz School of Medicine at Temple University, Philadelphia, Pennsylvania 19140, United States
    Center for Translational Medicine, Lewis Katz School of Medicine at Temple University, Philadelphia, Pennsylvania 19140, United States
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  • Edmund Carvalho
    Edmund Carvalho
    Department of Medical Genetics and Molecular Biochemistry, Lewis Katz School of Medicine at Temple University, Philadelphia, Pennsylvania 19140, United States
    Center for Translational Medicine, Lewis Katz School of Medicine at Temple University, Philadelphia, Pennsylvania 19140, United States
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  • Karthik Ramachandran
    Karthik Ramachandran
    Department of Medicine/Nephrology, Institute for Precision Medicine and Health, University of Texas Health San Antonio, San Antonio, Texas 78229, United States
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  • Subramanya Srikantan
    Subramanya Srikantan
    Department of Medicine/Nephrology, Institute for Precision Medicine and Health, University of Texas Health San Antonio, San Antonio, Texas 78229, United States
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  • Peter B. Stathopulos
    Peter B. Stathopulos
    Department of Physiology and Pharmacology, Western University, London, Ontario N6A 5C1, Canada
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  • Justin J. Wilson*
    Justin J. Wilson
    Department of Chemistry and Chemical Biology, Cornell University, Ithaca, New York 14853, United States
    *E-mail: [email protected]
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    Orcidhttp://orcid.org/0000-0002-4086-7982
  • Muniswamy Madesh*
    Muniswamy Madesh
    Department of Medical Genetics and Molecular Biochemistry, Lewis Katz School of Medicine at Temple University, Philadelphia, Pennsylvania 19140, United States
    Center for Translational Medicine, Lewis Katz School of Medicine at Temple University, Philadelphia, Pennsylvania 19140, United States
    Department of Medicine/Nephrology, Institute for Precision Medicine and Health, University of Texas Health San Antonio, San Antonio, Texas 78229, United States
    *E-mail: [email protected]
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Cite this: ACS Cent. Sci. 2019, 5, 1, 153–166
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https://doi.org/10.1021/acscentsci.8b00773
Published January 4, 2019

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Abstract

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Mitochondrial Ca2+ (mCa2+) uptake mediated by the mitochondrial calcium uniporter (MCU) plays a critical role in signal transduction, bioenergetics, and cell death, and its dysregulation is linked to several human diseases. In this study, we report a new ruthenium complex Ru265 that is cell-permeable, minimally toxic, and highly potent with respect to MCU inhibition. Cells treated with Ru265 show inhibited MCU activity without any effect on cytosolic Ca2+ dynamics and mitochondrial membrane potential (ΔΨm). Dose-dependent studies reveal that Ru265 is more potent than the currently employed MCU inhibitor Ru360. Site-directed mutagenesis of Cys97 in the N-terminal domain of human MCU ablates the inhibitory activity of Ru265, suggesting that this matrix-residing domain is its target site. Additionally, Ru265 prevented hypoxia/reoxygenation injury and subsequent mitochondrial dysfunction, demonstrating that this new inhibitor is a valuable tool for studying the functional role of the MCU in intact biological models.

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Synopsis

A new, cell-permeable inhibitor of mitochondrial calcium uptake, called Ru265, is reported, and its application in biological systems is described.

Introduction

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Intracellular calcium (Ca2+) ion concentration plays a crucial role in signal transduction and bioenergetics. Plasma membrane depolarization or the stimulation of receptors, such as the inositol triphosphate receptors (InsP3Rs) or ryanodine receptors (RYRs), produces a transient intracellular Ca2+ increase. This Ca2+ increase is achieved by both influx from the extracellular milieu in addition to Ca2+ release from the endoplasmic reticulum (ER) and sarcoplasmic reticulum (SR). (1−4) Under these conditions, the highly selective and inward-rectifying Ca2+ channel, known as the mitochondrial calcium uniporter (MCU), acts to clear excessive cytosolic Ca2+. (5−8) In this capacity, mitochondria act as Ca2+ sinks, shaping cytosolic Ca2+ (cCa2+) transients, while also utilizing these ions for cellular energy production.
The MCU complex comprises multiple functional domains with the MCU as the central pore-forming subunit (Figure 1A). (9−17) The MCU subunit is a 351-amino acid residue long motif with the N- and C-terminal domains both located in the inner mitochondrial matrix (IMM). (18) The transmembrane domains (TM1 and TM2) are connected through the solvent-accessible region with a highly conserved DXXE motif located in the upper helix of TM2. (19−21) The MCU pore is constructed from four of these subunits, giving a tetrameric structure. (20−23) MCU-mediated mitochondrial Ca2+ uptake is regulated by the proteins MCUR1, (16) EMRE, (12) MICU1, (17,24) and MICU2. (14) A 2–3 μM rise in cCa2+ concentration causes MICU1 and MICU2 to dissociate from the MCU, thus opening the pore for Ca2+ uptake (Figure 1B,C). (25) MCUR1, in contrast, acts as a positive regulator for mCa2+ uptake by binding to the coiled-coil region of the N-terminal domain (NTD, residues 72–189) (26,27) within the IMM. (26,28) Like most Ca2+ channels, the MCU is additionally autoregulated by divalent cations such as Mg2+ and Ca2+. Binding of divalent cations to the MCU-regulating acidic patch (MRAP) region in the β-grasp-like fold of the NTD destabilizes and shifts the self-association equilibrium of the MCU pore domain toward monomer formation. This equilibrium shift inhibits MCU function, thus fine-tuning Ca2+ entry into the mitochondria (Figure 1D). (27)

Figure 1

Figure 1. Topology diagram of the MCU. (A) Diagram showing the relevant regulator proteins EMRE, MCUR1, MICU1, and MICU2 and the orientation of the MCU in the IMM. Insets depict (left) the location of Cys97 in the X-ray crystal structure of the MCU-NTD (residues 72–189, PDB 5KUJ) (27) and (right) location of the DXXE motif in the solvent-accessible region of the MCU pore in the cryo-EM structure of the MCU (PDB 6DNF). (20) (B) At low Ca2+ concentration, MICU1 and MICU2 prevent Ca2+ from entering the mitochondria. (C) Elevated Ca2+ concentrations cause MICU1 and MICU2 to dissociate from the MCU, allowing Ca2+ transport across the IMM. (D) Binding of Ca2+ to the MRAP region in the MCU-NTD promotes a change in MCU conformation, preventing Ca2+ uptake.

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MCU-mediated Ca2+ uptake into the mitochondria is an electrogenic process that is driven by the highly negative electrochemical gradient across the IMM. (29) The surge of MCU-mediated mitochondrial Ca2+ (mCa2+) uptake allosterically stimulates the mitochondrial matrix pyruvate dehydrogenase complex, α-keto-glutarate dehydrogenase, and isocitrate enzymes to generate reducing equivalents (NADH) and promote ATP production. (30−32) Although MCU-mediated mitochondrial Ca2+ uptake is essential for bioenergetics, Ca2+ overload via this pathway triggers opening of the mitochondrial permeability transition pore (mPTP), which gives rise to mitochondrial swelling and rupture, creating a cellular bioenergetic crisis and activating degradative enzymes under pathological conditions, which leads to irreversible cell damage and death. (33−36) Dysregulation of Ca2+ uptake by the MCU plays a major role in numerous pathological conditions such as ischemic reperfusion injury (37−39) and neurodegenerative disease. (40,41) As such, pharmacological strategies to regulate MCU activity are of great importance and may provide further insight into the role of this channel in mediating human disease.
With the significant recent interest within this field, systematic efforts to find selective and cell-permeable MCU inhibitors have only recently been initiated. However, the structural criteria required for small molecules to be effective inhibitors of the MCU is not well-established. As such, combinatorial screening strategies have recently been employed to discover organic molecules that possess MCU-inhibitory properties. (42,43) Despite the discovery of several organic small-molecule MCU inhibitors, (42−46) these compounds generally have alternative biological activities, which result in undesirable toxicity. For example, mitoxantrone was recently discovered via a combinatorial approach to be a cell-permeable MCU inhibitor. (42) This compound, however, is an established topoisomerase II inhibitor that has been employed as a cytotoxic agent for the treatment of cancer. (47−50) In addition to these secondary biological applications, it also gives rise to cardiotoxicity, (51,52) which could hamper its in vivo applications.
The most commonly used and well-known MCU inhibitor is the inorganic binuclear oxo-bridged ruthenium complex ruthenium 360 (Ru360; Figure 2A), which was named for its strong absorbance at 360 nm. Although Ru360 is a highly potent and selective MCU inhibitor, (53−55) its widespread applicability in biological systems is limited by several factors. For example, in most cell lines, this compound is impermeant to the plasma membrane, hindering its use in intact cellular models. Furthermore, the synthesis of this complex is challenging and low-yielding, (56,57) diminishing its widespread availability. With structural data on the MCU recently becoming available, efforts to understand the mechanism of action of Ru360 have been slowly progressing. For example, site-directed mutagenesis of the S259 residue (located in the solvent exposed region of the MCU near the DXXE motif of human MCU) to an alanine gave rise to a variant of the MCU (MCUS259A) that retained functional activity, but was partially resistant to the inhibitory action of Ru360. (9) This result was taken to imply that Ru360 interacts with the DXXE motif located near the central pore opening of the MCU. Additional molecular dynamics simulations and solution-state NMR studies were consistent with Ru360 interacting with aspartate residues in the DXXE motif to directly block the MCU channel. (58) Despite these initial studies, the features that give Ru360 its potency and selectivity have not been fully elucidated, thereby warranting further investigation of Ru360 and its structural analogues.

Figure 2

Figure 2. (A) Chemical structures of the established and commercially available MCU inhibitor Ru360 (left) and the related analogue C-1. (B) Synthetic scheme for the preparation of C-2 and C-3. Crystal structures of (C) C-2 and (D) C-3. Outer sphere chlorides and solvent are omitted for clarity. Thermal ellipsoids are shown at the 50% probability level. Interatomic distances and angles are given in Tables S1–S3 in the Supporting Information (SI).

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In this study, we aimed to circumvent the limitations of Ru360 by introducing structural modifications with the goal of developing synthetically available and highly potent MCU inhibitors. We have fully characterized a new ruthenium-based MCU inhibitor that is cell-permeable and have investigated its biological properties and interactions with the MCU in detail. The results described herein provide guiding principles for the design of new and effective MCU inhibitors as tools for understanding mitochondrial Ca2+ dynamics in intact biological systems in addition to the development of a new class of therapeutic agents.

Results

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Synthesis and Characterization

A key structural feature of Ru360 is its linear Ru–O–Ru functionality. We hypothesized that this Ru–O–Ru motif, a feature that is relatively unique to Ru360, is an important factor that contributes to this molecule’s MCU-inhibitory activity. For example, we have previously reported on the synthesis and characterization of a very close structural analogue of Ru360 containing this motif, C-1 (Figure 2A), which is a more potent MCU inhibitor than its parent complex Ru360. (56,57) The syntheses of linear oxo-bridged transition metal complexes is not always straightforward, making it challenging to develop rational synthetic strategies. (59) Therefore, we targeted an analogous structural motif that would allow for rational synthetic strategies and facile development of new complexes.
Nitrido-bridged ruthenium complexes, which bear a structurally similar linear Ru–N–Ru core, can be readily accessed via ligand substitution reactions with the precursor complex K3[Ru2(μ-N)Cl8(OH2)2]. (60) Accordingly, the reaction of this complex anion with either aqueous ammonium hydroxide or ethylenediamine (en) afforded the expected compounds [Ru2(μ-N)(NH3)8Cl2]Cl3 (Ru265; referred to herein as C-2) and [Ru2(μ-N)(en)4Cl2]Cl3 (C-3) (Figure 2B). In contrast to the low-yielding synthesis of Ru360 and related analogues, the syntheses of these compounds proceed in moderate yield and require no tedious chromatographic purification. These compounds were fully characterized by NMR, IR, UV–vis spectroscopy, and single-crystal X-ray diffraction (Figure 2, and Figure S1, Supporting Information, SI). UV–vis spectra reveal strong charge transfer bands at 265 nm (ε = 34 000 ± 2000 M–1 cm–1) and 273 nm (ε = 29 000 ± 4000 M–1 cm–1) for C-2 and C-3, respectively, with a lower energy shoulder observed near 325 nm for both complexes. IR spectroscopy reveals distinct bands near 1050 cm–1 corresponding to the asymmetric Ru–N–Ru vibrational mode. Furthermore, 1H and 13C{1H} NMR spectroscopy showed relatively sharp signals for the NH3 and ethylenediamine ligands, confirming the diamagnetic character of the RuIV/RuIV system. (61,62)
Single-crystal X-ray crystallography was employed to fully elucidate the three-dimensional structures of C-2 and C-3 (Figure 2C,D). X-ray diffraction quality crystals of C-2 and C-3 were grown by vapor diffusion of dioxane into water and vapor diffusion of ethanol into dilute hydrochloric acid, respectively. These structures verify the presence of both the linear Ru–N–Ru motif (C-2Ru–N–Ru = 180°; C-3Ru–N–Ru = 176.3°) and the chlorido axial ligands. Relevant interatomic distances and angles as well as details for the crystal structure refinement of C-2 and C-3 are shown in Tables S1–S3 (SI). The Ru–N distances of the nitrido bridge agree well with previously synthesized compounds. (60,63−65) The NH3 ligands of the bridged RuIV centers of C-2 are arranged in an eclipsed configuration, whereas the ethylenediamine ligands of C-3 crystallize in a staggered conformation. This disparity may indicate that the rotation about the Ru–N–Ru axis is facile. With the new ruthenium compounds fully characterized, we proceeded to evaluate their MCU-inhibitory properties.

Mitochondrial Ca2+ Uptake Inhibition and Cell Permeability

With the new nitrido-bridged ruthenium compounds C-2 and C-3 in hand, we sought to evaluate their MCU-inhibitory properties and compare their activity to C-1, the oxo-bridged Ru360 analogue that we have previously reported. (56,57) We first measured the cytotoxicity of the complexes in HEK293 cells using the colorimetric thiazolyl blue tetrazolium bromide (MTT) assay. (66) This assay revealed that C-2 and C-3 are effectively nontoxic, as indicated by their IC50 values of 195 ± 8 and 226 ± 19 μM, respectively (Figure S2, SI). To measure the effect of these compounds on MCU activity, permeabilized HEK293T cells were treated with commercially available Ru360, C-1, C-2, or C-3 (5 μM) 200 s prior to the delivery of a 20 μM bolus of Ca2+ in the presence of 2 mM succinate as an energy source and the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) pump blocker thapsigargin to prevent ER Ca2+ uptake. (67) As anticipated, extramitochondrial Ca2+ ([Ca2+]out) was rapidly cleared in control cells as Ca2+ ions were sequestered in the mitochondria by the MCU (Figure 3A). In contrast, cells that were treated with C-1, C-2, C-3 (Figure 3A–D), and commercially available Ru360 (Figure S3, SI) showed a significant reduction in MCU-mediated mCa2+ uptake. Additionally, the mitochondrial membrane potential (ΔΨm) was monitored using 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide (JC-1) dye. (68) Cells treated with commercially available Ru360, C-1, C-2, or C-3 showed no transient ΔΨm loss (Figure 3A–C and Figure S3A,C, SI), a phenomenon that occurs when Ca2+ rapidly enters the mitochondria. (11) These results show that commercially available Ru360, C-1, C-2, and C-3 are all capable of inhibiting MCU-mediated mCa2+ uptake in permeabilized cells without negatively affecting the ΔΨm, preserving normal cell function.

Figure 3

Figure 3. (A–C) Changes in ΔΨm (JC-1, top panel) and extramitochondrial Ca2+ ([Ca2+]out) clearance (Fura-2-FF, bottom panel) in digitonin-permeabilized HEK293T cells in response to 20 μM of Ca2+ after treatment with 5 μM of C-1, C-2, and C-3. The transient drop in ΔΨm in control cells upon Ca2+ addition indicates accumulation of Ca2+ in the mitochondrial matrix. (D) Quantification of the rate of [Ca2+]m uptake as a function of decrease in [Ca2+]out after 20 μM Ca2+pulse. Data are represented as mean ± standard error of measurement (SEM); ***p < 0.001; n = 3–6.

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We next investigated the cell permeability of the compounds and their ability to inhibit MCU activity in intact cells. The cellular uptake of C-1, C-2, and C-3 was quantified as previously described. (69,70) HEK293 or HeLa cells were incubated with the complexes (50 μM) in culture media for 24 h before the cells were harvested and lysed. The amount of ruthenium in the cell lysate was determined using graphite furnace absorption spectroscopy (GFAAS) and was normalized to the protein content of each sample. We observed that C-2 is taken up 10 times more effectively than C-1 and over twice as effectively as C-3 in HEK293 cells and twice as effectively as C-1 and C-3 in HeLa cells (Figure S4, SI). To determine the abilities of the complexes to accumulate in the mitochondria, we treated HeLa cells with C-1, C-2, or C-3 (50 μM) in culture media for 24 h, and the mitochondria were isolated following modified literature procedures. (71−73) Both the mitochondrial and extramitochondrial fractions were analyzed for ruthenium content. Cells treated with C-1, C-2, or C-3 show 2–4 times greater uptake of ruthenium into the mitochondria compared to the rest of the cell, demonstrating the high selectivity of these compounds for the mitochondria (Figure S4, SI). Consistent with the cellular uptake studies, greater than 3-fold higher concentrations of C-2 are found in the mitochondria, compared to C-1 and C-3.
Encouraged by the high cell permeability and mitochondrial selectivity of C-2, we investigated the capability of this complex to inhibit MCU-mediated mCa2+ uptake in intact, nonpermeabilized cells. Briefly, HeLa cells were loaded with the cytosolic calcium concentration ([Ca2+]c) indicator Fluo-4 AM and the mitochondrial calcium concentration ([Ca2+]m) indicator Rhod-2 AM in the presence or absence of C-1, C-2, and C-3 (50 μM). Cells were stimulated with the GPCR agonist histamine (100 μM) to rapidly elevate cCa2+ levels to induce mCa2+ uptake. (74) Cells treated with C-2 showed significant inhibition of MCU-mediated mCa2+ uptake compared to untreated cells (Figure 4A,B). These results were further confirmed using genetically encoded cytosolic (RGECO) and mitochondrial (GCamP6-mt) Ca2+ sensors (Figure 4C,D). (75,76) In contrast, intact cells treated with C-1 or C-3 did not show appreciable inhibition of mCa2+ uptake. These results are consistent with our cell uptake and MCU Ca2+ uptake experiments, suggesting that C-2 is capable of inhibiting mCa2+ uptake in intact cells as a result of its enhanced permeability compared to C-1 and C-3.

Figure 4

Figure 4. (A, B) Cytosolic ([Ca2+]c, Fluo-4 AM) and mitochondrial ([Ca2+]m, Rhod-2 AM) calcium transients in HeLa cells after treatment with histamine (100 μM) that were pretreated with or without C-1, C-2, or C-3 (50 μM) for 30 min. (C, D) Cytosolic ([Ca2+]c, R-GECO) and mitochondrial ([Ca2+]m, GCamP6-mt) calcium transients in HeLa cells after treatment with histamine (100 μM) that were pretreated with or without C-1, C-2, or C-3 (50 μM) for 30 min. HeLa cells were cotransfected with R-GECO1 and GCamP6-mt and analyzed 48 h post-transfection. Data are represented as mean ± SEM; *p < 0.05; n = 3–6.

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Having identified C-2 as a cell-permeable MCU inhibitor, we performed a dose–response analysis to determine the potency of this compound toward mCa2+ uptake inhibition in a permeabilized cell system. Permeabilized HEK293T cells were treated with C-2 (1 nM to 1 μM). A 20 μM Ca2+ bolus was added after baseline recording, and the extramitochondrial calcium cleared was used as an indicator for MCU-mediated mCa2+ uptake using Fura-2-FF (Figure 5A–C). (16) We found the 50% maximal inhibitory concentration (IC50) for mCa2+ uptake in permeabilized cells to be 2.6 nM for C-2, which is an order of magnitude more effective than commercially available Ru360 (IC50 = 30 nM) (Figure 5C). Furthermore, we observed complete inhibition of mCa2+ uptake when cells were dosed with 10 nM C-2 while 500 nM of Ru360 was required for the same response (Figure 5A,B, and Figure S5, SI).

Figure 5

Figure 5. (A) Representative traces of [Ca2+]out clearance in permeabilized HEK293T cells challenged with a range of C-2 concentrations (1, 2, 3, 5, and 10 nM). (B) Comparison of MCU-inhibitory effect by Ru360 (30 nM) and C-2 (3 and 10 nM) at lower concentrations. (C) Calculation of dose-dependent inhibition by C-2 and Ru360. (D) Assessment of mitochondrial Ca2+ influx and efflux rates. Permeabilized HEK293T cells pulsed with 20 μM Ca2+ at 350 s to measure mitochondrial Ca2+ uptake, followed by the addition of the 1 μM C-2 or Ru360 at 550 s, 10 μM CGP37157 at 600 s, and 6 μM CCCP at 750 s. Representative trace depicts the comparison of C-2 and Ru360. (E) Quantification of Ca2+ influx rate. (F) Quantification of Ca2+ efflux rate after addition of C-2 or Ru360. (G) Quantification of CCCP-induced release of accumulated mitochondrial Ca2+. Data represent mean ± SEM; n.s., not significant n = 3–6.

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For C-2 to be an effective mCa2+ uptake inhibitor, C-2 should selectively inhibit MCU-mediated Ca2+ uptake and not interact with other cellular ion channels. Despite showing greatly reduced mCa2+ uptake, intact cells treated with C-2 showed normal cytosolic calcium dynamics when stimulated with histamine (Figure 5D,F). To further confirm the selectivity of C-2 for the MCU, we measured mCa2+ uptake, mCa2+ efflux rates, and matrix Ca2+ levels in permeabilized HEK293T cells before and after treatment with C-2 using the fluorescent Ca2+ indicator Fura-2-FF. (16) As expected, untreated cells displayed normal [Ca2+]out clearance rates (Figure 5D). We next added Ru360 or C-2 to inhibit MCU-mediated Ca2+ uptake and subsequently visualized [Ca2+]m efflux (Figure 5D). [Ca2+]m efflux rates were similar between both inhibitors suggesting that Ru360 and C-2 selectively inhibit the MCU and do not affect mCa2+ efflux channels (Figure 5E–G). To further confirm that C-2 did not affect mitochondrial calcium efflux, cells were treated with the sodium calcium exchanger (NCLX) inhibitor CGP-37157. (77) We observed no change in C-2 activity upon treatment with CGP-37157 and CCCP (Figure 5E–G). These results suggest that Ru360 and C-2 do not interact with other mitochondrial calcium transport proteins and selectively interact with the MCU.

Exploring the Mechanism of MCU Inhibition

Having observed the high potency of C-2 compared to Ru360, we next investigated its mechanism of action. Site-directed mutagenesis revealed that the serine residue S259 of the human MCU is required for maximum activity of Ru360. Mutating this serine residue to an alanine (S259A) renders partial resistance to Ru360-mediated MCU inhibition. (9) First, we tested if interaction with the S259 residue on the MCU is necessary for C-2 to be effective. HEK293T cells stably expressing Flag-tagged full-length human wild-type MCU (MCUWT) or the mutant S259A (MCUS259A) were permeabilized, and mCa2+ uptake was measured as above. Cells expressing the MCUS259A mutation displayed partial resistance to Ru360 inhibition of the MCU compared to MCUWT cells (Figure 6B). In contrast, cells expressing the MCUS259A mutation showed almost no change in the inhibitory activity of C-2 compared to the MCUWT cells (Figure 6A). These results suggest that C-2 could inhibit the MCU in a manner distinct from Ru360.

Figure 6

Figure 6. Representative traces of [Ca2+]out clearance and ΔΨm in permeabilized HEK293T cells stably expressing Flag-tagged MCUWT and MCUS259A mutant challenged with either (A) C-2 (10 nM) or (B) Ru360 (500 nM). Bar graphs represent the quantification of the rate of [Ca2+]m uptake. Representative traces of [Ca2+]out clearance and ΔΨm in HEK293T cells stably expressing Flag-tagged MCUWT and MCUC97A mutant challenged with either (C) C-2 (10 nM) or (D) Ru360 (500 nM). Bar graphs represent the quantification of the rate of [Ca2+]m uptake. Compound MCU-inhibitory effect was compared with cells reconstituted with appropriate mutants alone. Data represent mean ± SEM; ***p < 0.001; n = 3–6.; n = 4.

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A recent report revealed that the conserved human MCU cysteine residue C97 located in the MCU-NTD (residues 72–189) (26,27) in the mitochondrial matrix senses mitochondrial levels of reactive oxygen species and induces higher order oligomerization to regulate MCU channel activity (Figure 1). (74) Additionally, recent structural and biochemical studies have demonstrated the importance of the NTD in MCU oligomerization and activity. (20,21,27,74) To determine if C-2 interacts with the NTD, HEK293T cells stably expressing Flag-tagged full-length wild-type MCU (MCUWT) and the mutant C97A (MCUC97A) were permeabilized, and mCa2+ uptake was measured as above. Upon MCUC97A mutation, cells treated with C-2 showed significantly reduced MCU-inhibitory activity compared to MCUWT cells (Figure 6C). In contrast, the MCUC97A mutation had almost no effect on the activity of Ru360 (Figure 6D). Taken together, these studies suggest that C-2 and Ru360 may act on different regions of the MCU to inhibit mitochondrial calcium uptake.
To further elucidate the potential interaction of C-2 with the MCU-NTD, we investigated the interaction between C-2 and the uniformly 15N-labeled recombinant human MCU-NTD (residues 72–189) (27) using solution nuclear magnetic resonance (NMR) spectroscopy. The 1H–15N-heteronuclear single quantum coherence (HSQC) spectrum of the MCU-NTD in the absence of C-2 showed well-dispersed amide H(N) cross-peaks with homogeneous peak intensities, consistent with the well-folded and soluble domain (Figure 7A). Remarkably, upon addition of a 3-fold molar excess of C-2, the NMR sample showed a rapid formation of insoluble precipitate. A similar addition of C-2 to sample buffer alone showed neither precipitate formation nor a change in pH, suggesting that the insoluble aggregate contained MCU-NTD. Consistent with precipitation of the protein, the 1H–15N-HSQC spectrum of the 1:3 MCU-NTD:C-2 sample mixture showed a drastic loss in most H(N) cross-peak intensities (Figure 7A). It is noteworthy that some of the remaining H(N) peaks of the protein also exhibited small chemical shift perturbations.

Figure 7

Figure 7. (A) 1H–15N-heteronuclear single quantum coherence (HSQC) spectra of uniformly 15N-labeled human MCU N-terminal domain (residues 72–189; MCU72–189) in the absence (blue cross-peaks) and presence (magenta cross-peaks) of C-2. A large amount of the protein immediately precipitated out of solution with the addition of 3 molar excess C-2 to the sample with no detectable change in solvent pH or ionic strength. Consequently, most amide H(N) cross-peaks show a drastic loss in intensity compared to cross-peaks in the control spectrum. (B) Coomassie blue-stained 15% (w/v) SDS-PAGE gel visualizing the insoluble precipitate formed after mixing 125 μM MCU72–189 with 375 μM C-2 (Insol. + C-2) and 375 μM Ru360 (Insol. + Ru360) compared to untreated MCU72–189 protein (Sol.). (C) Cross-peak broadening map on the MCU72–189 high-resolution crystal structure (PDB 5KUJ). (27) Tentatively assigned residues which exhibited a ≥40% reduction in peak intensity are highlighted in red on the backbone ribbon view of MCU72–189 (top dashed box). The surface representation of the same view (bottom dashed box) shows that the most affected H(N) cross-peaks primarily cluster on one face of the domain.

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To determine if the MCU-NTD remained intact upon treatment with C-2, we resuspended the insoluble aggregate (Insol. + C-2) in loading dye and compared the migration of the protein to untreated, soluble MCU-NTD (Sol.) by SDS-PAGE analysis (Figure 7B). After Coomassie blue staining, the untreated MCU-NTD control showed clear bands indicating the presence of the monomer (∼13.9 kDa) in addition to low levels of the dimer and tetramer oligomers which is characteristic of the native MCU-NTD conformation. The protein precipitate that formed upon treatment with C-2 showed a distinct and systematic laddering corresponding to the presence of monomer, dimer, trimer, tetramer, and higher aggregate formation. The appearance of the trimer and higher order aggregates upon treatment with C-2 may suggest that the MCU-NTD conformation induced by C-2 is different from that of the native conformation. Similar MCU-NTD aggregation was observed when the MCU-NTD was treated with lower molar ratios (i.e., 1:2 and 1:1) of C-2.
To distinguish whether C-2 induced a global or more local structural effect on the MCU-NTD, peaks exhibiting an intensity reduction of ≥40% were mapped (see the SI for details) onto the crystal structure of the domain (PDB 5KUJ). (27) Remarkably, these most severely affected H(N) cross-peaks were found to cluster close together on the two β-sheets of the β-grasp-like fold despite the long distance between these residues in sequence space (Figure 7C). The clustering of these perturbations could be a result of (i) direct binding with the compound, (ii) indirect effects of the lower soluble protein concentration shifting the self-association equilibrium, or (iii) C-2-mediated modifications to the protein. Nevertheless, these data collectively suggest that C-2 interacts with the MCU-NTD to induce aggregation of the protein and inhibit Ca2+ uptake through the MCU channel. (20−23,26,27) Notably, we have found that commercially available Ru360 also interacts with the purified MCU-NTD in a cell-free system, giving rise to precipitation and systematic laddering (Insol. + Ru360; Figure 7B). Given the structural similarity of Ru360 and C-2, it is not surprising that these compounds induce analogous conformational perturbations in the NTD of the MCU in solution. However, site-mutagenesis studies with MCUC97A and MCUS259A (Figure 6) clearly show that the inhibitory activity of C-2 is dependent on the mutation-status of the NTD, whereas the inhibitory activity of Ru360 is not. Thus, we hypothesize that the NTD is a potent target for MCU inhibition that is only accessible to C-2 because of its enhanced membrane permeability compared to Ru360.

Protection from Ca2+-Induced PTP Opening and Hypoxia/Reoxygenation Injury

Given the Ca2+ uptake inhibiting properties and low toxicity of C-2, we hypothesized that C-2 could be used to protect cardiomyocytes from the mCa2+ overload that occurs during hypoxia/reoxygenation (H/R) injury and prevent mPTP opening and mitochondrial swelling. (34,37,78) Freshly isolated neonatal rat ventricular myocytes (NRVMs) treated with C-2 (50 μM) were subjected to 16 h of hypoxia (1% O2–5% CO2), followed by 8 h of reoxygenation (21% O2–5% CO2). Untreated NRVMs served as controls. After H/R injury, NRVMs were permeabilized and loaded with the JC-1 and Fura-FF to simultaneously measure ΔΨm and mCa2+ uptake. (16) Cells treated with C-2 maintained mitochondrial integrity with no loss of ΔΨm after H/R injury (Figure 8B, top panel; and Figure 8D). Additionally, treatment with C-2 resulted in complete ablation of mCa2+ uptake after H/R injury (Figure 8B, bottom panel; and 8E). On the contrary, untreated cells subjected to identical conditions showed no preservation of ΔΨm and did not show normal mCa2+ uptake (Figure 8A, top panel; and Figure 8D−E). In contrast to C-2, pretreatment with Ru360 did not prevent H/R-mediated ΔΨm dissipation (Figure 8C–E). Additionally, we monitored the swelling of mitochondria isolated from NRVMs subjected to H/R injury in the absence (Figure 8F; black trace) or presence (Figure 8F; blue trace) of C-2 or Ru360 (Figure 8F; red trace). After baseline measurement of the absorbance at 600 nm, a single bolus of Ca2+ (250 μM) was added to induce mPTP opening. The decrease in mitochondrial absorbance indicates mitochondrial swelling and subsequent mPTP opening. (34) When cells were treated with C-2 or Ru360, we observed no decrease in absorbance after Ca2+ addition, indicating a lack of mitochondrial swelling (Figure 8F). These results demonstrate that inhibition of MCU-mediated mCa2+ uptake by C-2 effectively prevents mPTP opening in response to H/R injury and when challenged with high Ca2+ concentrations.

Figure 8

Figure 8. Measurement of ΔΨm (top panels) and MCU-mediated Ca2+ uptake (bottom panels) in (A) control, (B) C-2, and (C) Ru360 treated NRVMs exposed to normoxia (black) and H/R (red). (D) Quantification of basal ΔΨm from parts A–C under normoxia and H/R before the addition of extramitochondrial Ca2+ pulses. (E) Quantification of the rate of MCU-mediated Ca2+ uptake from parts A–C as a function of decrease in bath Ca2+ fluorescence. (F) Swelling of isolated NRVM mitochondria subjected to H/R injury in the absence (black trace) or presence (blue trace) of C-2 or Ru360 (red trace). Data represent mean ± SEM; ***p < 0.001; n = 3–6.

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Discussion

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Given the importance of mCa2+ in mitochondrial bioenergetics, damage, and cell death, identification of effective MCU-mediated mCa2+ uptake inhibitors has attracted considerable interest. (41,79−81) In this report, we describe the synthesis, characterization, and biological activity of a cell-permeable, highly potent, and selective MCU inhibitor. Identifying potent and selective MCU inhibitors has proven to be challenging, with reported potential inhibitors generally exhibiting low permeability or poor selectivity and mitochondrial targeting. (42) Given high potency and selectivity of Ru360, we focused our design on structurally related compounds. The syntheses of the oxo-bridged complexes Ru360 and C-1 are low-yielding and require tedious chromatographic purification. (56,57) In contrast, C-2, which we name Ru265, and C-3 can be prepared in moderate yields without the need for extensive purification. Additionally, the nitrido-bridged complexes can be accessed through the common starting material, K3[Ru2(μ-N)Cl8(OH2)2]. This synthetic route allows for facile design of related analogues of Ru265 and C-3 through ligand substitution reactions. This straightforward chemical reaction is advantageous for studying structure–activity relationships to probe the biological activity of these complexes, which will be the focus of a future report.
Among the compounds studied, Ru265 showed the most effective MCU inhibition in nonpermeabilized cells. Furthermore, this compound is highly water-soluble (1 mg/mL) and exhibits low toxicity to human cells. The low toxicity of these complexes is significant because of the large number of known cytotoxic ruthenium compounds. (82−91) The minimal toxicity of Ru265 in contrast to many other ruthenium compounds highlights the importance of molecular structure and coordination geometry in the biological activity of metal-based compounds. (92−94) In our intact cellular models, Ru265 consistently inhibited MCU-mediated mCa2+ uptake without any effect on intracellular Ca2+ dynamics.
A series of recent reports have definitively elucidated the structure of the full-length MCU through cryo-EM and X-ray crystallographic techniques. (20−23) Most notably, these reports highlight the assembly of the NTD directly under the channel pore in the mitochondrial matrix. Our binding studies suggest that Ru265 interacts with the MCU-NTD and may perturb the assembly of NTD through aggregation, inhibiting normal channel function (Figure 7). Based on these data, it is tempting to speculate that the Ru265-dependent aggregation of the NTD could inhibit the dynamics of the NTD and immediately adjacent coiled–coiled domains required to regulate pore opening and closing. Our speculation is supported by the lack of rapid MCU Ca2+ uptake inhibition at lower Ru265 concentrations (Figure 5). It remains to be definitively determined whether Ru265-induced NTD aggregation regulates the Ca2+ binding and release mechanism; this study will be detailed in a future report. Alternatively, binding of Ru265 to the NTD could modulate MCU channel activity by promoting conformations which constrict the pore.
We have previously shown that human MCU-NTD contains a contiguous electronegative surface patch which can bind divalent cations and inhibit the MCU channel function via a shift of the self-association equilibrium of the domain toward monomer formation. (27) Given the charge, symmetry, and size of Ru265, it is possible that Ru265 also interacts with the MRAP of the MCU-NTD, with the ability to bridge MCU-NTD subunits. Using Ru265, we observed a robust inhibition of the MCU channel, which may be a consequence of the higher order oligomerization of the MCU-NTD that it induces. In the context of the full-length MCU, the bridging of dimers could prevent MCU-NTD dimer dynamics proposed to be involved in MCU gating by locking the domains in an assembly pattern, promoting a closed pore and inhibited Ca2+ uptake. Future high-resolution structural investigations using the full-length human protein are required to determine the precise structural mechanism by which Ru265 causes MCU inhibition.
In summary, the current study presents a ruthenium-based complex that not only inhibits the MCU selectively in vitro but also efficiently inhibits MCU activity and prevents mitochondrial membrane potential dissipation, mitochondrial swelling, and mPTP opening in cells exposed to simulated ischemic reperfusion injury. We show that Ru265 is over 10 times more effective at inhibiting mCa2+ uptake than Ru360, and potentially interacts with the matrix side of the MCU rather than the intermembrane Ca2+ binding sites. Furthermore, Ru265 inhibits MCU activity without disrupting normal cellular Ca2+ dynamics. This work presents a new class of compounds that have important implications in drug development strategies for diseases that are intimately associated with mitochondrial dysfunction.

Supporting Information

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The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acscentsci.8b00773.

  • Experimental details, complex characterization data, cell viability curves, cell uptake, UV–vis spectra, and dose–response data for Ca2+ uptake inhibition (PDF)

  • X-ray crystal data for C-2 and C-3 (CIF)

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Author Information

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  • Corresponding Authors
    • Justin J. Wilson - Department of Chemistry and Chemical Biology, Cornell University, Ithaca, New York 14853, United States;  Orcidhttp://orcid.org/0000-0002-4086-7982;  Email: [email protected]
    • Muniswamy Madesh - Department of Medical Genetics and Molecular Biochemistry, Lewis Katz School of Medicine at Temple University, Philadelphia, Pennsylvania 19140, United States;  Center for Translational Medicine, Lewis Katz School of Medicine at Temple University, Philadelphia, Pennsylvania 19140, United States;  Department of Medicine/Nephrology, Institute for Precision Medicine and Health, University of Texas Health San Antonio, San Antonio, Texas 78229, United States;  Email: [email protected]
  • Authors
    • Joshua J. Woods - Robert F. Smith School for Chemical and Biomolecular Engineering, Cornell University, Ithaca, New York 14853, United States;  Department of Chemistry and Chemical Biology, Cornell University, Ithaca, New York 14853, United States;  Orcidhttp://orcid.org/0000-0002-6213-4093
    • Neeharika Nemani - Department of Medical Genetics and Molecular Biochemistry, Lewis Katz School of Medicine at Temple University, Philadelphia, Pennsylvania 19140, United States;  Center for Translational Medicine, Lewis Katz School of Medicine at Temple University, Philadelphia, Pennsylvania 19140, United States
    • Santhanam Shanmughapriya - Department of Medical Genetics and Molecular Biochemistry, Lewis Katz School of Medicine at Temple University, Philadelphia, Pennsylvania 19140, United States;  Center for Translational Medicine, Lewis Katz School of Medicine at Temple University, Philadelphia, Pennsylvania 19140, United States
    • Akshay Kumar - Department of Medical Genetics and Molecular Biochemistry, Lewis Katz School of Medicine at Temple University, Philadelphia, Pennsylvania 19140, United States;  Center for Translational Medicine, Lewis Katz School of Medicine at Temple University, Philadelphia, Pennsylvania 19140, United States
    • MengQi Zhang - Department of Physiology and Pharmacology, Western University, London, Ontario N6A 5C1, Canada
    • Sarah R. Nathan - Department of Chemistry and Chemical Biology, Cornell University, Ithaca, New York 14853, United States
    • Manfred Thomas - Department of Medical Genetics and Molecular Biochemistry, Lewis Katz School of Medicine at Temple University, Philadelphia, Pennsylvania 19140, United States;  Center for Translational Medicine, Lewis Katz School of Medicine at Temple University, Philadelphia, Pennsylvania 19140, United States
    • Edmund Carvalho - Department of Medical Genetics and Molecular Biochemistry, Lewis Katz School of Medicine at Temple University, Philadelphia, Pennsylvania 19140, United States;  Center for Translational Medicine, Lewis Katz School of Medicine at Temple University, Philadelphia, Pennsylvania 19140, United States
    • Karthik Ramachandran - Department of Medicine/Nephrology, Institute for Precision Medicine and Health, University of Texas Health San Antonio, San Antonio, Texas 78229, United States
    • Subramanya Srikantan - Department of Medicine/Nephrology, Institute for Precision Medicine and Health, University of Texas Health San Antonio, San Antonio, Texas 78229, United States
    • Peter B. Stathopulos - Department of Physiology and Pharmacology, Western University, London, Ontario N6A 5C1, Canada
  • Author Contributions

    J. J. Woods and N. Nemani contributed equally. J. J. Woods, N. Nemani, S. Shanmughapriya, A. Kumar, S. R. Nathan, M. Zhang, M. Thomas, E. Carvalho, P. B. Stathopulos, J. J. Wilson, and M. Madesh performed and analyzed experiments involving chemical synthesis, biochemical, mitochondrial bioenergetics, and molecular and cellular experiments. J. J. Wilson, P. B. Stathopulos, and M. Madesh conceived, designed, analyzed, and interpreted experimental data. J. J. Woods, N. Nemani, P. B. Stathopulos, J. J. Wilson, and M. Madesh wrote the manuscript with contributions from S. Shanmughapriya and A. Kumar.

  • Notes
    The authors declare no competing financial interest.

Acknowledgments

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This research in the laboratory of M. Madesh was funded by the National Institutes of Health (R01GM109882, R01HL086699, R01HL142673, R01HL119306). Research in the laboratory of J. J. Wilson was supported by the National Science Foundation (NSF) (CHE-1750295). S. Shanmughapriya is supported by the NIH K99/R00 grant (1 K99 HL138268-01). E. Carvalho and N. Nemani are supported by the AHA fellowships (18POST33990217 and 17PRE33660720). J. J. Woods is supported by the NSF-GRFP (DGE-1650441). This work made use of the Cornell NMR facility, which is funded in part by the NSF (CHE-1531632). We thank Reggie Jacob for helpful comments on the manuscript and Samantha Davalos for assistance in preparing the artwork associated with this manuscript.

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    Mitochondrial Ca2+ homeostasis has a key role in the regulation of aerobic metab. and cell survival, but the mol. identity of the Ca2+ channel, the mitochondrial calcium uniporter, is still unknown. Here we have identified in silico a protein (named MCU) that shares tissue distribution with MICU1 (also known as CBARA1), a recently characterized uniporter regulator, is present in organisms in which mitochondrial Ca2+ uptake was demonstrated and whose sequence includes two transmembrane domains. Short interfering RNA (siRNA) silencing of MCU in HeLa cells markedly reduced mitochondrial Ca2+ uptake. MCU overexpression doubled the matrix Ca2+ concn. increase evoked by inositol 1,4,5-trisphosphate-generating agonists, thus significantly buffering the cytosolic elevation. The purified MCU protein showed channel activity in planar lipid bilayers, with electrophysiol. properties and inhibitor sensitivity of the uniporter. A mutant MCU, in which two neg. charged residues of the putative pore-forming region were replaced, had no channel activity and reduced agonist-dependent matrix Ca2+ concn. transients when overexpressed in HeLa cells. Overall, these data demonstrate that the 40-kDa protein identified is the channel responsible for ruthenium-red-sensitive mitochondrial Ca2+ uptake, thus providing a mol. basis for this process of utmost physiol. and pathol. relevance.
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  11. 11
    Mallilankaraman, K.; Doonan, P.; Cárdenas, C.; Chandramoorthy, H. C.; Müller, M.; Miller, R.; Hoffman, N. E.; Gandhirajan, R. K.; Molgó, J.; Birnbaum, M. J.; Rothberg, B. S.; Mak, D. O. D.; Foskett, J. K.; Madesh, M. MICU1 is an Essential Gatekeeper for MCU-Mediated Mitochondrial Ca2+ Uptake That Regulates Cell Survival. Cell 2012, 151, 630– 644,  DOI: 10.1016/j.cell.2012.10.011
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  12. 12
    Sancak, Y.; Markhard, A. L.; Kitami, T.; Kovacs-Bogdan, E.; Kamer, K. J.; Udeshi, N. D.; Carr, S. A.; Chaudhuri, D.; Clapham, D. E.; Li, A. A.; Calvo, S. E.; Goldberger, O.; Mootha, V. K. EMRE is an Essential Component of the Mitochondrial Calcium Uniporter Complex. Science 2013, 342, 1379– 1382,  DOI: 10.1126/science.1242993
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    12
    EMRE Is an Essential Component of the Mitochondrial Calcium Uniporter Complex
    Sancak, Yasemin; Markhard, Andrew L.; Kitami, Toshimori; Kovacs-Bogdan, Erika; Kamer, Kimberli J.; Udeshi, Namrata D.; Carr, Steven A.; Chaudhuri, Dipayan; Clapham, David E.; Li, Andrew A.; Calvo, Sarah E.; Goldberger, Olga; Mootha, Vamsi K.
    Science (Washington, DC, United States) (2013), 342 (6164), 1379-1382CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
    The mitochondrial uniporter is a highly selective calcium channel in the organelle's inner membrane. Its mol. components include the EF-hand-contg. calcium-binding proteins mitochondrial calcium uptake 1 (MICU1) and MICU2 and the pore-forming subunit mitochondrial calcium uniporter (MCU). We sought to achieve a full mol. characterization of the uniporter holocomplex (uniplex). Quant. mass spectrometry of affinity-purified uniplex recovered MICU1 and MICU2, MCU and its paralog MCUb, and a previously uncharacterized 10-kilodalton metazoan-specific single transmembrane domain protein designated EMRE ("essential MCU regulator"). In its absence, uniporter channel activity was lost despite intact MCU expression and oligomerization. EMRE was required for the interaction of MCU with MICU1 and MICU2. Hence, EMRE is essential for in vivo uniporter current and addnl. bridges the calcium-sensing role of MICU1 and MICU2 with the calcium-conducting role of MCU.
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  13. 13
    Perocchi, F.; Gohil, V. M.; Girgis, H. S.; Bao, X. R.; McCombs, J. E.; Palmer, A. E.; Mootha, V. K. MICU1 Encodes a Mitochondrial EF Hand Protein Required for Ca2+ uptake. Nature 2010, 467, 291– 296,  DOI: 10.1038/nature09358
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    There is no corresponding record for this reference.
  14. 14
    Plovanich, M.; Bogorad, R. L.; Sancak, Y.; Kamer, K. J.; Strittmatter, L.; Li, A. A.; Girgis, H. S.; Kuchimanchi, S.; De Groot, J.; Speciner, L.; Taneja, N.; OShea, J.; Koteliansky, V.; Mootha, V. K. MICU2, a Paralog of MICU1, Resides within the Mitochondrial Uniporter Complex to Regulate Calcium Handling. PLoS One 2013, 8, e55785,  DOI: 10.1371/journal.pone.0055785
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    There is no corresponding record for this reference.
  15. 15
    Csordás, G.; Golenár, T.; Seifert, E. L.; Kamer, K. J.; Sancak, Y.; Perocchi, F.; Moffat, C.; Weaver, D.; De la Fuente, S.; Bogorad, R.; Koteliansky, V.; Adijanto, J.; Mootha, V. K.; Hajnóczky, G. MICU1 Controls Both the Threshold and Cooperative Activation of the Mitochondrial Ca2+ Uniporter. Cell Metab. 2013, 17, 976– 987,  DOI: 10.1016/j.cmet.2013.04.020
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    15
    MICU1 Controls Both the Threshold and Cooperative Activation of the Mitochondrial Ca2+ Uniporter
    Csordas, Gyorgy; Golenar, Tunde; Seifert, Erin L.; Kamer, Kimberli J.; Sancak, Yasemin; Perocchi, Fabiana; Moffat, Cynthia; Weaver, David; Perez, Sergio de la Fuente; Bogorad, Roman; Koteliansky, Victor; Adijanto, Jeffrey; Mootha, Vamsi K.; Hajnoczky, Gyorgy
    Cell Metabolism (2013), 17 (6), 976-987CODEN: CMEEB5; ISSN:1550-4131. (Elsevier Inc.)
    Mitochondrial Ca2+ uptake via the uniporter is central to cell metab., signaling, and survival. Recent studies identified MCU as the uniporter's likely pore and MICU1, an EF-hand protein, as its crit. regulator. How this complex decodes dynamic cytoplasmic [Ca2+] ([Ca2+]c) signals, to tune out small [Ca2+]c increases yet permit pulse transmission, remains unknown. We report that loss of MICU1 in mouse liver and cultured cells causes mitochondrial Ca2+ accumulation during small [Ca2+]c elevations but an attenuated response to agonist-induced [Ca2+]c pulses. The latter reflects loss of pos. cooperativity, likely via the EF-hands. MICU1 faces the intermembrane space and responds to [Ca2+]c changes. Prolonged MICU1 loss leads to an adaptive increase in matrix Ca2+ binding, yet cells show impaired oxidative metab. and sensitization to Ca2+ overload. Collectively, the data indicate that MICU1 senses the [Ca2+]c to establish the uniporter's threshold and gain, thereby allowing mitochondria to properly decode different inputs.
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  16. 16
    Mallilankaraman, K.; Cárdenas, C.; Doonan, P. J.; Chandramoorthy, H. C.; Irrinki, K. M.; Golenár, T.; Csordás, G.; Madireddi, P.; Yang, J.; Müller, M.; Miller, R.; Kolesar, J. E.; Molgó, J.; Kaufman, B.; Hajnóczky, G.; Foskett, J. K.; Madesh, M. MCUR1 is an Essential Component of Mitochondrial Ca2+ Uptake that Regulates Cellular Metabolism. Nat. Cell Biol. 2012, 14, 1336– 1343,  DOI: 10.1038/ncb2622
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    16
    MCUR1 is an essential component of mitochondrial Ca2+ uptake that regulates cellular metabolism
    Mallilankaraman, Karthik; Cardenas, Cesar; Doonan, Patrick J.; Chandramoorthy, Harish C.; Irrinki, Krishna M.; Golenar, Tuende; Csordas, Gyoergy; Madireddi, Priyanka; Yang, Jun; Mueller, Marioly; Miller, Russell; Kolesar, Jill E.; Molgo, Jordi; Kaufman, Brett; Hajnoczky, Gyoergy; Foskett, J. Kevin; Madesh, Muniswamy
    Nature Cell Biology (2012), 14 (12), 1336-1343CODEN: NCBIFN; ISSN:1465-7392. (Nature Publishing Group)
    Ca2+ flux across the mitochondrial inner membrane regulates bioenergetics, cytoplasmic Ca2+ signals and activation of cell death pathways. Mitochondrial Ca2+ uptake occurs at regions of close apposition with intracellular Ca2+ release sites, driven by the inner membrane voltage generated by oxidative phosphorylation and mediated by a Ca2+ selective ion channel (MiCa; ref. ) called the uniporter whose complete mol. identity remains unknown. Mitochondrial calcium uniporter (MCU) was recently identified as the likely ion-conducting pore. In addn., MICU1 was identified as a mitochondrial regulator of uniporter-mediated Ca2+ uptake in HeLa cells. Here we identified CCDC90A, hereafter referred to as MCUR1 (mitochondrial calcium uniporter regulator 1), an integral membrane protein required for MCU-dependent mitochondrial Ca2+ uptake. MCUR1 binds to MCU and regulates ruthenium-red-sensitive MCU-dependent Ca2+ uptake. MCUR1 knockdown does not alter MCU localization, but abrogates Ca2+ uptake by energized mitochondria in intact and permeabilized cells. Ablation of MCUR1 disrupts oxidative phosphorylation, lowers cellular ATP and activates AMP kinase-dependent pro-survival autophagy. Thus, MCUR1 is a crit. component of a mitochondrial uniporter channel complex required for mitochondrial Ca2+ uptake and maintenance of normal cellular bioenergetics.
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  17. 17
    Patron, M.; Checchetto, V.; Raffaello, A.; Teardo, E.; VecellioReane, D.; Mantoan, M.; Granatiero, V.; Szabò, I.; DeStefani, D.; Rizzuto, R. MICU1 and MICU2 Finely Tune the Mitochondrial Ca2+ Uniporter by Exerting Opposite Effects on MCU Activity. Mol. Cell 2014, 53, 726– 737,  DOI: 10.1016/j.molcel.2014.01.013
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    17
    MICU1 and MICU2 Finely Tune the Mitochondrial Ca2+ Uniporter by Exerting Opposite Effects on MCU Activity
    Patron, Maria; Checchetto, Vanessa; Raffaello, Anna; Teardo, Enrico; Vecellio Reane, Denis; Mantoan, Maura; Granatiero, Veronica; Szabo, Ildiko; De Stefani, Diego; Rizzuto, Rosario
    Molecular Cell (2014), 53 (5), 726-737CODEN: MOCEFL; ISSN:1097-2765. (Elsevier Inc.)
    Mitochondrial calcium accumulation was recently shown to depend on a complex composed of an inner-membrane channel (MCU and MCUb) and regulatory subunits (MICU1, MCUR1, and EMRE). A fundamental property of MCU is low activity at resting cytosolic Ca2+ concns., preventing deleterious Ca2+ cycling and organelle overload. Here we demonstrate that these properties are ensured by a regulatory heterodimer composed of two proteins with opposite effects - MICU1 and MICU2 - which in both purified lipid bilayers and in intact cells stimulate and inhibit MCU activity, resp. Both MICU1 and MICU2 are regulated by calcium through their EF-hand domains, thus accounting for the sigmoidal response of MCU to [Ca2+] in situ and allowing tight physiol. control. At low [Ca2+], the dominant effect of MICU2 largely shuts down MCU activity; at higher [Ca2+], the stimulatory effect of MICU1 allows the prompt response of mitochondria to Ca2+ signals generated in the cytoplasm.
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  18. 18
    Oxenoid, K.; Dong, Y.; Cao, C.; Cui, T.; Sancak, Y.; Markhard, A. L.; Grabarek, Z.; Kong, L.; Liu, Z.; Ouyang, B.; Cong, Y.; Mootha, V. K.; Chou, J. J. Architecture of the Mitochondrial Calcium Uniporter. Nature 2016, 533, 269– 273,  DOI: 10.1038/nature17656
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    18
    Architecture of the mitochondrial calcium uniporter
    Oxenoid, Kirill; Dong, Ying; Cao, Chan; Cui, Tanxing; Sancak, Yasemin; Markhard, Andrew L.; Grabarek, Zenon; Kong, Liangliang; Liu, Zhijun; Ouyang, Bo; Cong, Yao; Mootha, Vamsi K.; Chou, James J.
    Nature (London, United Kingdom) (2016), 533 (7602), 269-273CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
    Mitochondria from many eukaryotic clades take up large amts. of Ca2+ via an inner membrane transporter called the mitochondrial calcium uniporter (MCU). Transport by MCU is membrane potential-dependent and sensitive to ruthenium red or its deriv., Ru360. Electrophysiol. studies have shown that MCU is an ion channel with remarkably high conductance and selectivity. Ca2+ entry into mitochondria is also known to activate the tricarboxylic acid cycle and seems to be crucial for matching the prodn. of ATP in mitochondria with its cytosolic demand. MCU is the pore-forming and Ca2+-conducting subunit of the uniporter holocomplex, but its primary sequence does not resemble any Ca2+ channel studied to date. Here, the authors report the structure of the pore domain of MCU from Caenorhabditis elegans, detd. using NMR spectroscopy and electron microscopy (EM). MCU is a homo-oligomer in which the 2nd transmembrane helix forms a hydrophilic pore across the membrane. The channel assembly represented a new soln. of ion channel architecture, and was stabilized by a coiled-coil motif protruding into the mitochondrial matrix. The crit. DXXE motif formed the pore entrance, which featured 2 carboxylate rings; based on the ring dimensions and functional mutagenesis, these rings appeared to form the selectivity filter. To the authors' knowledge, this is one of the largest membrane protein structures characterized by NMR, and provides a structural blueprint for understanding the function of this channel.
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  19. 19
    Bick, A. G.; Calvo, S. E.; Mootha, V. K. Evolutionary Diversity of the Mitochondrial Calcium Uniporter. Science 2012, 336, 886,  DOI: 10.1126/science.1214977
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    Baradaran, R.; Wang, C.; Siliciano, A. F.; Long, S. B. Cryo-EM Structures of Fungal and Metazoan Mitochondrial Calcium Uniporters. Nature 2018, 559, 580– 584,  DOI: 10.1038/s41586-018-0331-8
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    20
    Cryo-EM structures of fungal and metazoan mitochondrial calcium uniporters
    Baradaran, Rozbeh; Wang, Chongyuan; Siliciano, Andrew Francis; Long, Stephen Barstow
    Nature (London, United Kingdom) (2018), 559 (7715), 580-584CODEN: NATUAS; ISSN:0028-0836. (Nature Research)
    The mitochondrial calcium uniporter (MCU) is a highly selective calcium channel and a major route of calcium entry into mitochondria. How the channel catalyzes ion permeation and achieves ion selectivity are not well understood, partly because MCU is thought to have a distinct architecture in comparison to other cellular channels. Here we report cryo-electron microscopy reconstructions of MCU channels from zebrafish and Cyphellophora europaea at 8.5 Å and 3.2 Å resolns., resp. In contrast to a previous report of pentameric stoichiometry for MCU, both channels are tetramers. The at. model of C. europaea MCU shows that a conserved WDXXEP signature sequence forms the selectivity filter, in which calcium ions are arranged in single file. Coiled-coil legs connect the pore to N-terminal domains in the mitochondrial matrix. In C. europaea MCU, the N-terminal domains assemble as a dimer of dimers; in zebrafish MCU, they form an asym. crescent. The structures define principles that underlie ion permeation and calcium selectivity in this unusual channel.
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  21. 21
    Fan, C.; Fan, M.; Orlando, B. J.; Fastman, N. M.; Zhang, J.; Xu, Y.; Chambers, M. G.; Xu, X.; Perry, K.; Liao, M.; Feng, L. X-Ray and Cryo-EM Structures of the Mitochondrial Calcium Uniporter. Nature 2018, 559, 575– 579,  DOI: 10.1038/s41586-018-0330-9
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    21
    X-ray and cryo-EM structures of the mitochondrial calcium uniporter
    Fan, Chao; Fan, Minrui; Orlando, Benjamin J.; Fastman, Nathan M.; Zhang, Jinru; Xu, Yan; Chambers, Melissa G.; Xu, Xiaofang; Perry, Kay; Liao, Maofu; Feng, Liang
    Nature (London, United Kingdom) (2018), 559 (7715), 575-579CODEN: NATUAS; ISSN:0028-0836. (Nature Research)
    Mitochondrial Ca2+ uptake is crit. for regulating ATP prodn., intracellular Ca2+ signaling, and cell death. This uptake is mediated by a highly selective Ca2+ channel called the mitochondrial calcium uniporter (MCU). Here, we detd. the structures of the pore-forming MCU proteins from 2 fungi (Fusarium graminearum and Metarhizium acridum) by x-ray crystallog. and single-particle cryo-electron microscopy (cryo-EM). The stoichiometry, overall architecture, and individual subunit structure differed markedly from those described in the recent NMR structure of Caenorhabditis elegans MCU. We obsd. a dimer-of-dimer architecture across species and chem. environments, which was corroborated by biochem. expts. Structural analyses and functional characterization uncovered the roles of key residues in the pore. These results revealed a new ion channel architecture, provide insights into Ca2+ coordination, selectivity, and conduction, and established a structural framework for understanding the mechanism of MCU function.
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  22. 22
    Nguyen, N. X.; Armache, J. P.; Lee, C.; Yang, Y.; Zeng, W.; Mootha, V. K.; Cheng, Y.; Bai, X.; Jiang, Y. Cryo-EM Structure of a Fungal Mitochondrial Calcium Uniporter. Nature 2018, 559, 570– 574,  DOI: 10.1038/s41586-018-0333-6
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    22
    Cryo-EM structure of a fungal mitochondrial calcium uniporter
    Nguyen, Nam X.; Armache, Jean-Paul; Lee, Changkeun; Yang, Yi; Zeng, Weizhong; Mootha, Vamsi K.; Cheng, Yifan; Bai, Xiao-chen; Jiang, Youxing
    Nature (London, United Kingdom) (2018), 559 (7715), 570-574CODEN: NATUAS; ISSN:0028-0836. (Nature Research)
    The mitochondrial calcium uniporter (MCU) is a highly selective calcium channel localized to the inner mitochondrial membrane. Here, we describe the structure of an MCU ortholog from the fungus Neosartorya fischeri (NfMCU) detd. to 3.8 Å resoln. by phase-plate cryo-electron microscopy. The channel is a homotetramer with two-fold symmetry in its amino-terminal domain (NTD) that adopts a similar structure to that of human MCU. The NTD assembles as a dimer of dimers to form a tetrameric ring that connects to the transmembrane domain through an elongated coiled-coil domain. The ion-conducting pore domain maintains four-fold symmetry, with the selectivity filter positioned at the start of the pore-forming TM2 helix. The aspartate and glutamate sidechains of the conserved DIME motif are oriented towards the central axis and sepd. by one helical turn. The structure of NfMCU offers insights into channel assembly, selective calcium permeation, and inhibitor binding.
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  23. 23
    Yoo, J.; Wu, M.; Yin, Y.; Herzik, M. A.; Lander, G. C.; Lee, S.-Y. Cryo-EM Structure of a Mitochondrial Calcium Uniporter. Science 2018, 361, 506– 511,  DOI: 10.1126/science.aar4056
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    23
    Cryo-EM structure of a mitochondrial calcium uniporter
    Yoo, Jiho; Wu, Mengyu; Yin, Ying; Herzik, Mark A., Jr; Lander, Gabriel C.; Lee, Seok-Yong
    Science (Washington, DC, United States) (2018), 361 (6401), 506-511CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
    Calcium transport plays an important role in regulating mitochondrial physiol. and pathophysiol. The mitochondrial calcium uniporter (MCU) is a calcium-selective ion channel that is the primary mediator for calcium uptake into the mitochondrial matrix. Here, we present the cryo-electron microscopy structure of the full-length MCU from Neurospora crassa to an overall resoln. of ∼3.7 angstroms. Our structure reveals a tetrameric architecture, with the sol. and transmembrane domains adopting different sym. arrangements within the channel. The conserved W-D-Φ-Φ-E-P-V-T-Y sequence motif of MCU pore forms a selectivity filter comprising two acidic rings sepd. by one helical turn along the central axis of the channel pore. The structure combined with mutagenesis gives insight into the basis of calcium recognition.
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  24. 24
    Hoffman, N. E.; Chandramoorthy, H. C.; Shamugapriya, S.; Zhang, X.; Rajan, S.; Mallilankaraman, K.; Gandhirajan, R. K.; Vagnozzi, R. J.; Ferrer, L. M.; Sreekrishnanilayam, K.; Natarajaseenivasan, K.; Vallem, S.; Force, T.; Choi, E. T.; Cheung, J. Y.; Madesh, M. MICU1 Motifs Define Mitochondrial Calcium Uniporter Binding and Activity. Cell Rep. 2013, 5, 1576– 1588,  DOI: 10.1016/j.celrep.2013.11.026
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    24
    MICU1 Motifs Define Mitochondrial Calcium Uniporter Binding and Activity
    Hoffman, Nicholas E.; Chandramoorthy, Harish C.; Shamugapriya, Santhanam; Zhang, Xueqian; Rajan, Sudarsan; Mallilankaraman, Karthik; Gandhirajan, Rajesh Kumar; Vagnozzi, Ronald J.; Ferrer, Lucas M.; Sreekrishnanilayam, Krishnalatha; Natarajaseenivasan, Kalimuthusamy; Vallem, Sandhya; Force, Thomas; Choi, Eric T.; Cheung, Joseph Y.; Madesh, Muniswamy
    Cell Reports (2013), 5 (6), 1576-1588CODEN: CREED8; ISSN:2211-1247. (Cell Press)
    Resting mitochondrial matrix Ca2+ is maintained through a mitochondrial calcium uptake 1 (MICU1)-established threshold inhibition of mitochondrial calcium uniporter (MCU) activity. It is not known how MICU1 interacts with MCU to establish this Ca2+ threshold for mitochondrial Ca2+ uptake and MCU activity. Here, we show that MICU1 localizes to the mitochondrial matrix side of the inner mitochondrial membrane and MICU1/MCU binding is detd. by a MICU1 N-terminal polybasic domain and two interacting coiled-coil domains of MCU. Further investigation reveals that MICU1 forms homo-oligomers, and this oligomerization is independent of the polybasic region. However, the polybasic region confers MICU1 oligomeric binding to MCU and controls mitochondrial Ca2+ current (IMCU). Moreover, MICU1 EF hands regulate MCU channel activity, but do not det. MCU binding. Loss of MICU1 promotes MCU activation leading to oxidative burden and a halt to cell migration. These studies establish a mol. mechanism for MICU1 control of MCU-mediated mitochondrial Ca2+ accumulation, and dysregulation of this mechanism probably enhances vascular dysfunction.
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  25. 25
    Petrungaro, C.; Zimmermann, K. M.; Küttner, V.; Fischer, M.; Dengjel, J.; Bogeski, I.; Riemer, J. The Ca2+-Dependent Release of the Mia40-Induced MICU1-MICU2 Dimer from MCU Regulates Mitochondrial Ca2+ Uptake. Cell Metab. 2015, 22, 721– 733,  DOI: 10.1016/j.cmet.2015.08.019
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    25
    The Ca2+-Dependent Release of the Mia40-Induced MICU1-MICU2 Dimer from MCU Regulates Mitochondrial Ca2+ Uptake
    Petrungaro, Carmelina; Zimmermann, Katharina M.; Kuettner, Victoria; Fischer, Manuel; Dengjel, Joern; Bogeski, Ivan; Riemer, Jan
    Cell Metabolism (2015), 22 (4), 721-733CODEN: CMEEB5; ISSN:1550-4131. (Elsevier Inc.)
    The essential oxidoreductase Mia40/CHCHD4 mediates disulfide bond formation and protein folding in the mitochondrial intermembrane space. Here, we investigated the interactome of Mia40 thereby revealing links between thiol-oxidn. and apoptosis, energy metab., and Ca2+ signaling. Among the interaction partners of Mia40 is MICU1-the regulator of the mitochondrial Ca2+ uniporter (MCU), which transfers Ca2+ across the inner membrane. We examd. the biogenesis of MICU1 and find that Mia40 introduces an intermol. disulfide bond that links MICU1 and its inhibitory paralog MICU2 in a heterodimer. Absence of this disulfide bond results in increased receptor-induced mitochondrial Ca2+ uptake. In the presence of the disulfide bond, MICU1-MICU2 heterodimer binding to MCU is controlled by Ca2+ levels: the dimer assocs. with MCU at low levels of Ca2+ and dissocs. upon high Ca2+ concns. Our findings support a model in which mitochondrial Ca2+ uptake is regulated by a Ca2+-dependent remodeling of the uniporter complex.
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  26. 26
    Lee, Y.; Min, C. K.; Kim, T. G.; Song, H. K.; Lim, Y.; Kim, D.; Shin, K.; Kang, M.; Kang, J. Y.; Youn, H.-S.; Lee, J.-G.; An, J. Y.; Park, K. R.; Lim, J. J.; Kim, J. H.; Kim, J. H.; Park, Z. Y.; Kim, Y.-S.; Wang, J.; Kim, D. H.; Eom, S. H. Structure and Function of the N-Terminal Domain of the Human Mitochondrial Calcium Uniporter. EMBO Rep. 2015, 16, 1318– 1333,  DOI: 10.15252/embr.201540436
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    26
    Structure and function of the N-terminal domain of the human mitochondrial calcium uniporter
    Lee, Youngjin; Min, Choon Kee; Kim, Tae Gyun; Song, Hong Ki; Lim, Yunki; Kim, Dongwook; Shin, Kahee; Kang, Moonkyung; Kang, Jung Youn; Youn, Hyung-Seop; Lee, Jung-Gyu; An, Jun Yop; Park, Kyoung Ryoung; Lim, Jia Jia; Kim, Ji Hun; Kim, Ji Hye; Park, Zee Yong; Kim, Yeon-Soo; Wang, Jimin; Kim, Do Han; Eom, Soo Hyun
    EMBO Reports (2015), 16 (10), 1318-1333CODEN: ERMEAX; ISSN:1469-221X. (Wiley-VCH Verlag GmbH & Co. KGaA)
    The mitochondrial calcium uniporter (MCU) is responsible for mitochondrial calcium uptake and homeostasis. It is also a target for the regulation of cellular anti-/pro-apoptosis and necrosis by several oncogenes and tumor suppressors. Herein, we report the crystal structure of the MCU N-terminal domain (NTD) at a resoln. of 1.50 Å in a novel fold and the S92A MCU mutant at 2.75 Å resoln.; the residue S92 is a predicted CaMKII phosphorylation site. The assembly of the mitochondrial calcium uniporter complex (uniplex) and the interaction with the MCU regulators such as the mitochondrial calcium uptake-1 and mitochondrial calcium uptake-2 proteins (MICU1 and MICU2) are not affected by the deletion of MCU NTD. However, the expression of the S92A mutant or a NTD deletion mutant failed to restore mitochondrial Ca2+ uptake in a stable MCU knockdown HeLa cell line and exerted dominant-neg. effects in the wild-type MCU-expressing cell line. These results suggest that the NTD of MCU is essential for the modulation of MCU function, although it does not affect the uniplex formation.
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  27. 27
    Lee, S. K.; Shanmughapriya, S.; Mok, M. C. Y.; Dong, Z.; Tomar, D.; Carvalho, E.; Rajan, S.; Junop, M. S.; Madesh, M.; Stathopulos, P. B. Structural Insights into Mitochondrial Calcium Uniporter Regulation by Divalent Cations. Cell Chem. Biol. 2016, 23, 1157– 1169,  DOI: 10.1016/j.chembiol.2016.07.012
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    27
    Structural Insights into Mitochondrial Calcium Uniporter Regulation by Divalent Cations
    Lee, Samuel K.; Shanmughapriya, Santhanam; Mok, Mac C. Y.; Dong, Zhiwei; Tomar, Dhanendra; Carvalho, Edmund; Rajan, Sudarsan; Junop, Murray S.; Madesh, Muniswamy; Stathopulos, Peter B.
    Cell Chemical Biology (2016), 23 (9), 1157-1169CODEN: CCBEBM; ISSN:2451-9448. (Cell Press)
    Calcium (Ca2+) flux into the matrix is tightly controlled by the mitochondrial Ca2+ uniporter (MCU) due to vital roles in cell death and bioenergetics. However, the precise at. mechanisms of MCU regulation remain unclear. Here, we solved the crystal structure of the N-terminal matrix domain of human MCU, revealing a β-grasp-like fold with a cluster of neg. charged residues that interacts with divalent cations. Binding of Ca2+ or Mg2+ destabilizes and shifts the self-assocn. equil. of the domain toward monomer. Mutational disruption of the acidic face weakens oligomerization of the isolated matrix domain and full-length human protein similar to cation binding and markedly decreases MCU activity. Moreover, mitochondrial Mg2+ loading or blockade of mitochondrial Ca2+ extrusion suppresses MCU Ca2+-uptake rates. Collectively, our data reveal that the β-grasp-like matrix region harbors an MCU-regulating acidic patch that inhibits human MCU activity in response to Mg2+ and Ca2+ binding.
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  28. 28
    Tomar, D.; Dong, Z.; Shanmughapriya, S.; Koch, D. A.; Thomas, T.; Hoffman, N. E.; Timbalia, S. A.; Goldman, S. J.; Breves, S. L.; Corbally, D. P.; Nemani, N.; Fairweather, J. P.; Cutri, A. R.; Zhang, X.; Song, J.; Jaña, F.; Huang, J.; Barrero, C.; Rabinowitz, J. E.; Luongo, T. S.; Schumacher, S. M.; Rockman, M. E.; Dietrich, A.; Merali, S.; Caplan, J.; Stathopulos, P. B.; Ahima, R. S.; Cheung, J. Y.; Houser, S. R.; Koch, W. J.; Patel, V.; Gohil, V. M.; Elrod, J. W.; Rajan, S.; Madesh, M. MCUR1 is a Scaffold Factor for the MCU Complex Function and Promotes Mitochondrial Bioenergetics. Cell Rep. 2016, 15, 1673– 1685,  DOI: 10.1016/j.celrep.2016.04.050
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    MCUR1 Is a Scaffold Factor for the MCU Complex Function and Promotes Mitochondrial Bioenergetics
    Tomar, Dhanendra; Dong, Zhiwei; Shanmughapriya, Santhanam; Koch, Diana A.; Thomas, Toby; Hoffman, Nicholas E.; Timbalia, Shrishiv A.; Goldman, Samuel J.; Breves, Sarah L.; Corbally, Daniel P.; Nemani, Neeharika; Fairweather, Joseph P.; Cutri, Allison R.; Zhang, Xueqian; Song, Jianliang; Jana, Fabian; Huang, Jianhe; Barrero, Carlos; Rabinowitz, Joseph E.; Luongo, Timothy S.; Schumacher, Sarah M.; Rockman, Michael E.; Dietrich, Alexander; Merali, Salim; Caplan, Jeffrey; Stathopulos, Peter; Ahima, Rexford S.; Cheung, Joseph Y.; Houser, Steven R.; Koch, Walter J.; Patel, Vickas; Gohil, Vishal M.; Elrod, John W.; Rajan, Sudarsan; Madesh, Muniswamy
    Cell Reports (2016), 15 (8), 1673-1685CODEN: CREED8; ISSN:2211-1247. (Cell Press)
    Mitochondrial Ca2+ Uniporter (MCU)-dependent mitochondrial Ca2+ uptake is the primary mechanism for increasing matrix Ca2+ in most cell types. However, a limited understanding of the MCU complex assembly impedes the comprehension of the precise mechanisms underlying MCU activity. Here, we report that mouse cardiomyocytes and endothelial cells lacking MCU regulator 1 (MCUR1) have severely impaired [Ca2+]m uptake and IMCU current. MCUR1 binds to MCU and EMRE and function as a scaffold factor. Our protein binding analyses identified the minimal, highly conserved regions of coiled-coil domain of both MCU and MCUR1 that are necessary for heterooligomeric complex formation. Loss of MCUR1 perturbed MCU heterooligomeric complex and functions as a scaffold factor for the assembly of MCU complex. Vascular endothelial deletion of MCU and MCUR1 impaired mitochondrial bioenergetics, cell proliferation, and migration but elicited autophagy. These studies establish the existence of a MCU complex that assembles at the mitochondrial integral membrane and regulates Ca2+-dependent mitochondrial metab.
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    Gunter, T. E.; Gunter, K. K.; Sheu, S.-S.; Gavin, C. E. Mitochondrial Calcium Transport: Physiological and Pathological Relevance. Am. J. Physiol. 1994, 267, C313– 319,  DOI: 10.1152/ajpcell.1994.267.2.C313
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    Hajnóczky, G.; Robb-Gaspers, L. D.; Seitz, M. B.; Thomas, A. P. Decoding of Cytosolic Calcium Oscillations in the Mitochondria. Cell 1995, 82, 415– 424,  DOI: 10.1016/0092-8674(95)90430-1
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    Denton, R.; McCormack, J. G. Ca2+ as a Second Messenger Within Mitochondria Of The Heart And Other Tissues. Annu. Rev. Physiol. 1990, 52, 451– 466,  DOI: 10.1146/annurev.ph.52.030190.002315
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    Santo-Domingo, J.; Demaurex, N. Calcium Uptake Mechanisms of Mitochondria. Biochim. Biophys. Acta, Bioenerg. 2010, 1797, 907– 912,  DOI: 10.1016/j.bbabio.2010.01.005
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    Calcium uptake mechanisms of mitochondria
    Santo-Domingo, Jaime; Demaurex, Nicolas
    Biochimica et Biophysica Acta, Bioenergetics (2010), 1797 (6-7), 907-912CODEN: BBBEB4; ISSN:0005-2728. (Elsevier B. V.)
    A review. The ability of mitochondria to capture Ca2+ ions has important functional implications for cells, because mitochondria shape cellular Ca2+ signals by acting as a Ca2+ buffer and respond to Ca2+ elevations either by increasing the cell energy supply or by triggering the cell death program of apoptosis. A mitochondrial Ca2+ channel known as the uniporter drives the rapid and massive entry of Ca2+ ions into mitochondria. The uniporter operates at high, micromolar cytosolic Ca2+ concns. that are only reached transiently in cells, near Ca2+ release channels. Mitochondria can also take up Ca2+ at low, nanomolar concns., but this high affinity mode of Ca2+ uptake is not well characterized. Recently, the leucine zipper-EF hand-contg. transmembrane region (Letm1) was proposed to be an electrogenic 1:1 mitochondrial Ca2+/H+ antiporter that drives the uptake of Ca2+ into mitochondria at nanomolar cytosolic Ca2+ concns. Here, the authors review the properties of the Ca2+ import systems of mitochondria and discuss how Ca2+ uptake via an electrogenic 1:1 Ca2+/H+ antiport challenges the current thinking of the mitochondrial Ca2+ uptake mechanism.
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    Bernardi, P. Mitochondrial Transport of Cations: Channels, Exchangers, and Permeability Transition. Physiol. Rev. 1999, 79, 1127– 1155,  DOI: 10.1152/physrev.1999.79.4.1127
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    Mitochondrial transport of cations: channels, exchangers, and permeability transition
    Bernardi, Paolo
    Physiological Reviews (1999), 79 (4), 1127-1155CODEN: PHREA7; ISSN:0031-9333. (American Physiological Society)
    A review with 372 refs. This review provides a selective history of how studies of mitochondrial cation transport (K+, Na+, Ca2+) developed in relation to the major themes of research in bioenergetics. It then covers in some detail specific transport pathways for these cations, and it introduces and discusses open problems about their nature and physiol. function, particularly in relation to vol. regulation and Ca2+ homeostasis. The review should provide the basic elements needed to understand both earlier mitochondrial literature and current problems assocd. with mitochondrial transport of cations and hopefully will foster new interest in the mol. definition of mitochondrial cation channels and exchangers as well as their roles in cell physiol.
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  34. 34
    Shanmughapriya, S.; Rajan, S.; Hoffman, N. E.; Higgins, A. M.; Tomar, D.; Nemani, N.; Hines, K. J.; Smith, D. J.; Eguchi, A.; Vallem, S.; Shaikh, F.; Cheung, M.; Leonard, N. J.; Stolakis, R. S.; Wolfers, M. P.; Ibetti, J.; Chuprun, J. K.; Jog, N. R.; Houser, S. R.; Koch, W. J.; Elrod, J. W.; Madesh, M. SPG7 is an Essential and Conserved Component of the Mitochondrial Permeability Transition Pore. Mol. Cell 2015, 60, 47– 62,  DOI: 10.1016/j.molcel.2015.08.009
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    Orrenius, S.; Zhivotovsky, B.; Nicotera, P. Regulation of Cell Death: The Calcium-Apoptosis Link. Nat. Rev. Mol. Cell Biol. 2003, 4, 552– 565,  DOI: 10.1038/nrm1150
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    Calcium: Regulation of cell death: the calcium-apoptosis link
    Orrenius, Sten; Zhivotovsky, Boris; Nicotera, Pierluigi
    Nature Reviews Molecular Cell Biology (2003), 4 (7), 552-565CODEN: NRMCBP; ISSN:1471-0072. (Nature Publishing Group)
    A review and discussion. To live or to die. This crucial question eloquently reflects the dual role of Ca2+ in living organisms, as a survival factor or as a ruthless killer. It has long been known that Ca2+ signals govern a host of vital cell functions and so are necessary for cell survival. However, more recently it has become clear that cellular Ca2+ overload, or perturbation of intracellular Ca2+ compartmentalization, can cause cytotoxicity and trigger either apoptotic or necrotic cell death.
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    De Stefani, D.; Rizzuto, R.; Pozzan, T. Enjoy the Trip: Calcium in Mitochondria Back and Forth. Annu. Rev. Biochem. 2016, 85, 161– 192,  DOI: 10.1146/annurev-biochem-060614-034216
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    Enjoy the Trip: Calcium in Mitochondria Back and Forth
    De Stefani, Diego; Rizzuto, Rosario; Pozzan, Tullio
    Annual Review of Biochemistry (2016), 85 (), 161-192CODEN: ARBOAW; ISSN:0066-4154. (Annual Reviews)
    A review. In the last 5 years, most of the mols. that control mitochondrial Ca2+ homeostasis have been finally identified. Mitochondrial Ca2+ uptake is mediated by the Mitochondrial Calcium Uniporter (MCU) complex, a macromol. structure that guarantees Ca2+ accumulation inside mitochondrial matrix upon increases in cytosolic Ca2+. Conversely, Ca2+ release is under the control of the Na+/Ca2+ exchanger, encoded by the NCLX gene, and of a H+/Ca2+ antiporter, whose identity is still debated. The low affinity of the MCU complex, coupled to the activity of the efflux systems, protects cells from continuous futile cycles of Ca2+ across the inner mitochondrial membrane and consequent massive energy dissipation. In this review, we discuss the basic principles that govern mitochondrial Ca2+ homeostasis and the methods used to investigate the dynamics of Ca2+ concn. within the organelles. We discuss the functional and structural role of the different mols. involved in mitochondrial Ca2+ handling and their pathophysiol. role.
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    Marchi, S.; Pinton, P. The Mitochondrial Calcium Uniporter Complex: Molecular Components, Structure and Physiopathological Implications. J. Physiol. 2014, 592, 829– 839,  DOI: 10.1113/jphysiol.2013.268235
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    The mitochondrial calcium uniporter complex: molecular components, structure and physiopathological implications
    Marchi, Saverio; Pinton, Paolo
    Journal of Physiology (Oxford, United Kingdom) (2014), 592 (5), 829-839CODEN: JPHYA7; ISSN:0022-3751. (Wiley-Blackwell)
    A review. Although it has long been known that mitochondria take up Ca2+, the mol. identities of the channels and transporters involved in this process were revealed only recently. Here, we discuss the recent work that has led to the characterization of the mitochondrial calcium uniporter complex, which includes the channel-forming subunit MCU (mitochondrial calcium uniporter) and its regulators MICU1, MICU2, MCUb, EMRE, MCUR1 and miR-25. We review not only the biochem. identities and structures of the proteins required for mitochondrial Ca2+ uptake but also their implications in different physiopathol. contexts.
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  38. 38
    Luongo, T. S.; Lambert, J. P.; Yuan, A.; Zhang, X.; Gross, P.; Song, J.; Shanmughapriya, S.; Gao, E.; Jain, M.; Houser, S. R.; Koch, W. J.; Cheung, J. Y.; Madesh, M.; Elrod, J. W. The Mitochondrial Calcium Uniporter Matches Energetic Supply with Cardiac Workload during Stress and Modulates Permeability Transition. Cell Rep. 2015, 12, 23– 34,  DOI: 10.1016/j.celrep.2015.06.017
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    38
    The Mitochondrial Calcium Uniporter Matches Energetic Supply with Cardiac Workload during Stress and Modulates Permeability Transition
    Luongo, Timothy S.; Lambert, Jonathan P.; Yuan, Ancai; Zhang, Xueqian; Gross, Polina; Song, Jianliang; Shanmughapriya, Santhanam; Gao, Erhe; Jain, Mohit; Houser, Steven R.; Koch, Walter J.; Cheung, Joseph Y.; Madesh, Muniswamy; Elrod, John W.
    Cell Reports (2015), 12 (1), 23-34CODEN: CREED8; ISSN:2211-1247. (Cell Press)
    Cardiac contractility is mediated by a variable flux in intracellular calcium (Ca2+), thought to be integrated into mitochondria via the mitochondrial calcium uniporter (MCU) channel to match energetic demand. Here, we examine a conditional, cardiomyocyte-specific, mutant mouse lacking Mcu, the pore-forming subunit of the MCU channel, in adulthood. Mcu-/- mice display no overt baseline phenotype and are protected against mCa2+ overload in an in vivo myocardial ischemia-reperfusion injury model by preventing the activation of the mitochondrial permeability transition pore, decreasing infarct size, and preserving cardiac function. In addn., we find that Mcu-/- mice lack contractile responsiveness to acute β-adrenergic receptor stimulation and in parallel are unable to activate mitochondrial dehydrogenases and display reduced bioenergetic reserve capacity. These results support the hypothesis that MCU may be dispensable for homeostatic cardiac function but required to modulate Ca2+-dependent metab. during acute stress.
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  39. 39
    Kwong, J. Q.; Lu, X.; Correll, R. N.; Schwanekamp, J. A.; Vagnozzi, R. J.; Sargent, M. A.; York, A. J.; Zhang, J.; Bers, D. M.; Molkentin, J. D. The Mitochondrial Calcium Uniporter Selectively Matches Metabolic Output to Acute Contractile Stress in the Heart. Cell Rep. 2015, 12, 15– 22,  DOI: 10.1016/j.celrep.2015.06.002
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    The Mitochondrial Calcium Uniporter Selectively Matches Metabolic Output to Acute Contractile Stress in the Heart
    Kwong, Jennifer Q.; Lu, Xiyuan; Correll, Robert N.; Schwanekamp, Jennifer A.; Vagnozzi, Ronald J.; Sargent, Michelle A.; York, Allen J.; Zhang, Jianyi; Bers, Donald M.; Molkentin, Jeffery D.
    Cell Reports (2015), 12 (1), 15-22CODEN: CREED8; ISSN:2211-1247. (Cell Press)
    In the heart, augmented Ca2+ fluxing drives contractility and ATP generation through mitochondrial Ca2+ loading. Pathol. mitochondrial Ca2+ overload with ischemic injury triggers mitochondrial permeability transition pore (MPTP) opening and cardiomyocyte death. Mitochondrial Ca2+ uptake is primarily mediated by the mitochondrial Ca2+ uniporter (MCU). Here, we generated mice with adult and cardiomyocyte-specific deletion of Mcu, which produced mitochondria refractory to acute Ca2+ uptake, with impaired ATP prodn., and inhibited MPTP opening upon acute Ca2+ challenge. Mice lacking Mcu in the adult heart were also protected from acute ischemia-reperfusion injury. However, resting/basal mitochondrial Ca2+ levels were normal in hearts of Mcu-deleted mice, and mitochondria lacking MCU eventually loaded with Ca2+ after stress stimulation. Indeed, Mcu-deleted mice were unable to immediately sprint on a treadmill unless warmed up for 30 min. Hence, MCU is a dedicated regulator of short-term mitochondrial Ca2+ loading underlying a "fight-or-flight" response that acutely matches cardiac workload with ATP prodn.
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    Medvedeva, Y. V.; Weiss, J. H.; Weiss, J. H.; Medvedeva, Y. V. Intramitochondrial Zn2+ Accumulation via the Ca2+ Uniporter Contributes to Acute Ischemic Neurodegeneration. Neurobiol. Dis. 2014, 68, 137– 144,  DOI: 10.1016/j.nbd.2014.04.011
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    40
    Intramitochondrial Zn2 + accumulation via the Ca2 + uniporter contributes to acute ischemic neurodegeneration
    Medvedeva, Yuliya V.; Weiss, John H.
    Neurobiology of Disease (2014), 68 (), 137-144CODEN: NUDIEM; ISSN:0969-9961. (Elsevier Inc.)
    Ca2 + and Zn2 + have both been implicated in the induction of acute ischemic neurodegeneration. We recently examd. changes in intracellular Zn2 + and Ca2 + in CA1 pyramidal neurons subjected to oxygen glucose deprivation (OGD), and found that Zn2 + rises precede and contribute to the onset of terminal Ca2 + rises ("Ca2 + deregulation"), which are causatively linked to a lethal loss of membrane integrity. The present study seeks to examine the specific role of intramitochondrial Zn2 + accumulation in ischemic injury, using blockers of the mitochondrial Ca2 + uniporter (MCU), through which both Zn2 + and Ca2 + appear able to enter the mitochondrial matrix. In physiol. extracellular Ca2 +, treatment with the MCU blocker, Ruthenium Red (RR), accelerated the Ca2 + deregulation, most likely by disrupting mitochondrial Ca2 + buffering and thus accelerating the lethal cytosolic Ca2 + overload. However, when intracellular Ca2 + overload was slowed, either by adding blockers of major Ca2 + entry channels or by lowering the concn. of Ca2 + in the extracellular buffer, Ca2 + deregulation was delayed, and under these conditions either Zn2 + chelation or MCU blockade resulted in similar further delays of the Ca2 + deregulation. In parallel studies using the reactive oxygen species (ROS) indicator, hydroethidine, lowering Ca2 + surprisingly accelerated OGD induced ROS generation, and in these low Ca2 + conditions, either Zn2 + chelation or MCU block slowed the ROS generation. These studies suggest that, during acute ischemia, Zn2 + entry into mitochondria via the MCU induces mitochondrial dysfunction (including ROS generation) that occurs upstream of, and contributes to the terminal Ca2 + deregulation.
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    Giorgi, C.; Agnoletto, C.; Bononi, A.; Bonora, M.; De Marchi, E.; Marchi, S.; Missiroli, S.; Patergnani, S.; Poletti, F.; Rimessi, A.; Suski, J. M.; Wieckowski, M. R.; Pinton, P. Mitochondrial Calcium Homeostasis as Potential Target for Mitochondrial Medicine. Mitochondrion 2012, 12, 77– 85,  DOI: 10.1016/j.mito.2011.07.004
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    Mitochondrial calcium homeostasis as potential target for mitochondrial medicine
    Giorgi, Carlotta; Agnoletto, Chiara; Bononi, Angela; Bonora, Massimo; De Marchi, Elena; Marchi, Saverio; Missiroli, Sonia; Patergnani, Simone; Poletti, Federica; Rimessi, Alessandro; Suski, Jan M.; Wieckowski, Mariusz R.; Pinton, Paolo
    Mitochondrion (2012), 12 (1), 77-85CODEN: MITOCN; ISSN:1567-7249. (Elsevier B.V.)
    A review. Mitochondria are crucial in different intracellular pathways of signal transduction. Mitochondria are capable of decoding a variety of extracellular stimuli into markedly different intracellular actions, ranging from energy prodn. to cell death. The fine modulation of mitochondrial calcium (Ca2+) homeostasis plays a fundamental role in many of the processes involving this organelle. When mitochondrial Ca2+ homeostasis is compromised, different pathol. conditions can occur, depending on the cell type involved. Recent data have shed light on the mol. identity of the main proteins involved in the handling of mitochondrial Ca2+ traffic, opening fascinating and ambitious new avenues for mitochondria-based pharmacol. strategies.
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  42. 42
    Arduino, D. M.; Wettmarshausen, J.; Vais, H.; Navas-Navarro, P.; Cheng, Y.; Leimpek, A.; Ma, Z.; Delrio-Lorenzo, A.; Giordano, A.; Garcia-Perez, C.; Médard, G.; Kuster, B.; García-Sancho, J.; Mokranjac, D.; Foskett, J. K.; Alonso, M. T.; Perocchi, F. Systematic Identification of MCU Modulators by Orthogonal Interspecies Chemical Screening. Mol. Cell 2017, 67, 711– 723,  DOI: 10.1016/j.molcel.2017.07.019
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    Systematic Identification of MCU Modulators by Orthogonal Interspecies Chemical Screening
    Arduino, Daniela M.; Wettmarshausen, Jennifer; Vais, Horia; Navas-Navarro, Paloma; Cheng, Yiming; Leimpek, Anja; Ma, Zhongming; Delrio-Lorenzo, Alba; Giordano, Andrea; Garcia-Perez, Cecilia; Medard, Guillaume; Kuster, Bernhard; Garcia-Sancho, Javier; Mokranjac, Dejana; Foskett, J. Kevin; Alonso, M. Teresa; Perocchi, Fabiana
    Molecular Cell (2017), 67 (4), 711-723.e7CODEN: MOCEFL; ISSN:1097-2765. (Elsevier Inc.)
    The mitochondrial calcium uniporter complex is essential for calcium (Ca2+) uptake into mitochondria of all mammalian tissues, where it regulates bioenergetics, cell death, and Ca2+ signal transduction. Despite its involvement in several human diseases, we currently lack pharmacol. agents for targeting uniporter activity. Here we introduce a high-throughput assay that selects for human MCU-specific small-mol. modulators in primary drug screens. Using isolated yeast mitochondria, reconstituted with human MCU, its essential regulator EMRE, and aequorin, and exploiting a D-lactate- and mannitol/sucrose-based bioenergetic shunt that greatly minimizes false-pos. hits, we identify mitoxantrone out of more than 600 clin. approved drugs as a direct selective inhibitor of human MCU. We validate mitoxantrone in orthogonal mammalian cell-based assays, demonstrating that our screening approach is an effective and robust tool for MCU-specific drug discovery and, more generally, for the identification of compds. that target mitochondrial functions.
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    Kon, N.; Murakoshi, M.; Isobe, A.; Kagechika, K.; Miyoshi, N.; Nagayama, T. DS16570511 is a Small-Molecule Inhibitor of the Mitochondrial Calcium Uniporter. Cell Death Discov. 2017, 3, 17045,  DOI: 10.1038/cddiscovery.2017.45
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    DS16570511 is a small-molecule inhibitor of the mitochondrial calcium uniporter
    Kon, Naohiro; Murakoshi, Michiko; Isobe, Aya; Kagechika, Katsuji; Miyoshi, Naoki; Nagayama, Takahiro
    Cell Death Discovery (2017), 3 (), 17045CODEN: CDDEB5; ISSN:2058-7716. (Nature Publishing Group)
    In cardiac myocytes, regulation of mitochondrial Ca2+ is important for cellular signaling and cardiac contraction. Ca2+ entry into the mitochondria is mediated by a highly selective Ca2+ channel called the mitochondrial calcium uniporter, which consists of a pore-forming subunit MCU and regulatory subunits such as MICU1. Although pharmacol. regulation of the mitochondrial Ca2+ influx is a promising approach to controlling the cellular functions, a cell-permeable and specific inhibitor of the mitochondrial calcium uniporter has not yet been developed. Here, we identify a novel cell-permeable inhibitor of the uniporter by a high-throughput screening of 120 000 small-mol. compds. In our study, DS16570511 dose-dependently inhibited serum-induced mitochondrial Ca2+ influx in HEK293A cells with an IC50 of 7 μM. DS16570511 inhibited Ca2+ uptake of isolated mitochondria from human cells, rat heart and pig heart. Overexpression of hMCU or hMICU1 in HEK293A cells increased mitochondrial Ca2+ influx, and the increases were completely suppressed by the pretreatment with DS16570511. DS16570511 also blocks mitochondrial Ca2+ overload in a Langendorff perfused beating rat heart. Interestingly, DS16570511 increased cardiac contractility without affecting heart rate in the perfused heart. These results show that DS16570511 is a novel cell-permeable inhibitor of the mitochondrial calcium uniporter and applicable for control of the cardiac functions.
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  44. 44
    Thu, V. T.; Kim, H. K.; Long, L. T.; Lee, S. R.; Hanh, T. M.; Ko, T. H.; Heo, H. J.; Kim, N.; Kim, S. H.; Ko, K. S.; Rhee, B. D.; Han, J. NecroX-5 Prevents Hypoxia/Reoxygenation Injury by Inhibiting the Mitochondrial Calcium Uniporter. Cardiovasc. Res. 2012, 94, 342– 350,  DOI: 10.1093/cvr/cvs122
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    44
    NecroX-5 prevents hypoxia/reoxygenation injury by inhibiting the mitochondrial calcium uniporter
    Thu, Vu Thi; Kim, Hyoung-Kyu; Long, Le Thanh; Lee, Sung-Ryul; Hanh, Tran My; Ko, Tae Hee; Heo, Hye-Jin; Kim, Nari; Kim, Soon Ha; Ko, Kyung Soo; Rhee, Byoung Doo; Han, Jin
    Cardiovascular Research (2012), 94 (2), 342-350CODEN: CVREAU; ISSN:0008-6363. (Oxford University Press)
    Aims: Preservation of mitochondrial function is essential to limit myocardial damage in ischemic heart disease. We examd. the protective effects and mechanism of a new compd., NecroX-5, on rat heart mitochondria in a hypoxia/reoxygenation (HR) model. Methods and results: NecroX-5 reduced mitochondrial oxidative stress, prevented the collapse in mitochondrial membrane potential, improved mitochondrial oxygen consumption, and suppressed mitochondrial Ca2+ overload during reoxygenation in an in vitro rat heart HR model. Furthermore, NecroX-5 reduced the ouabain- or histamine-induced increase in mitochondrial Ca2+. Conclusion: These findings suggest that NecroX-5 may act as a mitochondrial Ca2+ uniporter inhibitor to protect cardiac mitochondria against HR damage.
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  45. 45
    Antonenko, Y. N.; Rokitskaya, T. I.; Cooper, A. J. L.; Krasnikov, B. F. Minocycline Chelates Ca2+, Binds to Membranes, and Depolarizes Mitochondria by Formation of Ca2+-Dependent Ion Channels. J. Bioenerg. Biomembr. 2010, 42, 151– 163,  DOI: 10.1007/s10863-010-9271-1
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    Santo-Domingo, J.; Vay, L.; Hernández-Sanmiguel, E.; Lobatón, C. D.; Moreno, A.; Montero, M.; Alvarez, J. The Plasma Membrane Na+/Ca2+ Exchange Inhibitor KB-R7943 is Also a Potent Inhibitor of the Mitochondrial Ca2+ Uniporter. Br. J. Pharmacol. 2007, 151, 647– 654,  DOI: 10.1038/sj.bjp.0707260
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    46
    The plasma membrane Na+/Ca2+ exchange inhibitor KB-R7943 is also a potent inhibitor of the mitochondrial Ca2+ uniporter
    Santo-Domingo, J.; Vay, L.; Hernandez-SanMiguel, E.; Lobaton, C. D.; Moreno, A.; Montero, M.; Alvarez, J.
    British Journal of Pharmacology (2007), 151 (5), 647-654CODEN: BJPCBM; ISSN:0007-1188. (Nature Publishing Group)
    Background and purpose: The thiourea deriv. KB-R7943, originally developed as inhibitor of the plasma membrane Na+/Ca2+ exchanger, has been shown to protect against myocardial ischemia-reperfusion injury. We have studied here its effects on mitochondrial Ca2+ fluxes. [Ca2+] in cytosol, mitochondria and endoplasmic reticulum (ER), and mitochondrial membrane potential were monitored using both luminescent (targeted aequorins) and fluorescent (fura-2, tetramethylrhodamine Et ester) probes in HeLa cells. KB-R7943 was also a potent inhibitor of the mitochondrial Ca2+ uniporter (MCU). In permeabilized HeLa cells, KB-R7943 inhibited mitochondrial Ca2+ uptake with a Ki of 5.5±1.3 μM (mean±S.D.). In intact cells, 10μM KB-R7943 reduced by 80% the mitochondrial [Ca2+] peak induced by histamine. KB-R7943 did not modify the mitochondrial membrane potential and had no effect on the mitochondrial Na+/Ca2+ exchanger. KB-R7943 inhibited histamine-induced ER-Ca2+ release in intact cells, but not in cells loaded with a Ca2+-chelator to damp cytosolic [Ca2+] changes. Therefore, inhibition of ER-Ca2+-release by KB-R7943 was probably due to the increased feedback Ca2+-inhibition of inositol 1,4,5-trisphosphate receptors after MCU block. This mechanism also explains why KB-R7943 reversibly blocked histamine-induced cytosolic [Ca2+] oscillations in the same range of concns. required to inhibit MCU. Inhibition of MCU by KB-R7943 may contribute to its cardioprotective activity by preventing mitochondrial Ca2+-overload during ischemia-reperfusion. In addn., the effects of KB-R7943 on Ca2+ homeostasis provide new evidence for the role of mitochondria modulating Ca2+-release and regenerative Ca2+-oscillations. Search for permeable and selective MCU inhibitors may yield useful pharmacol. tools in the future.
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    Kapuscinski, J.; Darzynkiewicz, Z. Interactions of Antitumor Agents Ametantrone and Mitoxantrone (Novatrone) with Double-Stranded DNA. Biochem. Pharmacol. 1985, 34, 4203– 4213,  DOI: 10.1016/0006-2952(85)90275-8
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    47
    Interactions of antitumor agents Ametantrone and mitoxantrone (Novatrone) with double-stranded DNA
    Kapuscinski, Jan; Darzynkiewicz, Zbigniew
    Biochemical Pharmacology (1985), 34 (24), 4203-13CODEN: BCPCA6; ISSN:0006-2952.
    Interactions of mitoxantrone and ametantrone with natural and synthetic nucleic acids in aq. medium [0.15 NaCl, 5 mM 4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid (Hepes), pH 7.0. 25%] were studied using computer-aided spectrophotometric techniques. Absorption spectra of the drugs in monomeric and dimeric form and their complexes with DNAs at low drug/phosphate ratios (D/P) were established. The latter were red-shifted and had lower amplitude as compared with the spectra of the free ligand's monomer; the change is consistent with the already well-established intercalative mode of drug-nucleic acid interaction. Drug-DNA equil. were studied using the McGhee-von Hippel model of noncooperative ligand-polymer interaction, with the correction for dimerization of drugs. Although mitoxantrone was 2 orders of magnitude more potent an antitumor drug than Ametantrone, the intrinsic assocn. consts. (Ki) of both drugs were of similar magnitude. Also, no significant DNA-base specificity for either of the drugs (measured as Ki value for various homopolymers) was obsd. Therefore, no correlation was apparent between the intercalative mode of binding to DNA, regardless of base compn., and the pharmacol. activity of these drugs. At higher D/P ratios, a secondary mode of binding was detected by both spectroscopy and light-scattering measurement. Homopolymer-pairs and polymers contg. only dI and dC were esp. susceptible to this secondary type of binding. The possibility that this secondary type of binding may be responsible for the antitumor properties of the drugs is considered.
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  48. 48
    Mazerski, J.; Martelli, S.; Borowski, E. The Geometry of Intercalation Complex of Antitumor Mitoxantrone and Ametantrone with DNA: Molecular Dynamics Simulations. Acta Biochim. Polym. 1998, 45, 1– 11
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    The geometry of intercalation complex of antitumor mitoxantrone and ametantrone with DNA: molecular dynamics simulations
    Mazerski, Jan; Martelli, Sante; Borowski, Edward
    Acta Biochimica Polonica (1998), 45 (1), 1-11CODEN: ABPLAF; ISSN:0001-527X. (Polish Biochemical Society)
    Intercalative binding of the antitumor drugs ametantrone and mitoxantrone to the dodecamer duplex d(CGCGAGCTCGCG)2 was studied by applying mol. dynamics in water with the GROMOS 87 force field. A no. of reasonable binding orientations were tested by short pre-simulations. It was shown that in energetically favorable orientation the anthraquinone chromophore is perpendicular to the direction of inter-base hydrogen bonds. Helically shaped side-chains of the drugs fit to the minor groove. The best orientation obtained in pre-simulations was applied in the main simulations. Small but significant differences were found between structures of inter-calation complexes of the two drugs with the dodecamer duplex, the mitoxantrone complex possessing more favorable energy. The mol. nature of interactions responsible for those differences has been discussed.
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    Boland, M. P.; Fitzgerald, K. A.; O’Neill, L. A. J. Topoisomerase II Is Required for Mitoxantrone to Signal Nuclear Factor ΚB Activation in HL60 Cells. J. Biol. Chem. 2000, 275, 25231– 25238,  DOI: 10.1074/jbc.275.33.25231
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    Smith, P. J.; Morgan, S. A.; Fox, M. E.; Watson, J. V. Mitoxantrone-DNA Binding and the Induction of Topoisomerase II Associated DNA Damage in Multi-Drug Resistant Small Cell Lung Cancer Cells. Biochem. Pharmacol. 1990, 40, 2069– 2078,  DOI: 10.1016/0006-2952(90)90237-F
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    Nägele, H.; Castel, M. A.; Deutsch, O.; Wagner, F. M.; Reichenspurner, H. Heart Transplantation in a Patient with Multiple Sclerosis and Mitoxantrone-Induced Cardiomyopathy. J. Heart Lung Transplant. 2004, 23, 641– 643,  DOI: 10.1016/S1053-2498(03)00307-3
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    Heart transplantation in a patient with multiple sclerosis and mitoxantrone-induced cardiomyopathy
    Nagele H; Castel M A; Deutsch O; Wagner F M; Reichenspurner H
    The Journal of heart and lung transplantation : the official publication of the International Society for Heart Transplantation (2004), 23 (5), 641-3 ISSN:1053-2498.
    We describe a 30-year-old man with end-stage heart failure after therapy with mitoxantrone for multiple sclerosis. A successful orthotopic heart transplantation was performed when intensified medical therapy failed to improve the patient's hemodynamics. In spite of the severe underlying disease he did well on dual immunosuppression with methylprednisone and cyclosporine. Neurologic symptoms remained stable throughout the procedure and, after 2 months, he resumed preoperative ambulatory status. Eight years after the operation, the patient is now in New York Heart Association (NYHA) Class I status. Using canes, he is able to walk short distances. Repeated urinary tract infections caused by Escherichia coli became a problem, but have been controlled by long-term oral antibiotic prophylaxis with trimethoprim.
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    Goebel, M.; Kaplan, E. Anthracycline-Induced Cardiotoxicity – A Review. Oncol. Res. Treat. 2004, 15, 198– 204,  DOI: 10.1159/000217360
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    Ying, W.-L.; Emerson, J.; Clarke, M. J.; Sanadi, D. R. Inhibition of Mitochondrial Calcium Ion Transport by an Oxo-Bridged Dinuclear Ruthenium Ammine Complex. Biochemistry 1991, 30, 4949– 4952,  DOI: 10.1021/bi00234a016
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    Inhibition of mitochondrial calcium ion transport by an oxo-bridged dinuclear ruthenium ammine complex
    Ying, Wen Long; Emerson, Jeffrey; Clarke, Michael J.; Sanadi, D. Rao
    Biochemistry (1991), 30 (20), 4949-52CODEN: BICHAW; ISSN:0006-2960.
    Ruthenium red is a well-known and effective inhibitor of the mitochondria Ca2+ uniporter; however, K.C. Reed and F. Bygrave (1974) tentatively attributed this inhibition to a colorless impurity present in com. samples of ruthenium red (RR). This component has now been isolated and a deriv., (μ-O)[(HCO2)(NH3)4Ru]2Cl3, structurally characterized. The active species in soln. appears to be the sym. oxo-bridged ion, [X(NH3)4Ru-O-Ru(NH3)4X]3+, where X = Cl- or OH-. Its absorption spectrum shows a max. at 360 nm. The dinuclear ruthenium ammine complex inhibits Ca2+-stimulated respiration of rat liver mitochondria with an I50 of 3.5 pmol/mg of protein compared to the value of 60 pmol of RR/mg of protein. The inhibition by the dinuclear compd. is noncompetitive with Ca2+. Respiration-linked swelling of mitochondria induced by Cd2+ also responds similarly to both the dinuclear complex and RR. A close correlation was obsd. between binding to mitochondria as monitored with 103Ru-labeled dinuclear complex and inhibition of Ca2+ transport. A Scatchard plot yielded ests. of max. specific binding and dissocn. const. of 7.5 pmol/mg of protein and 1.3 nM, resp. The inhibitor has the characteristics of a satisfactory affinity ligand for purifn. of the uniporter.
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  54. 54
    Matlib, M. A.; Zhou, Z.; Knight, S.; Ahmed, S.; Choi, K. M.; Krause-Bauer, J.; Phillips, R.; Altschuld, R.; Katsube, Y.; Sperelakis, N.; Bers, D. M. Oxygen-Bridged Dinuclear Ruthenium Amine Complex Specifically Inhibits Ca2+ Uptake into Mitochondria in Vitro and in Situ in Single Cardiac Myocytes. J. Biol. Chem. 1998, 273, 10223– 10231,  DOI: 10.1074/jbc.273.17.10223
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    54
    Oxygen-bridged dinuclear ruthenium amine complex specifically inhibits Ca2+ uptake into mitochondria in vitro and in situ in single cardiac myocytes
    Matlib, Mohammed A.; Zhou, Zhuan; Knight, Selena; Ahmed, Saadia; Choi, Kin M.; Krause-Bauer, Jeanette; Phillips, Ronald; Altschuld, Ruth; Katsube, Yasuhiro; Sperelakis, Nicholas; Bers, Donald M.
    Journal of Biological Chemistry (1998), 273 (17), 10223-10231CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
    Ruthenium red is a well known inhibitor of Ca2+ uptake into mitochondria in vitro. However, its utility as an inhibitor of Ca2+ uptake into mitochondria in vivo or in situ in intact cells is limited because of its inhibitory effects on sarcoplasmic reticulum Ca2+ release channel and other cellular processes. We have synthesized a ruthenium deriv. and found it to be an oxygen-bridged dinuclear ruthenium amine complex. It has the same chem. structure as Ru360 reported previously (Emerson, J., Clarke, M. J., Ying, W-L., and Sanadi, D. R. (1993) J. Am. Chem. Soc. 115, 11799-11805). Ru360 has been shown to be a potent inhibitor of Ca2+-stimulated respiration of liver mitochondria in vitro. However, the specificity of Ru360 on Ca2+ uptake into mitochondria in vitro or in intact cells has not been detd. The present study reports in detail the potency, the effectiveness, and the mechanism of inhibition of mitochondrial Ca2+ uptake by Ru360 and its specificity in vitro in isolated mitochondria and in situ in isolated cardiac myocytes. Ru360 was more potent (IC50 = 0.184 nM) than ruthenium red (IC50 = 6.85 nM) in inhibiting Ca2+ uptake into mitochondria. 103Ru360 was found to bind to isolated mitochondria with high affinity (Kd = 0.34 nM, Bmax = 80 fmol/mg of mitochondrial protein). The IC50 of 103Ru360 for the inhibition of Ca2+ uptake into mitochondria was also 0.2 nM, indicating that satn. of a specific binding site is responsible for the inhibition of Ca2+ uptake. Ru360, as high as 10 μM, produced no effect on sarcoplasmic reticulum Ca2+ uptake or release, sarcolemmal Na+/Ca2+ exchange, actomyosin ATPase activity, L-type Ca2+ channel current, cytosolic Ca2+ transients, or cell shortening. 103Ru360 was taken up by isolated myocytes in a time-dependent biphasic manner. Ru360 (10 μM) applied outside intact voltage-clamped ventricular myocytes prevented Ca2+ uptake into mitochondria in situ where the cells were progressively loaded with Ca2+ via sarcolemmal Na+/Ca2+ exchange by depolarization to +110 mV. We conclude that Ru360 specifically blocks Ca2+ uptake into mitochondria and can be used in intact cells.
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  55. 55
    Emerson, J.; Clarke, M. J.; Ying, W. L.; Sanadi, D. R. The Component of “Ruthenium Red” Responsible for Inhibition of Mitochondrial Calcium Ion Transport. Spectra, Electrochemistry, and Aquation Kinetics. Crystal Structure of μ-O-[(HCO)2(NH3)4Ru]2Cl3. J. Am. Chem. Soc. 1993, 115, 11799– 11805,  DOI: 10.1021/ja00078a019
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    Nathan, S. R.; Pino, N. W.; Arduino, D. M.; Perocchi, F.; MacMillan, S. N.; Wilson, J. J. Synthetic Methods for the Preparation of a Functional Analogue of Ru360, a Potent Inhibitor of Mitochondrial Calcium Uptake. Inorg. Chem. 2017, 56, 3123– 3126,  DOI: 10.1021/acs.inorgchem.6b03108
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    56
    Synthetic Methods for the Preparation of a Functional Analogue of Ru360, a Potent Inhibitor of Mitochondrial Calcium Uptake
    Nathan, Sarah R.; Pino, Nicholas W.; Arduino, Daniela M.; Perocchi, Fabiana; MacMillan, Samantha N.; Wilson, Justin J.
    Inorganic Chemistry (2017), 56 (6), 3123-3126CODEN: INOCAJ; ISSN:0020-1669. (American Chemical Society)
    The mixed-valent oxo-bridged Ru complex [(HCO2)(NH3)4Ru(μ-O)Ru(NH3)4(O2CH)]3+, known as Ru360, is a selective inhibitor of mitochondrial Ca uptake. Although this compd. is useful for studying the role of mitochondrial Ca in biol. processes, its widespread availability is limited because of challenges in purifn. and characterization. Here, the authors describe the authors' studies of three different synthetic methods for the prepn. of a functional analog of this valuable compd. This analog, isolated from the authors' procedures, exhibits potent mitochondrial Ca uptake inhibitory properties in permeabilized HeLa cells and in isolated mitochondria.
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    Nathan, S. R.; Wilson, J. J. Synthesis and Evaluation of a Ruthenium-Based Mitochondrial Calcium Uptake Inhibitor. J. Visualized Exp. 2017, 128, e56527,  DOI: 10.3791/56527
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    Cao, C.; Wang, S.; Cui, T.; Su, X.-C.; Chou, J. J. Ion and Inhibitor Binding of the Double-Ring Ion Selectivity Filter of the Mitochondrial Calcium Uniporter. Proc. Natl. Acad. Sci. U. S. A. 2017, 114, E2846– E2851,  DOI: 10.1073/pnas.1620316114
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    Ion and inhibitor binding of the double-ring ion selectivity filter of the mitochondrial calcium uniporter
    Cao, Chan; Wang, Shuqing; Cui, Tanxing; Su, Xun-Cheng; Chou, James J.
    Proceedings of the National Academy of Sciences of the United States of America (2017), 114 (14), E2846-E2851CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    The mitochondrial Ca2+ uniporter (MCU) is a holocomplex consisting of the Ca2+-conducting channel and several accessory and regulatory components. A previous electrophysiol. study found that the uniporter had high Ca2+ selectivity and conductance and this depended critically on the conserved amino acid sequence motif, DXXE (Asp-X-X-Glu) of MCU. A recent NMR structure of the MCU channel from Caenorhabditis elegans revealed that the DXXE formed 2 parallel carboxylate rings at the channel entrance that seem to serve as the ion selectivity filter, although direct ion interaction of this structural motif has not been addressed. Here, we used a paramagnetic probe, Mn2+, to investigate ion and inhibitor binding of this putative selectivity filter. The paramagnetic NMR data showed that mutants with a single carboxylate ring, NXXE (Asn-X-X-Glu) and DXXQ (Asp-X-X-Gln), each could bind Mn2+ specifically, whereas in the wild type the 2 rings bound Mn2+ cooperatively, resulting in ∼1000-fold higher apparent affinity. Ca2+ could specifically displace bound Mn2+ at the DXXE site in the channel. Furthermore, titrating the sample with the known channel inhibitor, ruthenium 360 (Ru360), could displace Mn2+ binding from the solvent-accessible Asp site but not the inner Glu site. The NMR titrn. data, together with structural anal. of the DXXE motif and mol. dynamics simulations, indicated that the double carboxylate rings at the apex of the MCU pore constitute the ion selectivity filter and that Ru360 directly blocks ion entry into the filter by binding to the outer carboxylate ring.
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    Gushchin, A. L.; Laricheva, Y. A.; Abramov, P. A.; Sokolov, M. N.; Abramov, P. A. Synthesis and Electrochemical Properties of [RuIV2O(PhCN)4Cl6]. Inorg. Chem. Commun. 2018, 95, 1387– 7003,  DOI: 10.1016/j.inoche.2018.07.033
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    Urgiles, J.; Nathan, S. R.; MacMillan, S. N.; Wilson, J. J. Dinuclear Nitrido-Bridged Ruthenium Complexes Bearing Diimine Ligands. Dalton Trans. 2017, 46, 14256– 14263,  DOI: 10.1039/C7DT03085A
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    Dinuclear nitrido-bridged ruthenium complexes bearing diimine ligands
    Urgiles, Julie; Nathan, Sarah R.; MacMillan, Samantha N.; Wilson, Justin J.
    Dalton Transactions (2017), 46 (41), 14256-14263CODEN: DTARAF; ISSN:1477-9226. (Royal Society of Chemistry)
    Reactions of K3[Ru2NCl8(H2O)2] with 2,2'-bipyridine (bpy), 4,4'-dimethyl-2,2'-bipyridine (dmbpy), and 4,4'-dimethoxy-2,2'-bipyridine (dmobpy) yielded the nitrido-bridged dinuclear complexes [Ru2N(L)2Cl5(DMF)] where L = bpy (1), dmbpy (2), and dmobpy (3). The crystal structures of these complexes reveal a linear Ru-N-Ru moiety with each Ru center bearing a bidentate diimine ligand. The complexes were further characterized by NMR, IR, and UV-visible spectroscopic methods and cyclic voltammetry. Because the compds. bear some structural similarities with the mitochondrial Ca uptake inhibitor Ru360, the ability of these complexes to act in this capacity was evaluated. 1-3 All fail to block mitochondrial Ca uptake, revealing new facets of the structure-activity relations for Ru-based mitochondrial Ca uptake inhibitors.
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    Dunitz, J. D.; Orgel, L. E. Application of Molecular-Orbital Theory to Some Binuclear Coordination Compounds. J. Chem. Soc. 1953, 2594– 2596,  DOI: 10.1039/jr9530002594
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    Cleare, M. J.; Griffith, W. P. Binuclear Nitrido-Complexes of Ruthenium. Chem. Commun. 1968, 21, 1302,  DOI: 10.1039/c19680001302
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    Griffith, W. P.; McManus, N. T.; Skapski, A. C. X-Ray Crystal Structure of [Ru2N(ethylenediamine)5]Cl5· H2O; a Novel Complex Containing Both Nitrido and Ethylenediamine Bridges. J. Chem. Soc., Chem. Commun. 1984, 7, 434,  DOI: 10.1039/c39840000434
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    Ng, H.-Y.; Cheung, W.-M.; Kwan Huang, E.; Wong, K.-L.; Sung, H. H.-Y.; Williams, I. D.; Leung, W.-H. Ruthenium Chalcogenonitrosyl and Bridged Nitrido Complexes Containing Chelating Sulfur and Oxygen Ligands. Dalton Trans. 2015, 44, 18459– 18468,  DOI: 10.1039/C5DT02513C
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    Ruthenium chalcogenonitrosyl and bridged nitrido complexes containing chelating sulfur and oxygen ligands
    Ng, Ho-Yuen; Cheung, Wai-Man; Kwan Huang, Enrique; Wong, Kang-Long; Sung, Herman H.-Y.; Williams, Ian D.; Leung, Wa-Hung
    Dalton Transactions (2015), 44 (42), 18459-18468CODEN: DTARAF; ISSN:1477-9226. (Royal Society of Chemistry)
    Ruthenium thio- and seleno-nitrosyl complexes contg. chelating S and O ligands were synthesized and their de-chalcogenation reactions were studied. The reaction of mer-[Ru(N)Cl3(AsPh3)2] with elemental S and Se in THF at reflux afforded the chalcogenonitrosyl complexes mer-[Ru(NX)Cl3(AsPh3)2] [X = S (1), Se (2)]. Treatment of 1 with KN(R2PS)2 afforded trans-[Ru(NS)Cl{N(R2PS)2}2] [R = Ph (3), Pri (4), But (5)]. Alternatively, the thionitrosyl complex 5 was obtained from [Bu4N][Ru(N)Cl4] and KN(But2PS)2, presumably via S atom transfer from [N(But2PS)2]- to the nitride. Reactions of 1 and 2 with NaLOEt (LOEt- = [Co(η5-C5H5){P(O)(LOEt)2}3]-) gave [Ru(NX)LOEtCl2] (X = S (8), Se (9)). Treatment of [Bu4N][Ru(N)Cl4] with KN(R2PS)2 produced RuIV-RuIV μ-nitrido complexes [Ru2(μ-N){N(R2PS)2}4Cl] [R = Ph (6), Pri (7)]. Reactions of 3 and 9 with PPh3 afforded 6 and [Ru(NPPh3)LOEtCl2], resp. The desulfurization of 5 with [Ni(cod)2] (cod = 1,5-cyclooctadiene) gave the mixed valance RuIII-RuIV μ-nitrido complex [Ru2(μ-N){N(But2PS)2}4] (10) that was oxidized by [Cp2Fe](PF6) to give the RuIV-RuIV complex [Ru2(μ-N){N(But2PS)2}4](PF6) ([10]PF6). The crystal structures of 1, 2, 3, 7, 9 and 10 were detd.
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    Ciechanowicz, M.; Skapski, A. C. Crystal Structure of Potassium μ-Nitrido-Bis[Aquotetrachlororuthenate(IV)]. J. Chem. Soc. A 1971, 84, 1792– 1794,  DOI: 10.1039/J19710001792
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    Shanmughapriya, S.; Rajan, S.; Hoffman, N. E.; Zhang, X.; Guo, S.; Kolesar, J. E.; Hines, K. J.; Ragheb, J.; Jog, N. R.; Caricchio, R.; Baba, Y.; Zhou, Y.; Kaufman, B. A.; Cheung, J. Y.; Kurosaki, T.; Gill, D. L.; Madesh, M. Ca2+ Signals Regulate Mitochondrial Metabolism by Stimulating CREB-Mediated Expression of the Mitochondrial Ca2+ Uniporter Gene MCU. Sci. Signaling 2015, 8, ra23,  DOI: 10.1126/scisignal.2005673
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    Reers, M.; Smith, T. W.; Chen, L. B. J-aggregate formation of a carbocyanine as a quantitative fluorescent indicator of membrane potential. Biochemistry 1991, 30, 4480– 4486,  DOI: 10.1021/bi00232a015
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    J-aggregate formation of a carbocyanine as a quantitative fluorescent indicator of membrane potential
    Reers, Martin; Smith, Thomas W.; Chen, Lan Bo
    Biochemistry (1991), 30 (18), 4480-6CODEN: BICHAW; ISSN:0006-2960.
    The spectral properties of a novel membrane potential sensitive probe (JC-1) were characterized in aq. buffers and in isolated cardiac mitochondria. JC-1 is a carbocyanine with a delocalized pos. charge. It formed under favorable conditions a concn.-dependent fluorescent nematic phase consisting of J-aggregates. When excited at 490 nm, the monomers exhibited an emission max. at 527 nm and J-aggregates at 590 nm. Increasing concns. of JC-1 above a certain concn. caused a linear rise in the J-aggregate fluorescence, while the monomer fluorescence remained const. The membrane potential of energized mitochondria (neg. inside) promoted a directional uptake of JC-1 into the matrix, also with subsequent formation of J-aggregates. The J-aggregate fluorescence was sensitive to transient membrane potential changes induced by ADP and to metabolic inhibitors of oxidative phosphorylation. The J-aggregate fluorescence was pH independent within the physiol. pH range of 7.15-8.0 and could be linearly calibrated with valinomycin-induced K+ diffusion potentials. The advantage of JC-1 over rhodamines and other carbocyanines is that its color altered reversibly from green to red with increasing membrane potentials. This can be exploited for imaging live mitochondria on the stage of a microscope.
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    King, A. P.; Gellineau, H. A.; Ahn, J. E.; MacMillan, S. N.; Wilson, J. J. Bis(Thiosemicarbazone) Complexes of Cobalt(III). Synthesis, Characterization, and Anticancer Potential. Inorg. Chem. 2017, 56, 6609– 6623,  DOI: 10.1021/acs.inorgchem.7b00710
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    Bis(thiosemicarbazone) Complexes of Cobalt(III). Synthesis, Characterization, and Anticancer Potential
    King, A. Paden; Gellineau, Hendryck A.; Ahn, Jung-Eun; MacMillan, Samantha N.; Wilson, Justin J.
    Inorganic Chemistry (2017), 56 (11), 6609-6623CODEN: INOCAJ; ISSN:0020-1669. (American Chemical Society)
    Nine bis(thiosemicarbazone) (BTSC) cobalt(III) complexes of the general formula [Co(BTSC)(L)2]NO3 were synthesized, where BTSC = diacetyl bis(thiosemicarbazone) (ATS), pyruvaldehyde bis(thiosemicarbazone) (PTS), or glyoxal bis(thiosemicarbazone) (GTS) and L = ammonia, imidazole (Im), or benzylamine (BnA). These compds. were characterized by multinuclear NMR spectroscopy, mass spectrometry, cyclic voltammetry, and x-ray crystallog. Their stability in phosphate-buffered saline was investigated and is highly dependent on the nature of the axial ligand, L. These studies revealed that complex stability is primarily dictated by the axial ligand following the sequence NH3 > Im > BnA. The cellular uptake and cytotoxicity in cancer cells were also detd. Both the cellular uptake and cytotoxicity were significantly affected by the nature of the equatorial BTSC. Complexes of ATS were taken up much more effectively than those of PTS and GTS. The cytotoxicity of the complexes was correlated to that of the free ligand. Cell uptake and cytotoxicity were also detd. under hypoxic conditions. Only minor differences in the hypoxia activity and uptake were obsd. Treatment of the cancer cells with the copper-depleting agent tetrathiomolybdate decreased the cytotoxic potency of the complexes, indicating that they may operate via a copper-dependent mechanism. These results provide a structure-activity relation for this class of compds., which may be applied for the rational design of new cobalt(III) anticancer agents.
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    Egger, A. E.; Rappel, C.; Jakupec, M. A.; Hartinger, C. G.; Heffeter, P.; Keppler, B. K. Development of an Experimental Protocol for Uptake Studies of Metal Compounds in Adherent Tumor Cells. J. Anal. At. Spectrom. 2009, 24, 51– 61,  DOI: 10.1039/B810481F
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    Development of an experimental protocol for uptake studies of metal compounds in adherent tumor cells
    Egger, Alexander E.; Rappel, Christina; Jakupec, Michael A.; Hartinger, Christian G.; Heffeter, Petra; Keppler, Bernhard K.
    Journal of Analytical Atomic Spectrometry (2009), 24 (1), 51-61CODEN: JASPE2; ISSN:0267-9477. (Royal Society of Chemistry)
    Cellular uptake is being widely investigated in the context of diverse biol. activities of metal compds. on the cellular level. However, the applied techniques differ considerably, and a validated methodol. is not at hand. Therefore, we have varied numerous aspects of sample prepn. of the human colon carcinoma cell line SW480 exposed in vitro to the tumor-inhibiting metal complexes cisplatin and indazolium trans-[tetrachlorobis(1H-indazole)ruthenate(III)] (KP1019) prior to anal. with ICP-MS, and the results were found to be tremendously influenced by adsorption to the culture dishes. Adsorption to culture plates increases linearly with the concn. of KP1019, depends on the protein content of the medium, the duration of contact to protein-contg. medium prior to drug addn. and the hydrophilicity/lipophilicity of the compd. For varying degrees of cell confluence, adsorption of Ru hardly differs from cell-free expts. Desorption from the plates contributes to total Ru detected in dependence on the cell harvesting method. Desorption kinetics for lysis in HNO3 and tetramethylammonium hydroxide (TMAH) are comparable, but TMAH is a more potent desorbant. Sample storage conditions prior to anal. influence significantly the recovery of analyte. Protocols using cell lysis in the culture plate without proper corrections run the risk of producing artifacts resulting from metal adsorption/desorption to an extent comparable with the actual cellular content. However, exptl. protocols reported in the literature frequently do not contain information whether adsorption or blank correction were performed and should be regarded with caution, esp. if lysis was performed directly in the culture dishes.
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    Komor, A. C.; Schneider, C. J.; Weidmann, A. G.; Barton, J. K. Cell-Selective Biological Activity of Rhodium Metalloinsertors Correlates with Subcellular Localization. J. Am. Chem. Soc. 2012, 134, 19223– 19233,  DOI: 10.1021/ja3090687
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    Cell-Selective Biological Activity of Rhodium Metalloinsertors Correlates with Subcellular Localization
    Komor, Alexis C.; Schneider, Curtis J.; Weidmann, Alyson G.; Barton, Jacqueline K.
    Journal of the American Chemical Society (2012), 134 (46), 19223-19233CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    Deficiencies in the mismatch repair (MMR) pathway are assocd. with several types of cancers, as well as resistance to commonly used chemotherapeutics. Rhodium metalloinsertors have been found to bind DNA mismatches with high affinity and specificity in vitro, and also exhibit cell-selective cytotoxicity, targeting MMR-deficient cells over MMR-proficient cells. Ten distinct metalloinsertors with varying lipophilicities have been synthesized and their mismatch binding affinities and biol. activities detd. Although DNA photocleavage expts. demonstrate that their binding affinities are quite similar, their cell-selective antiproliferative and cytotoxic activities vary significantly. Inductively coupled plasma mass spectrometry (ICP-MS) expts. have uncovered a relation between the subcellular distribution of these metalloinsertors and their biol. activities. Specifically, we find that all of our metalloinsertors localize in the nucleus at sufficient concns. for binding to DNA mismatches. However, the metalloinsertors with high rhodium localization in the mitochondria show toxicity that is not selective for MMR-deficient cells, whereas metalloinsertors with less mitochondrial rhodium show activity that is highly selective for MMR-deficient vs. proficient cells. This work supports the notion that specific targeting of the metalloinsertors to nuclear DNA gives rise to their cell-selective cytotoxic and antiproliferative activities. The selectivity in cellular targeting depends upon binding to mismatches in genomic DNA.
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    Boyle, K. M.; Barton, J. K. A Family of Rhodium Complexes with Selective Toxicity toward Mismatch Repair-Deficient Cancers. J. Am. Chem. Soc. 2018, 140, 5612– 5624,  DOI: 10.1021/jacs.8b02271
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    72
    A Family of Rhodium Complexes with Selective Toxicity toward Mismatch Repair-Deficient Cancers
    Boyle, Kelsey M.; Barton, Jacqueline K.
    Journal of the American Chemical Society (2018), 140 (16), 5612-5624CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    Rhodium metalloinsertors are a unique set of metal complexes that bind specifically to DNA base pair mismatches in vitro and kill mismatch repair (MMR)-deficient cells at lower concns. than their MMR-proficient counterparts. A family of metalloinsertors contg. rhodium-oxygen ligand coordination, termed "Rh-O" metalloinsertors, has been prepd. and shown to have a significant increase in both overall potency and selectivity toward MMR-deficient cells regardless of structural changes in the ancillary ligands. Here we describe DNA-binding and cellular studies with the second generation of Rh-O metalloinsertors in which an ancillary ligand is varied in both steric bulk and lipophilicity. These complexes, of the form [Rh(L)(chrysi)(PPO)]2+, all include the O-contg. PPO ligand (PPO = 2-(pyridine-2-yl)propan-2-ol) and the arom. inserting ligand chrysi (5,6-chrysene quinone diimine) but differ in the identity of their ancillary ligand L, where L is a phenanthroline or bipyridyl deriv. The Rh-O metalloinsertors in this family all show micromolar binding affinities for a 29-mer DNA hairpin contg. a single CC mismatch. The complexes display comparable lipophilic tendencies and pKa values of 8.1-9.1 for dissocn. of an imine proton on the chrysi ligand. In cellular proliferation and cytotoxicity assays with MMR-deficient cells (HCT116O) and MMR-proficient cells (HCT116N), the complexes contg. the phenanthroline-derived ligands show highly selective cytotoxic preference for the MMR-deficient cells at nanomolar concns. Using mass spectral analyses, it is shown that the complexes are taken into cells through a passive mechanism and exhibit low accumulation in mitochondria, an off-target organelle that, when targeted by parent metalloinsertors, can lead to nonselective cytotoxicity. Overall, these Rh-O metalloinsertors have distinct and improved behavior compared to previous generations of parent metalloinsertors, making them ideal candidates for further therapeutic assessment.
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    Ahmad, K. A.; Iskandar, K. B.; Hirpara, J. L.; Clement, M. V.; Pervaiz, S. Hydrogen Peroxide-Mediated Cytosolic Acidification is a Signal for Mitochondrial Translocation of Bax during Drug-Induced Apoptosis of Tumor Cells. Cancer Res. 2004, 64, 7867– 7878,  DOI: 10.1158/0008-5472.CAN-04-0648
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    Dong, Z.; Shanmughapriya, S.; Tomar, D.; Siddiqui, N.; Lynch, S.; Nemani, N.; Breves, S. L.; Zhang, X.; Tripathi, A.; Palaniappan, P.; Riitano, M. F.; Worth, A. M.; Seelam, A.; Carvalho, E.; Subbiah, R.; Jaña, F.; Soboloff, J.; Peng, Y.; Cheung, J. Y.; Joseph, S. K.; Caplan, J.; Rajan, S.; Stathopulos, P. B.; Madesh, M. Mitochondrial Ca2+ Uniporter is a Mitochondrial Luminal Redox Sensor That Augments MCU Channel Activity. Mol. Cell 2017, 65, 1014– 1028,  DOI: 10.1016/j.molcel.2017.01.032
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    74
    Mitochondrial Ca2+ Uniporter Is a Mitochondrial Luminal Redox Sensor that Augments MCU Channel Activity
    Dong, Zhiwei; Shanmughapriya, Santhanam; Tomar, Dhanendra; Siddiqui, Naveed; Lynch, Solomon; Nemani, Neeharika; Breves, Sarah L.; Zhang, Xueqian; Tripathi, Aparna; Palaniappan, Palaniappan; Riitano, Massimo F.; Worth, Alison M.; Seelam, Ajay; Carvalho, Edmund; Subbiah, Ramasamy; Jana, Fabian; Soboloff, Jonathan; Peng, Yizhi; Cheung, Joseph Y.; Joseph, Suresh K.; Caplan, Jeffrey; Rajan, Sudarsan; Stathopulos, Peter B.; Madesh, Muniswamy
    Molecular Cell (2017), 65 (6), 1014-1028.e7CODEN: MOCEFL; ISSN:1097-2765. (Elsevier Inc.)
    Ca2+ dynamics and oxidative signaling are fundamental mechanisms for mitochondrial bioenergetics and cell function. The MCU complex is the major pathway by which these signals are integrated in mitochondria. Whether and how these coactive elements interact with MCU have not been established. As an approach toward understanding the regulation of MCU channel by oxidative milieu, we adapted inflammatory and hypoxia models. We identified the conserved cysteine 97 (Cys-97) to be the only reactive thiol in human MCU that undergoes S-glutathionylation. Furthermore, biochem., structural, and superresoln. imaging anal. revealed that MCU oxidn. promotes MCU higher order oligomer formation. Both oxidn. and mutation of MCU Cys-97 exhibited persistent MCU channel activity with higher [Ca2+]m uptake rate, elevated mROS, and enhanced [Ca2+]m overload-induced cell death. In contrast, these effects were largely independent of MCU interaction with its regulators. These findings reveal a distinct functional role for Cys-97 in ROS sensing and regulation of MCU activity.
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    Zhao, Y.; Araki, S.; Wu, J.; Teramoto, T.; Chang, Y.-F.; Nakano, M.; Abdelfattah, A. S.; Fujiwara, M.; Ishihara, T.; Nagai, T.; Campbell, R. E. An Expanded Palette of Genetically Encoded Ca2+ Indicators. Science 2011, 333, 1888– 1891,  DOI: 10.1126/science.1208592
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    An Expanded Palette of Genetically Encoded Ca2+ Indicators
    Zhao, Yongxin; Araki, Satoko; Wu, Jiahui; Teramoto, Takayuki; Chang, Yu-Fen; Nakano, Masahiro; Abdelfattah, Ahmed S.; Fujiwara, Manabi; Ishihara, Takeshi; Nagai, Takeharu; Campbell, Robert E.
    Science (Washington, DC, United States) (2011), 333 (6051), 1888-1891CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
    Engineered fluorescent protein (FP) chimeras that modulate their fluorescence in response to changes in calcium ion (Ca2+) concn. are powerful tools for visualizing intracellular signaling activity. However, despite a decade of availability, the palette of single FP-based Ca2+ indicators has remained limited to a single green hue. The authors have expanded this palette by developing blue, improved green, and red intensiometric indicators, as well as an emission ratiometric indicator with an 11,000% ratio change. This series enables improved single-color Ca2+ imaging in neurons and transgenic Caenorhabditis elegans. In HeLa cells, Ca2+ was imaged in three subcellular compartments, and, in conjunction with a cyan FP-yellow FP-based indicator, Ca2+ and ATP were simultaneously imaged. This palette of indicators paints the way to a colorful new era of Ca2+ imaging.
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    Nakai, J.; Ohkura, M.; Imoto, K. A High Signal-to-Noise Ca2+ Probe Composed of a Single Green Fluorescent Protein. Nat. Biotechnol. 2001, 19, 137– 141,  DOI: 10.1038/84397
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    A high signal-to-noise Ca2+ probe composed of a single green fluorescent protein
    Nakai, Junichi; Ohkura, Masamichi; Imoto, Keiji
    Nature Biotechnology (2001), 19 (2), 137-141CODEN: NABIF9; ISSN:1087-0156. (Nature America Inc.)
    Recently, several groups have developed green fluorescent protein (GFP)-based Ca2+ probes. When applied in cells, however, these probes are difficult to use because of a low signal-to-noise ratio. Here we report the development of a high-affinity Ca2+ probe composed of a single GFP (named G-CaMP). G-CaMP showed an apparent Kd for Ca2+ of 235 μM. Assocn. kinetics of Ca2+ binding were faster at higher Ca2+ concns., with time consts. decreasing from 230 ms at 0.2 μM Ca2+ to 2.5 ms at 1 μM Ca2+. Dissocn. kinetics (τ -200 ms) are independent of Ca2+ concns. In HEK-293 cells and mouse myotubes expressing G-CaMP, large fluorescent changes were obsd. in response to application of drugs or elec. stimulations. G-CaMP will be a useful tool for visualizing intracellular Ca2+ in living cells. Mutational anal., together with previous structural information, suggests the residues that may alter the fluorescence of GFP.
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    Chiesi, M.; Schwaller, R.; Eichenberger, K. Structural Dependency of the Inhibitory Action of Benzodiazepines and Related Compounds on the Mitochondrial Na+-Ca2+ Exchanger. Biochem. Pharmacol. 1988, 37, 4399– 4403,  DOI: 10.1016/0006-2952(88)90623-5
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    Luongo, T. S.; Lambert, J. P.; Gross, P.; Nwokedi, M.; Lombardi, A. A.; Shanmughapriya, S.; Carpenter, A. C.; Kolmetzky, D.; Gao, E.; van Berlo, J. H.; Tsai, E. J.; Molkentin, J. D.; Chen, X.; Madesh, M.; Houser, S. R.; Elrod, J. W. The Mitochondrial Na+/Ca2+ Exchanger is Essential for Ca2+ Homeostasis and Viability. Nature 2017, 545, 93– 97,  DOI: 10.1038/nature22082
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    The mitochondrial Na+/Ca2+ exchanger is essential for Ca2+ homeostasis and viability
    Luongo, Timothy S.; Lambert, Jonathan P.; Gross, Polina; Nwokedi, Mary; Lombardi, Alyssa A.; Shanmughapriya, Santhanam; Carpenter, April C.; Kolmetzky, Devin; Gao, Erhe; van Berlo, Jop H.; Tsai, Emily J.; Molkentin, Jeffery D.; Chen, Xiongwen; Madesh, Muniswamy; Houser, Steven R.; Elrod, John W.
    Nature (London, United Kingdom) (2017), 545 (7652), 93-97CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
    Mitochondrial calcium (mCa2+) has a central role in both metabolic regulation and cell death signaling, however its role in homeostatic function and disease is controversial. Slc8b1 encodes the mitochondrial Na+/Ca2+ exchanger (NCLX), which is proposed to be the primary mechanism for mCa2+ extrusion in excitable cells. Here we show that tamoxifen-induced deletion of Slc8b1 in adult mouse hearts causes sudden death, with less than 13% of affected mice surviving after 14 days. Lethality correlated with severe myocardial dysfunction and fulminant heart failure. Mechanistically, cardiac pathol. was attributed to mCa2+ overload driving increased generation of superoxide and necrotic cell death, which was rescued by genetic inhibition of mitochondrial permeability transition pore activation. Corroborating these findings, overexpression of NCLX in the mouse heart by conditional transgenesis had the beneficial effect of augmenting mCa2+ clearance, preventing permeability transition and protecting against ischemia-induced cardiomyocyte necrosis and heart failure. These results demonstrate the essential nature of mCa2+ efflux in cellular function and suggest that augmenting mCa2+ efflux may be a viable therapeutic strategy in disease.
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    Storey, N. M.; Lambert, D. G. Mitochondrial Pharmacology Turns Its Sights on the Ca2+ Uniporter. Cell Death Discov. 2017, 3, 17064,  DOI: 10.1038/cddiscovery.2017.64
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    Mitochondrial pharmacology turns its sights on the Ca(2+) uniporter
    Storey Nina M; Lambert David G
    Cell death discovery (2017), 3 (), 17064 ISSN:2058-7716.
    There is no expanded citation for this reference.
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    Sebag, S. C.; Koval, O. M.; Paschke, J. D.; Winters, C. J.; Comellas, A. P.; Grumbach, I. M. Inhibition of the Mitochondrial Calcium Uniporter Prevents IL-13 and Allergen-Mediated Airway Epithelial Apoptosis and Loss of Barrier Function. Exp. Cell Res. 2018, 362, 400– 411,  DOI: 10.1016/j.yexcr.2017.12.003
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    Inhibition of the mitochondrial calcium uniporter prevents IL-13 and allergen-mediated airway epithelial apoptosis and loss of barrier function
    Sebag, Sara C.; Koval, Olha M.; Paschke, John D.; Winters, Christopher J.; Comellas, Alejandro P.; Grumbach, Isabella M.
    Experimental Cell Research (2018), 362 (2), 400-411CODEN: ECREAL; ISSN:0014-4827. (Elsevier B.V.)
    Mitochondria are increasingly recognized as key mediators of acute cellular stress responses in asthma. However, the distinct roles of regulators of mitochondrial physiol. on allergic asthma phenotypes are currently unknown. The mitochondrial Ca2+ uniporter (MCU) resides in the inner mitochondrial membrane and controls mitochondrial Ca2+ uptake into the mitochondrial matrix. To understand the function of MCU in models of allergic asthma, in vitro and in vivo studies were performed using models of functional deficiency or knockout of MCU. In primary human respiratory epithelial cells, MCU inhibition abrogated mitochondrial Ca2+ uptake and reactive oxygen species (ROS) prodn., preserved the mitochondrial membrane potential and protected from apoptosis in response to the pleiotropic Th2 cytokine IL-13. Consequently, epithelial barrier function was maintained with MCU inhibition. Similarly, the endothelial barrier was preserved in respiratory epithelium isolated from MCU-/- mice after exposure to IL-13. In the ovalbumin-model of allergic airway disease, MCU deficiency resulted in decreased apoptosis within the large airway epithelial cells. Concordantly, expression of the tight junction protein ZO-1 was preserved, indicative of maintenance of epithelial barrier function. These data implicate mitochondrial Ca2+ uptake through MCU as a key controller of epithelial cell viability in acute allergic asthma.
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    Xie, N.; Wu, C.; Wang, C.; Cheng, X.; Zhang, L.; Zhang, H.; Lian, Y. Inhibition of the Mitochondrial Calcium Uniporter Inhibits Aβ-Induced Apoptosis by Reducing Reactive Oxygen Species-Mediated Endoplasmic Reticulum Stress in Cultured Microglia. Brain Res. 2017, 1676, 100– 106,  DOI: 10.1016/j.brainres.2017.08.035
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    Inhibition of the mitochondrial calcium uniporter inhibits Aβ-induced apoptosis by reducing reactive oxygen species-mediated endoplasmic reticulum stress in cultured microglia
    Xie, Nanchang; Wu, Chuanjie; Wang, Cui; Cheng, Xuan; Zhang, Lu; Zhang, Haifeng; Lian, Yajun
    Brain Research (2017), 1676 (), 100-106CODEN: BRREAP; ISSN:0006-8993. (Elsevier B.V.)
    Amyloid-beta (Aβ) has been shown to induce microglial apoptosis, which is itself sensitive to disturbed mitochondrial calcium (Ca2+) homeostasis. The mitochondrial calcium uniporter (MCU) plays an important regulatory role in mitochondrial Ca2+ homeostasis, but its role in Aβ-induced microglia apoptosis is unknown. In this study, we found increased mitochondrial Ca2+ concn. in Aβ-treated primary microglia and BV-2 cells; also, the MCU inhibitor Ru360 significantly attenuated Aβ-induced microglial apoptosis, whereas the MCU activator spermine augmented it. In addn., Ru360 significantly attenuated Aβ-induced mitochondrial reactive oxygen species (ROS) prodn., as well as endoplasmic reticulum (ER) stress characterized by glucose-regulated protein 78 (GRP78) and C/-EBP homologous protein (CHOP) expression. Spermine, however, exerted the opposite effects on mitochondrial ROS prodn. and ER stress. We also found that mitochondria-targeted antioxidant (Mito-TEMPO) treatment decreased GRP78 and CHOP expression in Aβ-treated microglia. Moreover, blocking endogenous CHOP expression using a CHOP small interfering RNA (siRNA) attenuated Aβ-induced cell death. Altogether, our data suggested that (1) inhibition of MCU exerts a neuroprotective effect on Aβ-induced microglia apoptosis, and (2) that the underlying mechanism may be related to reducing mitochondrial ROS-mediated ER stress.
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    Alessio, E. Thirty Years of the Drug Candidate NAMI-A and the Myths in the Field of Ruthenium Anticancer Compounds: A Personal Perspective. Eur. J. Inorg. Chem. 2017, 2017, 1549– 1560,  DOI: 10.1002/ejic.201600986
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    Thirty Years of the Drug Candidate NAMI-A and the Myths in the Field of Ruthenium Anticancer Compounds: A Personal Perspective
    Alessio, Enzo
    European Journal of Inorganic Chemistry (2017), 2017 (12), 1549-1560CODEN: EJICFO; ISSN:1434-1948. (Wiley-VCH Verlag GmbH & Co. KGaA)
    As anticipated in the title, this contribution is basically divided into two, strictly connected, parts. The first is a personal overview of the ruthenium drug candidate NAMI-A, almost 30 years after its synthesis and the discovery of its unprecedented antimetastatic properties in animal models at nontoxic dosages. The sections relating to the chem. and biol. behavior of the complex, and the hypotheses on its mechanism(s) of action, are kept to a min., whereas more space is devoted to discussion of the results of the clin. investigations. The second part deals in detail with a no. of undemonstrated misconceptions (or myths) that, over the years, have thrived around NAMI-A and other ruthenium drug candidates, thus neg. affecting the whole field of Ru anticancer drugs.
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    Incorporation of biquinoline ligands into Ru(ii) polypyridyl complexes produces light-activated systems that eject a ligand and photobind DNA after irradn. with visible and near-IR light. Structural anal. shows that distortion facilitates the photochem., and gel shift and cytotoxicity studies prove the compds. act as anti-cancer photodynamic therapy (PDT) agents in the tissue penetrant region.
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    A review. Neutral or cationic arene ruthenium complexes providing both hydrophilic as well as hydrophobic properties due to the robustness of the ruthenium-arene unit hold a high potential for the development of metal-based anticancer drugs. Mononuclear arene ruthenium complexes contg. P- or N-donor ligands or N,N-, N,O- or O,O-chelating ligands, dinuclear arene ruthenium systems with adjustable org. linkers, trinuclear arene ruthenium clusters contg. an oxo cap, tetranuclear arene ruthenium porphyrin derivs. that are photoactive, as well as hexanuclear ruthenium cages that are either empty or filled with other mols. have been shown to be active against a variety of cancer cells.
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    Wang, F.; Chen, H.; Parsons, S.; Oswald, I. D. H.; Davidson, J. E.; Sadler, P. J. Kinetics of Aquation and Anation of Ruthenium(II) Arene Anticancer Complexes, Acidity and X-Ray Structures of Aqua Adducts. Chem. - Eur. J. 2003, 9, 5810– 5820,  DOI: 10.1002/chem.200304724
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    Kinetics of aquation and anation of ruthenium(II) arene anticancer complexes, acidity and X-ray structures of aqua adducts
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    Chemistry - A European Journal (2003), 9 (23), 5810-5820CODEN: CEUJED; ISSN:0947-6539. (Wiley-VCH Verlag GmbH & Co. KGaA)
    The aqua adducts of the anticancer complexes [(η6-X)Ru(en)Cl] [PF6] (X = biphenyl (Bip) 1, X = 5,8,9,10-tetrahydroanthracene (THA) 2, X = 9,10-dihydroanthracene (DHA) 3; en = ethylenediamime) were sepd. by HPLC and characterized by mass spectrometry as the products of hydrolysis in H2O. The x-ray structures of the aqua complexes [(η6-X)Ru(en)Y] [PF6]n, X = Bip, Y = 0.5H2O/0.5OH, n = 1.5 (4), X = THA, Y = 0.5H2O/0.5OH, n = 1.5 (5A), X = THA, Y = H2O, n = 2 (5B), and X = DHA, Y = H2O, n = 2(6), are reported. In complex 4 there is a large propeller twist of 45° of the pendant Ph ring with respect to the coordinated Ph ring. Although the THA ligand in 5A and 5B is relatively flat, the DHA ring system in 6 is markedly bent (hinge bend ∼35°) as in the chloro complex 3 (41°). The rates of aquation of 1-3 detd. by UV/visible spectroscopy at various ionic strengths and temps. (1.23-2.59 × 10-3s-1 at 298 K, I = 0.1M) are >20× faster than that of cisplatin. The reverse, anation reactions were very rapid on addn. of 100 mM NaCl (a similar concn. to that in blood plasma). The aquation and anation reactions were about two times faster for the DHA and THA complexes compared to the biphenyl complex. The hydrolysis reactions appear to occur by an associative pathway. The pKa values of the aqua adducts were detd. by 1H NMR spectroscopy as 7.71 for 4, 8.01 for 5 and 7.89 for 6. At physiol.-relevant concns. (0.5-5 μM) and temp. (310 K), the complexes will exist in blood plasma as >89% chloro complex, whereas in the cell nucleus significant amts. (45-65%) of the more reactive aqua adducts would be formed together with smaller amts. of the hydroxo complexes (9-25%, pH 7.4, [Cl-] = 4 mM).
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    Chemistry & Biodiversity (2008), 5 (10), 2140-2155CODEN: CBHIAM; ISSN:1612-1872. (Verlag Helvetica Chimica Acta)
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    Peacock, A. F. A.; Habtemariam, A.; Fernández, R.; Walland, V.; Fabbiani, F. P. A.; Parsons, S.; Aird, R. E.; Jodrell, D. I.; Sadler, P. J. Tuning the Reactivity of Osmium(II) and Ruthenium(II) Arene Complexes under Physiological Conditions. J. Am. Chem. Soc. 2006, 128, 1739– 1748,  DOI: 10.1021/ja055886r
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    Tuning the Reactivity of Osmium(II) and Ruthenium(II) Arene Complexes under Physiological Conditions
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    The OsII arene ethylenediamine (en) complexes [(η6-biphenyl)Os(en)Cl][Z], Z = BPh4 (4) and BF4 (5), are inactive toward A2780 ovarian cancer cells despite 4 being isostructural with an active RuII analog, 4R. Hydrolysis of 5 occurred 40 times more slowly than 4R. The aqua adduct 5A has a low pKa (6.3) compared to that of [(η6-biphenyl)Ru(en)(OH2)]2+ (7.7) and is therefore largely in the hydroxo form at physiol. pH. The rate and extent of reaction of 5 with 9-ethylguanine were also less than those of 4R. The authors replaced the neutral en ligand by anionic acetylacetonate (acac). The complexes [(η6-arene)Os(acac)Cl], arene = biphenyl (6), benzene (7), and p-cymene (8), adopt piano-stool structures similar to those of the RuII analogs and form weak dimers through intermol. (arene)C-H···O(acac) H-bonds. Remarkably, these OsII acac complexes undergo rapid hydrolysis to produce not only the aqua adduct, [(η6-arene)Os(acac)(OH2)]+, but also the hydroxo-bridged dimer, [(η6-arene)Os(μ2-OH)3Os(η6-arene)]+. The pKa values for the aqua adducts 6A, 7A, and 8A (7.1, 7.3, and 7.6, resp.) are lower than that for [(η6-p-cymene)Ru(acac)(OH2)]+ (9.4). Complex 8A rapidly forms adducts with 9-ethylguanine and adenosine, but not with cytidine or thymidine. Despite their reactivity toward nucleobases, complexes 6-8 were inactive toward A549 lung cancer cells. This is attributable to rapid hydrolysis and formation of unreactive hydroxo-bridged dimers which, surprisingly, were the only species present in aq. soln. at biol. relevant concns. Hence, the choice of chelating ligand in OsII (and RuII) arene complexes can have a dramatic effect on hydrolysis behavior and nucleobase binding and provides a means of tuning the reactivity and the potential for discovery of anticancer complexes.
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    Mühlgassner, G.; Bartel, C.; Schmid, W. F.; Jakupec, M. A.; Arion, V. B.; Keppler, B. K. Biological Activity of Ruthenium and Osmium Arene Complexes with Modified Paullones in Human Cancer Cells. J. Inorg. Biochem. 2012, 116, 180– 187,  DOI: 10.1016/j.jinorgbio.2012.06.003
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    Biological activity of ruthenium and osmium arene complexes with modified paullones in human cancer cells
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    Journal of Inorganic Biochemistry (2012), 116 (), 180-187CODEN: JIBIDJ; ISSN:0162-0134. (Elsevier)
    In an attempt to combine the ability of indolobenzazepines (paullones) to inhibit cyclin-dependent kinases (Cdks) and that of platinum-group metal ions to interact with proteins and DNA, ruthenium(II) and osmium(II) arene complexes with paullones were prepd., expecting synergies and an increase of soly. of paullones. Complexes with the general formula [MIICl(η6-p-cymene)L]Cl, where M = Ru (1, 3) or Os (2, 4), and L = L1 (1, 2) or L2 (3, 4), L1 = N-(9-bromo-7,12-dihydroindolo[3,2-d][1]-benzazepin-6(5H)-yliden-N'-(2-hydroxybenzylidene)azine and L2 = N-(9-bromo-7,12-dihydroindolo[3,2-d][1]benzazepin-6-yl)-N'-[3-hydroxy-5-(hydroxymethyl)-2-methylpyridin-4-yl-methylene]azinium chloride (L2*HCl), were now investigated regarding cytotoxicity and accumulation in cancer cells, impact on the cell cycle, capacity of inhibiting DNA synthesis and inducing apoptosis as well as their ability to inhibit Cdk activity. The MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) assay yielded IC50 values in the nanomolar to low micromolar range. In accordance with cytotoxicity data, the BrdU assay showed that 1 is the most and 4 the least effective of these compds. regarding inhibition of DNA synthesis. Effects on the cell cycle are minor, although concn.-dependent inhibition of Cdk2/cyclin E activity was obsd. in cell-free expts. Induction of apoptosis is most pronounced for complex 1, accompanied by a low fraction of necrotic cells, as obsd. by annexin V-fluorescein isothiocyanate/propidium iodide staining and flow cytometric anal.
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    Lameijer, L. N.; Ernst, D.; Hopkins, S. L.; Meijer, M. S.; Askes, S. H. C.; Le Dévédec, S. E.; Bonnet, S. A Red-Light-Activated Ruthenium-Caged NAMPT Inhibitor Remains Phototoxic in Hypoxic Cancer Cells. Angew. Chem., Int. Ed. 2017, 56, 11549– 11553,  DOI: 10.1002/anie.201703890
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    A Red-Light-Activated Ruthenium-Caged NAMPT Inhibitor Remains Phototoxic in Hypoxic Cancer Cells
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    We describe two water-sol. ruthenium complexes, [1]Cl2 and [2]Cl2, that photodissociate to release a cytotoxic nicotinamide phosphoribosyltransferase (NAMPT) inhibitor with a low dose (21 J cm-2) of red light in an oxygen-independent manner. Using a specific NAMPT activity assay, up to an 18-fold increase in inhibition potency was measured upon red-light activation of [2]Cl2, while [1]Cl2 was thermally unstable. For the first time, the dark and red-light-induced cytotoxicity of these photocaged compds. could be tested under hypoxia (1 % O2). In skin (A431) and lung (A549) cancer cells, a 3- to 4-fold increase in cytotoxicity was found upon red-light irradn. for [2]Cl2, whether the cells were cultured and irradiated with 1 % or 21 % O2. These results demonstrate the potential of photoactivated chemotherapy for hypoxic cancer cells, in which classical photodynamic therapy, which relies on oxygen activation, is poorly efficient.
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    Importance of the π-Ligand: Remarkable Effect of the Cyclopentadienyl Ring on the Cytotoxicity of Ruthenium PTA Compounds
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    The water-sol. complexes [(Cp'OR)RuCl(PTA)2] (Cp'OR = η5-1-alkoxy-2,4-di-tert-butyl-3-neopentylcyclopentadienyl; R = Me, Et; PTA = 1,3,5-triaza-7-phosphaadamantane) are considerably more cytotoxic (∼2 orders of magnitude) than the cyclopentadienyl analog [CpRuCl(PTA)2] (i.e., IC50 = 4-10 vs. >1000 μM, depending on the cell line). The structure of [(Cp'OMe)RuCl(PTA)2] is reported, together with that of the precursor [(Cp'OEt)Ru(μ-Cl)]2.
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    Metal-based drugs that break the rules
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    Cisplatin and other platinum compds. have had a huge impact in the treatment of cancers and are applied in the majority of anticancer chemotherapeutic regimens. The success of these compds. has biased the approaches used to discover new metal-based anticancer drugs. In this perspective we highlight compds. that are apparently incompatible with the more classical (platinum-derived) concepts employed in the development of metal-based anticancer drugs, with respect to both compd. design and the approaches used to validate their utility. Possible design approaches for the future are also suggested.
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    A review. Metal complexes are increasingly being used to inhibit enzymes. The reasons for this increased interest arise from the special features that metal complexes offer, e.g. the facile construction of 3D architectures that tightly fill enzyme active sites increasing selectivity and the possibility of facile coordination to protein residues that enhances enzyme inhibition. In this review we classify the main modes of enzyme inhibition by metal-based complexes and correlate the enzyme inhibition activity to macroscopic properties such as anticancer activity.
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    Barry, N. P. E.; Sadler, P. J. Exploration of the Medical Periodic Table: Towards New Targets. Chem. Commun. 2013, 49, 5106– 5131,  DOI: 10.1039/c3cc41143e
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    A review. Metallodrugs offer potential for unique mechanisms of drug action based on the choice of the metal, its oxidn. state, the types and no. of coordinated ligands and the coordination geometry. We discuss recent progress in identifying new target sites and elucidating the mechanisms of action of anti-cancer, anti-bacterial, anti-viral, anti-parasitic, anti-inflammatory, and anti-neurodegenerative agents, as well as in the design of metal-based diagnostic agents. Progress in identifying and defining target sites has been accelerated recently by advances in proteomics, genomics and metal speciation anal. Examples of metal compds. and chelating agents (enzyme inhibitors) currently in clin. use, clin. trials or preclin. development are highlighted.
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Cite this: ACS Cent. Sci. 2019, 5, 1, 153–166
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https://doi.org/10.1021/acscentsci.8b00773
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  • Abstract

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    Figure 1

    Figure 1. Topology diagram of the MCU. (A) Diagram showing the relevant regulator proteins EMRE, MCUR1, MICU1, and MICU2 and the orientation of the MCU in the IMM. Insets depict (left) the location of Cys97 in the X-ray crystal structure of the MCU-NTD (residues 72–189, PDB 5KUJ) (27) and (right) location of the DXXE motif in the solvent-accessible region of the MCU pore in the cryo-EM structure of the MCU (PDB 6DNF). (20) (B) At low Ca2+ concentration, MICU1 and MICU2 prevent Ca2+ from entering the mitochondria. (C) Elevated Ca2+ concentrations cause MICU1 and MICU2 to dissociate from the MCU, allowing Ca2+ transport across the IMM. (D) Binding of Ca2+ to the MRAP region in the MCU-NTD promotes a change in MCU conformation, preventing Ca2+ uptake.

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    Figure 2

    Figure 2. (A) Chemical structures of the established and commercially available MCU inhibitor Ru360 (left) and the related analogue C-1. (B) Synthetic scheme for the preparation of C-2 and C-3. Crystal structures of (C) C-2 and (D) C-3. Outer sphere chlorides and solvent are omitted for clarity. Thermal ellipsoids are shown at the 50% probability level. Interatomic distances and angles are given in Tables S1–S3 in the Supporting Information (SI).

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    Figure 3

    Figure 3. (A–C) Changes in ΔΨm (JC-1, top panel) and extramitochondrial Ca2+ ([Ca2+]out) clearance (Fura-2-FF, bottom panel) in digitonin-permeabilized HEK293T cells in response to 20 μM of Ca2+ after treatment with 5 μM of C-1, C-2, and C-3. The transient drop in ΔΨm in control cells upon Ca2+ addition indicates accumulation of Ca2+ in the mitochondrial matrix. (D) Quantification of the rate of [Ca2+]m uptake as a function of decrease in [Ca2+]out after 20 μM Ca2+pulse. Data are represented as mean ± standard error of measurement (SEM); ***p < 0.001; n = 3–6.

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    Figure 4

    Figure 4. (A, B) Cytosolic ([Ca2+]c, Fluo-4 AM) and mitochondrial ([Ca2+]m, Rhod-2 AM) calcium transients in HeLa cells after treatment with histamine (100 μM) that were pretreated with or without C-1, C-2, or C-3 (50 μM) for 30 min. (C, D) Cytosolic ([Ca2+]c, R-GECO) and mitochondrial ([Ca2+]m, GCamP6-mt) calcium transients in HeLa cells after treatment with histamine (100 μM) that were pretreated with or without C-1, C-2, or C-3 (50 μM) for 30 min. HeLa cells were cotransfected with R-GECO1 and GCamP6-mt and analyzed 48 h post-transfection. Data are represented as mean ± SEM; *p < 0.05; n = 3–6.

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    Figure 5

    Figure 5. (A) Representative traces of [Ca2+]out clearance in permeabilized HEK293T cells challenged with a range of C-2 concentrations (1, 2, 3, 5, and 10 nM). (B) Comparison of MCU-inhibitory effect by Ru360 (30 nM) and C-2 (3 and 10 nM) at lower concentrations. (C) Calculation of dose-dependent inhibition by C-2 and Ru360. (D) Assessment of mitochondrial Ca2+ influx and efflux rates. Permeabilized HEK293T cells pulsed with 20 μM Ca2+ at 350 s to measure mitochondrial Ca2+ uptake, followed by the addition of the 1 μM C-2 or Ru360 at 550 s, 10 μM CGP37157 at 600 s, and 6 μM CCCP at 750 s. Representative trace depicts the comparison of C-2 and Ru360. (E) Quantification of Ca2+ influx rate. (F) Quantification of Ca2+ efflux rate after addition of C-2 or Ru360. (G) Quantification of CCCP-induced release of accumulated mitochondrial Ca2+. Data represent mean ± SEM; n.s., not significant n = 3–6.

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    Figure 6

    Figure 6. Representative traces of [Ca2+]out clearance and ΔΨm in permeabilized HEK293T cells stably expressing Flag-tagged MCUWT and MCUS259A mutant challenged with either (A) C-2 (10 nM) or (B) Ru360 (500 nM). Bar graphs represent the quantification of the rate of [Ca2+]m uptake. Representative traces of [Ca2+]out clearance and ΔΨm in HEK293T cells stably expressing Flag-tagged MCUWT and MCUC97A mutant challenged with either (C) C-2 (10 nM) or (D) Ru360 (500 nM). Bar graphs represent the quantification of the rate of [Ca2+]m uptake. Compound MCU-inhibitory effect was compared with cells reconstituted with appropriate mutants alone. Data represent mean ± SEM; ***p < 0.001; n = 3–6.; n = 4.

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    Figure 7

    Figure 7. (A) 1H–15N-heteronuclear single quantum coherence (HSQC) spectra of uniformly 15N-labeled human MCU N-terminal domain (residues 72–189; MCU72–189) in the absence (blue cross-peaks) and presence (magenta cross-peaks) of C-2. A large amount of the protein immediately precipitated out of solution with the addition of 3 molar excess C-2 to the sample with no detectable change in solvent pH or ionic strength. Consequently, most amide H(N) cross-peaks show a drastic loss in intensity compared to cross-peaks in the control spectrum. (B) Coomassie blue-stained 15% (w/v) SDS-PAGE gel visualizing the insoluble precipitate formed after mixing 125 μM MCU72–189 with 375 μM C-2 (Insol. + C-2) and 375 μM Ru360 (Insol. + Ru360) compared to untreated MCU72–189 protein (Sol.). (C) Cross-peak broadening map on the MCU72–189 high-resolution crystal structure (PDB 5KUJ). (27) Tentatively assigned residues which exhibited a ≥40% reduction in peak intensity are highlighted in red on the backbone ribbon view of MCU72–189 (top dashed box). The surface representation of the same view (bottom dashed box) shows that the most affected H(N) cross-peaks primarily cluster on one face of the domain.

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    Figure 8

    Figure 8. Measurement of ΔΨm (top panels) and MCU-mediated Ca2+ uptake (bottom panels) in (A) control, (B) C-2, and (C) Ru360 treated NRVMs exposed to normoxia (black) and H/R (red). (D) Quantification of basal ΔΨm from parts A–C under normoxia and H/R before the addition of extramitochondrial Ca2+ pulses. (E) Quantification of the rate of MCU-mediated Ca2+ uptake from parts A–C as a function of decrease in bath Ca2+ fluorescence. (F) Swelling of isolated NRVM mitochondria subjected to H/R injury in the absence (black trace) or presence (blue trace) of C-2 or Ru360 (red trace). Data represent mean ± SEM; ***p < 0.001; n = 3–6.

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      12
      EMRE Is an Essential Component of the Mitochondrial Calcium Uniporter Complex
      Sancak, Yasemin; Markhard, Andrew L.; Kitami, Toshimori; Kovacs-Bogdan, Erika; Kamer, Kimberli J.; Udeshi, Namrata D.; Carr, Steven A.; Chaudhuri, Dipayan; Clapham, David E.; Li, Andrew A.; Calvo, Sarah E.; Goldberger, Olga; Mootha, Vamsi K.
      Science (Washington, DC, United States) (2013), 342 (6164), 1379-1382CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
      The mitochondrial uniporter is a highly selective calcium channel in the organelle's inner membrane. Its mol. components include the EF-hand-contg. calcium-binding proteins mitochondrial calcium uptake 1 (MICU1) and MICU2 and the pore-forming subunit mitochondrial calcium uniporter (MCU). We sought to achieve a full mol. characterization of the uniporter holocomplex (uniplex). Quant. mass spectrometry of affinity-purified uniplex recovered MICU1 and MICU2, MCU and its paralog MCUb, and a previously uncharacterized 10-kilodalton metazoan-specific single transmembrane domain protein designated EMRE ("essential MCU regulator"). In its absence, uniporter channel activity was lost despite intact MCU expression and oligomerization. EMRE was required for the interaction of MCU with MICU1 and MICU2. Hence, EMRE is essential for in vivo uniporter current and addnl. bridges the calcium-sensing role of MICU1 and MICU2 with the calcium-conducting role of MCU.
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    13. 13
      Perocchi, F.; Gohil, V. M.; Girgis, H. S.; Bao, X. R.; McCombs, J. E.; Palmer, A. E.; Mootha, V. K. MICU1 Encodes a Mitochondrial EF Hand Protein Required for Ca2+ uptake. Nature 2010, 467, 291– 296,  DOI: 10.1038/nature09358
      There is no corresponding record for this reference.
    14. 14
      Plovanich, M.; Bogorad, R. L.; Sancak, Y.; Kamer, K. J.; Strittmatter, L.; Li, A. A.; Girgis, H. S.; Kuchimanchi, S.; De Groot, J.; Speciner, L.; Taneja, N.; OShea, J.; Koteliansky, V.; Mootha, V. K. MICU2, a Paralog of MICU1, Resides within the Mitochondrial Uniporter Complex to Regulate Calcium Handling. PLoS One 2013, 8, e55785,  DOI: 10.1371/journal.pone.0055785
      There is no corresponding record for this reference.
    15. 15
      Csordás, G.; Golenár, T.; Seifert, E. L.; Kamer, K. J.; Sancak, Y.; Perocchi, F.; Moffat, C.; Weaver, D.; De la Fuente, S.; Bogorad, R.; Koteliansky, V.; Adijanto, J.; Mootha, V. K.; Hajnóczky, G. MICU1 Controls Both the Threshold and Cooperative Activation of the Mitochondrial Ca2+ Uniporter. Cell Metab. 2013, 17, 976– 987,  DOI: 10.1016/j.cmet.2013.04.020
      15
      MICU1 Controls Both the Threshold and Cooperative Activation of the Mitochondrial Ca2+ Uniporter
      Csordas, Gyorgy; Golenar, Tunde; Seifert, Erin L.; Kamer, Kimberli J.; Sancak, Yasemin; Perocchi, Fabiana; Moffat, Cynthia; Weaver, David; Perez, Sergio de la Fuente; Bogorad, Roman; Koteliansky, Victor; Adijanto, Jeffrey; Mootha, Vamsi K.; Hajnoczky, Gyorgy
      Cell Metabolism (2013), 17 (6), 976-987CODEN: CMEEB5; ISSN:1550-4131. (Elsevier Inc.)
      Mitochondrial Ca2+ uptake via the uniporter is central to cell metab., signaling, and survival. Recent studies identified MCU as the uniporter's likely pore and MICU1, an EF-hand protein, as its crit. regulator. How this complex decodes dynamic cytoplasmic [Ca2+] ([Ca2+]c) signals, to tune out small [Ca2+]c increases yet permit pulse transmission, remains unknown. We report that loss of MICU1 in mouse liver and cultured cells causes mitochondrial Ca2+ accumulation during small [Ca2+]c elevations but an attenuated response to agonist-induced [Ca2+]c pulses. The latter reflects loss of pos. cooperativity, likely via the EF-hands. MICU1 faces the intermembrane space and responds to [Ca2+]c changes. Prolonged MICU1 loss leads to an adaptive increase in matrix Ca2+ binding, yet cells show impaired oxidative metab. and sensitization to Ca2+ overload. Collectively, the data indicate that MICU1 senses the [Ca2+]c to establish the uniporter's threshold and gain, thereby allowing mitochondria to properly decode different inputs.
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    16. 16
      Mallilankaraman, K.; Cárdenas, C.; Doonan, P. J.; Chandramoorthy, H. C.; Irrinki, K. M.; Golenár, T.; Csordás, G.; Madireddi, P.; Yang, J.; Müller, M.; Miller, R.; Kolesar, J. E.; Molgó, J.; Kaufman, B.; Hajnóczky, G.; Foskett, J. K.; Madesh, M. MCUR1 is an Essential Component of Mitochondrial Ca2+ Uptake that Regulates Cellular Metabolism. Nat. Cell Biol. 2012, 14, 1336– 1343,  DOI: 10.1038/ncb2622
      16
      MCUR1 is an essential component of mitochondrial Ca2+ uptake that regulates cellular metabolism
      Mallilankaraman, Karthik; Cardenas, Cesar; Doonan, Patrick J.; Chandramoorthy, Harish C.; Irrinki, Krishna M.; Golenar, Tuende; Csordas, Gyoergy; Madireddi, Priyanka; Yang, Jun; Mueller, Marioly; Miller, Russell; Kolesar, Jill E.; Molgo, Jordi; Kaufman, Brett; Hajnoczky, Gyoergy; Foskett, J. Kevin; Madesh, Muniswamy
      Nature Cell Biology (2012), 14 (12), 1336-1343CODEN: NCBIFN; ISSN:1465-7392. (Nature Publishing Group)
      Ca2+ flux across the mitochondrial inner membrane regulates bioenergetics, cytoplasmic Ca2+ signals and activation of cell death pathways. Mitochondrial Ca2+ uptake occurs at regions of close apposition with intracellular Ca2+ release sites, driven by the inner membrane voltage generated by oxidative phosphorylation and mediated by a Ca2+ selective ion channel (MiCa; ref. ) called the uniporter whose complete mol. identity remains unknown. Mitochondrial calcium uniporter (MCU) was recently identified as the likely ion-conducting pore. In addn., MICU1 was identified as a mitochondrial regulator of uniporter-mediated Ca2+ uptake in HeLa cells. Here we identified CCDC90A, hereafter referred to as MCUR1 (mitochondrial calcium uniporter regulator 1), an integral membrane protein required for MCU-dependent mitochondrial Ca2+ uptake. MCUR1 binds to MCU and regulates ruthenium-red-sensitive MCU-dependent Ca2+ uptake. MCUR1 knockdown does not alter MCU localization, but abrogates Ca2+ uptake by energized mitochondria in intact and permeabilized cells. Ablation of MCUR1 disrupts oxidative phosphorylation, lowers cellular ATP and activates AMP kinase-dependent pro-survival autophagy. Thus, MCUR1 is a crit. component of a mitochondrial uniporter channel complex required for mitochondrial Ca2+ uptake and maintenance of normal cellular bioenergetics.
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    17. 17
      Patron, M.; Checchetto, V.; Raffaello, A.; Teardo, E.; VecellioReane, D.; Mantoan, M.; Granatiero, V.; Szabò, I.; DeStefani, D.; Rizzuto, R. MICU1 and MICU2 Finely Tune the Mitochondrial Ca2+ Uniporter by Exerting Opposite Effects on MCU Activity. Mol. Cell 2014, 53, 726– 737,  DOI: 10.1016/j.molcel.2014.01.013
      17
      MICU1 and MICU2 Finely Tune the Mitochondrial Ca2+ Uniporter by Exerting Opposite Effects on MCU Activity
      Patron, Maria; Checchetto, Vanessa; Raffaello, Anna; Teardo, Enrico; Vecellio Reane, Denis; Mantoan, Maura; Granatiero, Veronica; Szabo, Ildiko; De Stefani, Diego; Rizzuto, Rosario
      Molecular Cell (2014), 53 (5), 726-737CODEN: MOCEFL; ISSN:1097-2765. (Elsevier Inc.)
      Mitochondrial calcium accumulation was recently shown to depend on a complex composed of an inner-membrane channel (MCU and MCUb) and regulatory subunits (MICU1, MCUR1, and EMRE). A fundamental property of MCU is low activity at resting cytosolic Ca2+ concns., preventing deleterious Ca2+ cycling and organelle overload. Here we demonstrate that these properties are ensured by a regulatory heterodimer composed of two proteins with opposite effects - MICU1 and MICU2 - which in both purified lipid bilayers and in intact cells stimulate and inhibit MCU activity, resp. Both MICU1 and MICU2 are regulated by calcium through their EF-hand domains, thus accounting for the sigmoidal response of MCU to [Ca2+] in situ and allowing tight physiol. control. At low [Ca2+], the dominant effect of MICU2 largely shuts down MCU activity; at higher [Ca2+], the stimulatory effect of MICU1 allows the prompt response of mitochondria to Ca2+ signals generated in the cytoplasm.
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    18. 18
      Oxenoid, K.; Dong, Y.; Cao, C.; Cui, T.; Sancak, Y.; Markhard, A. L.; Grabarek, Z.; Kong, L.; Liu, Z.; Ouyang, B.; Cong, Y.; Mootha, V. K.; Chou, J. J. Architecture of the Mitochondrial Calcium Uniporter. Nature 2016, 533, 269– 273,  DOI: 10.1038/nature17656
      18
      Architecture of the mitochondrial calcium uniporter
      Oxenoid, Kirill; Dong, Ying; Cao, Chan; Cui, Tanxing; Sancak, Yasemin; Markhard, Andrew L.; Grabarek, Zenon; Kong, Liangliang; Liu, Zhijun; Ouyang, Bo; Cong, Yao; Mootha, Vamsi K.; Chou, James J.
      Nature (London, United Kingdom) (2016), 533 (7602), 269-273CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
      Mitochondria from many eukaryotic clades take up large amts. of Ca2+ via an inner membrane transporter called the mitochondrial calcium uniporter (MCU). Transport by MCU is membrane potential-dependent and sensitive to ruthenium red or its deriv., Ru360. Electrophysiol. studies have shown that MCU is an ion channel with remarkably high conductance and selectivity. Ca2+ entry into mitochondria is also known to activate the tricarboxylic acid cycle and seems to be crucial for matching the prodn. of ATP in mitochondria with its cytosolic demand. MCU is the pore-forming and Ca2+-conducting subunit of the uniporter holocomplex, but its primary sequence does not resemble any Ca2+ channel studied to date. Here, the authors report the structure of the pore domain of MCU from Caenorhabditis elegans, detd. using NMR spectroscopy and electron microscopy (EM). MCU is a homo-oligomer in which the 2nd transmembrane helix forms a hydrophilic pore across the membrane. The channel assembly represented a new soln. of ion channel architecture, and was stabilized by a coiled-coil motif protruding into the mitochondrial matrix. The crit. DXXE motif formed the pore entrance, which featured 2 carboxylate rings; based on the ring dimensions and functional mutagenesis, these rings appeared to form the selectivity filter. To the authors' knowledge, this is one of the largest membrane protein structures characterized by NMR, and provides a structural blueprint for understanding the function of this channel.
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    19. 19
      Bick, A. G.; Calvo, S. E.; Mootha, V. K. Evolutionary Diversity of the Mitochondrial Calcium Uniporter. Science 2012, 336, 886,  DOI: 10.1126/science.1214977
      There is no corresponding record for this reference.
    20. 20
      Baradaran, R.; Wang, C.; Siliciano, A. F.; Long, S. B. Cryo-EM Structures of Fungal and Metazoan Mitochondrial Calcium Uniporters. Nature 2018, 559, 580– 584,  DOI: 10.1038/s41586-018-0331-8
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      Cryo-EM structures of fungal and metazoan mitochondrial calcium uniporters
      Baradaran, Rozbeh; Wang, Chongyuan; Siliciano, Andrew Francis; Long, Stephen Barstow
      Nature (London, United Kingdom) (2018), 559 (7715), 580-584CODEN: NATUAS; ISSN:0028-0836. (Nature Research)
      The mitochondrial calcium uniporter (MCU) is a highly selective calcium channel and a major route of calcium entry into mitochondria. How the channel catalyzes ion permeation and achieves ion selectivity are not well understood, partly because MCU is thought to have a distinct architecture in comparison to other cellular channels. Here we report cryo-electron microscopy reconstructions of MCU channels from zebrafish and Cyphellophora europaea at 8.5 Å and 3.2 Å resolns., resp. In contrast to a previous report of pentameric stoichiometry for MCU, both channels are tetramers. The at. model of C. europaea MCU shows that a conserved WDXXEP signature sequence forms the selectivity filter, in which calcium ions are arranged in single file. Coiled-coil legs connect the pore to N-terminal domains in the mitochondrial matrix. In C. europaea MCU, the N-terminal domains assemble as a dimer of dimers; in zebrafish MCU, they form an asym. crescent. The structures define principles that underlie ion permeation and calcium selectivity in this unusual channel.
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    21. 21
      Fan, C.; Fan, M.; Orlando, B. J.; Fastman, N. M.; Zhang, J.; Xu, Y.; Chambers, M. G.; Xu, X.; Perry, K.; Liao, M.; Feng, L. X-Ray and Cryo-EM Structures of the Mitochondrial Calcium Uniporter. Nature 2018, 559, 575– 579,  DOI: 10.1038/s41586-018-0330-9
      21
      X-ray and cryo-EM structures of the mitochondrial calcium uniporter
      Fan, Chao; Fan, Minrui; Orlando, Benjamin J.; Fastman, Nathan M.; Zhang, Jinru; Xu, Yan; Chambers, Melissa G.; Xu, Xiaofang; Perry, Kay; Liao, Maofu; Feng, Liang
      Nature (London, United Kingdom) (2018), 559 (7715), 575-579CODEN: NATUAS; ISSN:0028-0836. (Nature Research)
      Mitochondrial Ca2+ uptake is crit. for regulating ATP prodn., intracellular Ca2+ signaling, and cell death. This uptake is mediated by a highly selective Ca2+ channel called the mitochondrial calcium uniporter (MCU). Here, we detd. the structures of the pore-forming MCU proteins from 2 fungi (Fusarium graminearum and Metarhizium acridum) by x-ray crystallog. and single-particle cryo-electron microscopy (cryo-EM). The stoichiometry, overall architecture, and individual subunit structure differed markedly from those described in the recent NMR structure of Caenorhabditis elegans MCU. We obsd. a dimer-of-dimer architecture across species and chem. environments, which was corroborated by biochem. expts. Structural analyses and functional characterization uncovered the roles of key residues in the pore. These results revealed a new ion channel architecture, provide insights into Ca2+ coordination, selectivity, and conduction, and established a structural framework for understanding the mechanism of MCU function.
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    22. 22
      Nguyen, N. X.; Armache, J. P.; Lee, C.; Yang, Y.; Zeng, W.; Mootha, V. K.; Cheng, Y.; Bai, X.; Jiang, Y. Cryo-EM Structure of a Fungal Mitochondrial Calcium Uniporter. Nature 2018, 559, 570– 574,  DOI: 10.1038/s41586-018-0333-6
      22
      Cryo-EM structure of a fungal mitochondrial calcium uniporter
      Nguyen, Nam X.; Armache, Jean-Paul; Lee, Changkeun; Yang, Yi; Zeng, Weizhong; Mootha, Vamsi K.; Cheng, Yifan; Bai, Xiao-chen; Jiang, Youxing
      Nature (London, United Kingdom) (2018), 559 (7715), 570-574CODEN: NATUAS; ISSN:0028-0836. (Nature Research)
      The mitochondrial calcium uniporter (MCU) is a highly selective calcium channel localized to the inner mitochondrial membrane. Here, we describe the structure of an MCU ortholog from the fungus Neosartorya fischeri (NfMCU) detd. to 3.8 Å resoln. by phase-plate cryo-electron microscopy. The channel is a homotetramer with two-fold symmetry in its amino-terminal domain (NTD) that adopts a similar structure to that of human MCU. The NTD assembles as a dimer of dimers to form a tetrameric ring that connects to the transmembrane domain through an elongated coiled-coil domain. The ion-conducting pore domain maintains four-fold symmetry, with the selectivity filter positioned at the start of the pore-forming TM2 helix. The aspartate and glutamate sidechains of the conserved DIME motif are oriented towards the central axis and sepd. by one helical turn. The structure of NfMCU offers insights into channel assembly, selective calcium permeation, and inhibitor binding.
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    23. 23
      Yoo, J.; Wu, M.; Yin, Y.; Herzik, M. A.; Lander, G. C.; Lee, S.-Y. Cryo-EM Structure of a Mitochondrial Calcium Uniporter. Science 2018, 361, 506– 511,  DOI: 10.1126/science.aar4056
      23
      Cryo-EM structure of a mitochondrial calcium uniporter
      Yoo, Jiho; Wu, Mengyu; Yin, Ying; Herzik, Mark A., Jr; Lander, Gabriel C.; Lee, Seok-Yong
      Science (Washington, DC, United States) (2018), 361 (6401), 506-511CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
      Calcium transport plays an important role in regulating mitochondrial physiol. and pathophysiol. The mitochondrial calcium uniporter (MCU) is a calcium-selective ion channel that is the primary mediator for calcium uptake into the mitochondrial matrix. Here, we present the cryo-electron microscopy structure of the full-length MCU from Neurospora crassa to an overall resoln. of ∼3.7 angstroms. Our structure reveals a tetrameric architecture, with the sol. and transmembrane domains adopting different sym. arrangements within the channel. The conserved W-D-Φ-Φ-E-P-V-T-Y sequence motif of MCU pore forms a selectivity filter comprising two acidic rings sepd. by one helical turn along the central axis of the channel pore. The structure combined with mutagenesis gives insight into the basis of calcium recognition.
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    24. 24
      Hoffman, N. E.; Chandramoorthy, H. C.; Shamugapriya, S.; Zhang, X.; Rajan, S.; Mallilankaraman, K.; Gandhirajan, R. K.; Vagnozzi, R. J.; Ferrer, L. M.; Sreekrishnanilayam, K.; Natarajaseenivasan, K.; Vallem, S.; Force, T.; Choi, E. T.; Cheung, J. Y.; Madesh, M. MICU1 Motifs Define Mitochondrial Calcium Uniporter Binding and Activity. Cell Rep. 2013, 5, 1576– 1588,  DOI: 10.1016/j.celrep.2013.11.026
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      MICU1 Motifs Define Mitochondrial Calcium Uniporter Binding and Activity
      Hoffman, Nicholas E.; Chandramoorthy, Harish C.; Shamugapriya, Santhanam; Zhang, Xueqian; Rajan, Sudarsan; Mallilankaraman, Karthik; Gandhirajan, Rajesh Kumar; Vagnozzi, Ronald J.; Ferrer, Lucas M.; Sreekrishnanilayam, Krishnalatha; Natarajaseenivasan, Kalimuthusamy; Vallem, Sandhya; Force, Thomas; Choi, Eric T.; Cheung, Joseph Y.; Madesh, Muniswamy
      Cell Reports (2013), 5 (6), 1576-1588CODEN: CREED8; ISSN:2211-1247. (Cell Press)
      Resting mitochondrial matrix Ca2+ is maintained through a mitochondrial calcium uptake 1 (MICU1)-established threshold inhibition of mitochondrial calcium uniporter (MCU) activity. It is not known how MICU1 interacts with MCU to establish this Ca2+ threshold for mitochondrial Ca2+ uptake and MCU activity. Here, we show that MICU1 localizes to the mitochondrial matrix side of the inner mitochondrial membrane and MICU1/MCU binding is detd. by a MICU1 N-terminal polybasic domain and two interacting coiled-coil domains of MCU. Further investigation reveals that MICU1 forms homo-oligomers, and this oligomerization is independent of the polybasic region. However, the polybasic region confers MICU1 oligomeric binding to MCU and controls mitochondrial Ca2+ current (IMCU). Moreover, MICU1 EF hands regulate MCU channel activity, but do not det. MCU binding. Loss of MICU1 promotes MCU activation leading to oxidative burden and a halt to cell migration. These studies establish a mol. mechanism for MICU1 control of MCU-mediated mitochondrial Ca2+ accumulation, and dysregulation of this mechanism probably enhances vascular dysfunction.
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    25. 25
      Petrungaro, C.; Zimmermann, K. M.; Küttner, V.; Fischer, M.; Dengjel, J.; Bogeski, I.; Riemer, J. The Ca2+-Dependent Release of the Mia40-Induced MICU1-MICU2 Dimer from MCU Regulates Mitochondrial Ca2+ Uptake. Cell Metab. 2015, 22, 721– 733,  DOI: 10.1016/j.cmet.2015.08.019
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      The Ca2+-Dependent Release of the Mia40-Induced MICU1-MICU2 Dimer from MCU Regulates Mitochondrial Ca2+ Uptake
      Petrungaro, Carmelina; Zimmermann, Katharina M.; Kuettner, Victoria; Fischer, Manuel; Dengjel, Joern; Bogeski, Ivan; Riemer, Jan
      Cell Metabolism (2015), 22 (4), 721-733CODEN: CMEEB5; ISSN:1550-4131. (Elsevier Inc.)
      The essential oxidoreductase Mia40/CHCHD4 mediates disulfide bond formation and protein folding in the mitochondrial intermembrane space. Here, we investigated the interactome of Mia40 thereby revealing links between thiol-oxidn. and apoptosis, energy metab., and Ca2+ signaling. Among the interaction partners of Mia40 is MICU1-the regulator of the mitochondrial Ca2+ uniporter (MCU), which transfers Ca2+ across the inner membrane. We examd. the biogenesis of MICU1 and find that Mia40 introduces an intermol. disulfide bond that links MICU1 and its inhibitory paralog MICU2 in a heterodimer. Absence of this disulfide bond results in increased receptor-induced mitochondrial Ca2+ uptake. In the presence of the disulfide bond, MICU1-MICU2 heterodimer binding to MCU is controlled by Ca2+ levels: the dimer assocs. with MCU at low levels of Ca2+ and dissocs. upon high Ca2+ concns. Our findings support a model in which mitochondrial Ca2+ uptake is regulated by a Ca2+-dependent remodeling of the uniporter complex.
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    26. 26
      Lee, Y.; Min, C. K.; Kim, T. G.; Song, H. K.; Lim, Y.; Kim, D.; Shin, K.; Kang, M.; Kang, J. Y.; Youn, H.-S.; Lee, J.-G.; An, J. Y.; Park, K. R.; Lim, J. J.; Kim, J. H.; Kim, J. H.; Park, Z. Y.; Kim, Y.-S.; Wang, J.; Kim, D. H.; Eom, S. H. Structure and Function of the N-Terminal Domain of the Human Mitochondrial Calcium Uniporter. EMBO Rep. 2015, 16, 1318– 1333,  DOI: 10.15252/embr.201540436
      26
      Structure and function of the N-terminal domain of the human mitochondrial calcium uniporter
      Lee, Youngjin; Min, Choon Kee; Kim, Tae Gyun; Song, Hong Ki; Lim, Yunki; Kim, Dongwook; Shin, Kahee; Kang, Moonkyung; Kang, Jung Youn; Youn, Hyung-Seop; Lee, Jung-Gyu; An, Jun Yop; Park, Kyoung Ryoung; Lim, Jia Jia; Kim, Ji Hun; Kim, Ji Hye; Park, Zee Yong; Kim, Yeon-Soo; Wang, Jimin; Kim, Do Han; Eom, Soo Hyun
      EMBO Reports (2015), 16 (10), 1318-1333CODEN: ERMEAX; ISSN:1469-221X. (Wiley-VCH Verlag GmbH & Co. KGaA)
      The mitochondrial calcium uniporter (MCU) is responsible for mitochondrial calcium uptake and homeostasis. It is also a target for the regulation of cellular anti-/pro-apoptosis and necrosis by several oncogenes and tumor suppressors. Herein, we report the crystal structure of the MCU N-terminal domain (NTD) at a resoln. of 1.50 Å in a novel fold and the S92A MCU mutant at 2.75 Å resoln.; the residue S92 is a predicted CaMKII phosphorylation site. The assembly of the mitochondrial calcium uniporter complex (uniplex) and the interaction with the MCU regulators such as the mitochondrial calcium uptake-1 and mitochondrial calcium uptake-2 proteins (MICU1 and MICU2) are not affected by the deletion of MCU NTD. However, the expression of the S92A mutant or a NTD deletion mutant failed to restore mitochondrial Ca2+ uptake in a stable MCU knockdown HeLa cell line and exerted dominant-neg. effects in the wild-type MCU-expressing cell line. These results suggest that the NTD of MCU is essential for the modulation of MCU function, although it does not affect the uniplex formation.
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    27. 27
      Lee, S. K.; Shanmughapriya, S.; Mok, M. C. Y.; Dong, Z.; Tomar, D.; Carvalho, E.; Rajan, S.; Junop, M. S.; Madesh, M.; Stathopulos, P. B. Structural Insights into Mitochondrial Calcium Uniporter Regulation by Divalent Cations. Cell Chem. Biol. 2016, 23, 1157– 1169,  DOI: 10.1016/j.chembiol.2016.07.012
      27
      Structural Insights into Mitochondrial Calcium Uniporter Regulation by Divalent Cations
      Lee, Samuel K.; Shanmughapriya, Santhanam; Mok, Mac C. Y.; Dong, Zhiwei; Tomar, Dhanendra; Carvalho, Edmund; Rajan, Sudarsan; Junop, Murray S.; Madesh, Muniswamy; Stathopulos, Peter B.
      Cell Chemical Biology (2016), 23 (9), 1157-1169CODEN: CCBEBM; ISSN:2451-9448. (Cell Press)
      Calcium (Ca2+) flux into the matrix is tightly controlled by the mitochondrial Ca2+ uniporter (MCU) due to vital roles in cell death and bioenergetics. However, the precise at. mechanisms of MCU regulation remain unclear. Here, we solved the crystal structure of the N-terminal matrix domain of human MCU, revealing a β-grasp-like fold with a cluster of neg. charged residues that interacts with divalent cations. Binding of Ca2+ or Mg2+ destabilizes and shifts the self-assocn. equil. of the domain toward monomer. Mutational disruption of the acidic face weakens oligomerization of the isolated matrix domain and full-length human protein similar to cation binding and markedly decreases MCU activity. Moreover, mitochondrial Mg2+ loading or blockade of mitochondrial Ca2+ extrusion suppresses MCU Ca2+-uptake rates. Collectively, our data reveal that the β-grasp-like matrix region harbors an MCU-regulating acidic patch that inhibits human MCU activity in response to Mg2+ and Ca2+ binding.
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    28. 28
      Tomar, D.; Dong, Z.; Shanmughapriya, S.; Koch, D. A.; Thomas, T.; Hoffman, N. E.; Timbalia, S. A.; Goldman, S. J.; Breves, S. L.; Corbally, D. P.; Nemani, N.; Fairweather, J. P.; Cutri, A. R.; Zhang, X.; Song, J.; Jaña, F.; Huang, J.; Barrero, C.; Rabinowitz, J. E.; Luongo, T. S.; Schumacher, S. M.; Rockman, M. E.; Dietrich, A.; Merali, S.; Caplan, J.; Stathopulos, P. B.; Ahima, R. S.; Cheung, J. Y.; Houser, S. R.; Koch, W. J.; Patel, V.; Gohil, V. M.; Elrod, J. W.; Rajan, S.; Madesh, M. MCUR1 is a Scaffold Factor for the MCU Complex Function and Promotes Mitochondrial Bioenergetics. Cell Rep. 2016, 15, 1673– 1685,  DOI: 10.1016/j.celrep.2016.04.050
      28
      MCUR1 Is a Scaffold Factor for the MCU Complex Function and Promotes Mitochondrial Bioenergetics
      Tomar, Dhanendra; Dong, Zhiwei; Shanmughapriya, Santhanam; Koch, Diana A.; Thomas, Toby; Hoffman, Nicholas E.; Timbalia, Shrishiv A.; Goldman, Samuel J.; Breves, Sarah L.; Corbally, Daniel P.; Nemani, Neeharika; Fairweather, Joseph P.; Cutri, Allison R.; Zhang, Xueqian; Song, Jianliang; Jana, Fabian; Huang, Jianhe; Barrero, Carlos; Rabinowitz, Joseph E.; Luongo, Timothy S.; Schumacher, Sarah M.; Rockman, Michael E.; Dietrich, Alexander; Merali, Salim; Caplan, Jeffrey; Stathopulos, Peter; Ahima, Rexford S.; Cheung, Joseph Y.; Houser, Steven R.; Koch, Walter J.; Patel, Vickas; Gohil, Vishal M.; Elrod, John W.; Rajan, Sudarsan; Madesh, Muniswamy
      Cell Reports (2016), 15 (8), 1673-1685CODEN: CREED8; ISSN:2211-1247. (Cell Press)
      Mitochondrial Ca2+ Uniporter (MCU)-dependent mitochondrial Ca2+ uptake is the primary mechanism for increasing matrix Ca2+ in most cell types. However, a limited understanding of the MCU complex assembly impedes the comprehension of the precise mechanisms underlying MCU activity. Here, we report that mouse cardiomyocytes and endothelial cells lacking MCU regulator 1 (MCUR1) have severely impaired [Ca2+]m uptake and IMCU current. MCUR1 binds to MCU and EMRE and function as a scaffold factor. Our protein binding analyses identified the minimal, highly conserved regions of coiled-coil domain of both MCU and MCUR1 that are necessary for heterooligomeric complex formation. Loss of MCUR1 perturbed MCU heterooligomeric complex and functions as a scaffold factor for the assembly of MCU complex. Vascular endothelial deletion of MCU and MCUR1 impaired mitochondrial bioenergetics, cell proliferation, and migration but elicited autophagy. These studies establish the existence of a MCU complex that assembles at the mitochondrial integral membrane and regulates Ca2+-dependent mitochondrial metab.
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    29. 29
      Gunter, T. E.; Gunter, K. K.; Sheu, S.-S.; Gavin, C. E. Mitochondrial Calcium Transport: Physiological and Pathological Relevance. Am. J. Physiol. 1994, 267, C313– 319,  DOI: 10.1152/ajpcell.1994.267.2.C313
      There is no corresponding record for this reference.
    30. 30
      Hajnóczky, G.; Robb-Gaspers, L. D.; Seitz, M. B.; Thomas, A. P. Decoding of Cytosolic Calcium Oscillations in the Mitochondria. Cell 1995, 82, 415– 424,  DOI: 10.1016/0092-8674(95)90430-1
      There is no corresponding record for this reference.
    31. 31
      Denton, R.; McCormack, J. G. Ca2+ as a Second Messenger Within Mitochondria Of The Heart And Other Tissues. Annu. Rev. Physiol. 1990, 52, 451– 466,  DOI: 10.1146/annurev.ph.52.030190.002315
      There is no corresponding record for this reference.
    32. 32
      Santo-Domingo, J.; Demaurex, N. Calcium Uptake Mechanisms of Mitochondria. Biochim. Biophys. Acta, Bioenerg. 2010, 1797, 907– 912,  DOI: 10.1016/j.bbabio.2010.01.005
      32
      Calcium uptake mechanisms of mitochondria
      Santo-Domingo, Jaime; Demaurex, Nicolas
      Biochimica et Biophysica Acta, Bioenergetics (2010), 1797 (6-7), 907-912CODEN: BBBEB4; ISSN:0005-2728. (Elsevier B. V.)
      A review. The ability of mitochondria to capture Ca2+ ions has important functional implications for cells, because mitochondria shape cellular Ca2+ signals by acting as a Ca2+ buffer and respond to Ca2+ elevations either by increasing the cell energy supply or by triggering the cell death program of apoptosis. A mitochondrial Ca2+ channel known as the uniporter drives the rapid and massive entry of Ca2+ ions into mitochondria. The uniporter operates at high, micromolar cytosolic Ca2+ concns. that are only reached transiently in cells, near Ca2+ release channels. Mitochondria can also take up Ca2+ at low, nanomolar concns., but this high affinity mode of Ca2+ uptake is not well characterized. Recently, the leucine zipper-EF hand-contg. transmembrane region (Letm1) was proposed to be an electrogenic 1:1 mitochondrial Ca2+/H+ antiporter that drives the uptake of Ca2+ into mitochondria at nanomolar cytosolic Ca2+ concns. Here, the authors review the properties of the Ca2+ import systems of mitochondria and discuss how Ca2+ uptake via an electrogenic 1:1 Ca2+/H+ antiport challenges the current thinking of the mitochondrial Ca2+ uptake mechanism.
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    33. 33
      Bernardi, P. Mitochondrial Transport of Cations: Channels, Exchangers, and Permeability Transition. Physiol. Rev. 1999, 79, 1127– 1155,  DOI: 10.1152/physrev.1999.79.4.1127
      33
      Mitochondrial transport of cations: channels, exchangers, and permeability transition
      Bernardi, Paolo
      Physiological Reviews (1999), 79 (4), 1127-1155CODEN: PHREA7; ISSN:0031-9333. (American Physiological Society)
      A review with 372 refs. This review provides a selective history of how studies of mitochondrial cation transport (K+, Na+, Ca2+) developed in relation to the major themes of research in bioenergetics. It then covers in some detail specific transport pathways for these cations, and it introduces and discusses open problems about their nature and physiol. function, particularly in relation to vol. regulation and Ca2+ homeostasis. The review should provide the basic elements needed to understand both earlier mitochondrial literature and current problems assocd. with mitochondrial transport of cations and hopefully will foster new interest in the mol. definition of mitochondrial cation channels and exchangers as well as their roles in cell physiol.
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    34. 34
      Shanmughapriya, S.; Rajan, S.; Hoffman, N. E.; Higgins, A. M.; Tomar, D.; Nemani, N.; Hines, K. J.; Smith, D. J.; Eguchi, A.; Vallem, S.; Shaikh, F.; Cheung, M.; Leonard, N. J.; Stolakis, R. S.; Wolfers, M. P.; Ibetti, J.; Chuprun, J. K.; Jog, N. R.; Houser, S. R.; Koch, W. J.; Elrod, J. W.; Madesh, M. SPG7 is an Essential and Conserved Component of the Mitochondrial Permeability Transition Pore. Mol. Cell 2015, 60, 47– 62,  DOI: 10.1016/j.molcel.2015.08.009
      There is no corresponding record for this reference.
    35. 35
      Orrenius, S.; Zhivotovsky, B.; Nicotera, P. Regulation of Cell Death: The Calcium-Apoptosis Link. Nat. Rev. Mol. Cell Biol. 2003, 4, 552– 565,  DOI: 10.1038/nrm1150
      35
      Calcium: Regulation of cell death: the calcium-apoptosis link
      Orrenius, Sten; Zhivotovsky, Boris; Nicotera, Pierluigi
      Nature Reviews Molecular Cell Biology (2003), 4 (7), 552-565CODEN: NRMCBP; ISSN:1471-0072. (Nature Publishing Group)
      A review and discussion. To live or to die. This crucial question eloquently reflects the dual role of Ca2+ in living organisms, as a survival factor or as a ruthless killer. It has long been known that Ca2+ signals govern a host of vital cell functions and so are necessary for cell survival. However, more recently it has become clear that cellular Ca2+ overload, or perturbation of intracellular Ca2+ compartmentalization, can cause cytotoxicity and trigger either apoptotic or necrotic cell death.
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    36. 36
      De Stefani, D.; Rizzuto, R.; Pozzan, T. Enjoy the Trip: Calcium in Mitochondria Back and Forth. Annu. Rev. Biochem. 2016, 85, 161– 192,  DOI: 10.1146/annurev-biochem-060614-034216
      36
      Enjoy the Trip: Calcium in Mitochondria Back and Forth
      De Stefani, Diego; Rizzuto, Rosario; Pozzan, Tullio
      Annual Review of Biochemistry (2016), 85 (), 161-192CODEN: ARBOAW; ISSN:0066-4154. (Annual Reviews)
      A review. In the last 5 years, most of the mols. that control mitochondrial Ca2+ homeostasis have been finally identified. Mitochondrial Ca2+ uptake is mediated by the Mitochondrial Calcium Uniporter (MCU) complex, a macromol. structure that guarantees Ca2+ accumulation inside mitochondrial matrix upon increases in cytosolic Ca2+. Conversely, Ca2+ release is under the control of the Na+/Ca2+ exchanger, encoded by the NCLX gene, and of a H+/Ca2+ antiporter, whose identity is still debated. The low affinity of the MCU complex, coupled to the activity of the efflux systems, protects cells from continuous futile cycles of Ca2+ across the inner mitochondrial membrane and consequent massive energy dissipation. In this review, we discuss the basic principles that govern mitochondrial Ca2+ homeostasis and the methods used to investigate the dynamics of Ca2+ concn. within the organelles. We discuss the functional and structural role of the different mols. involved in mitochondrial Ca2+ handling and their pathophysiol. role.
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    37. 37
      Marchi, S.; Pinton, P. The Mitochondrial Calcium Uniporter Complex: Molecular Components, Structure and Physiopathological Implications. J. Physiol. 2014, 592, 829– 839,  DOI: 10.1113/jphysiol.2013.268235
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      The mitochondrial calcium uniporter complex: molecular components, structure and physiopathological implications
      Marchi, Saverio; Pinton, Paolo
      Journal of Physiology (Oxford, United Kingdom) (2014), 592 (5), 829-839CODEN: JPHYA7; ISSN:0022-3751. (Wiley-Blackwell)
      A review. Although it has long been known that mitochondria take up Ca2+, the mol. identities of the channels and transporters involved in this process were revealed only recently. Here, we discuss the recent work that has led to the characterization of the mitochondrial calcium uniporter complex, which includes the channel-forming subunit MCU (mitochondrial calcium uniporter) and its regulators MICU1, MICU2, MCUb, EMRE, MCUR1 and miR-25. We review not only the biochem. identities and structures of the proteins required for mitochondrial Ca2+ uptake but also their implications in different physiopathol. contexts.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXjtlOkuro%253D&md5=5f8c78976edf6e35e484a9a40e107784
    38. 38
      Luongo, T. S.; Lambert, J. P.; Yuan, A.; Zhang, X.; Gross, P.; Song, J.; Shanmughapriya, S.; Gao, E.; Jain, M.; Houser, S. R.; Koch, W. J.; Cheung, J. Y.; Madesh, M.; Elrod, J. W. The Mitochondrial Calcium Uniporter Matches Energetic Supply with Cardiac Workload during Stress and Modulates Permeability Transition. Cell Rep. 2015, 12, 23– 34,  DOI: 10.1016/j.celrep.2015.06.017
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      The Mitochondrial Calcium Uniporter Matches Energetic Supply with Cardiac Workload during Stress and Modulates Permeability Transition
      Luongo, Timothy S.; Lambert, Jonathan P.; Yuan, Ancai; Zhang, Xueqian; Gross, Polina; Song, Jianliang; Shanmughapriya, Santhanam; Gao, Erhe; Jain, Mohit; Houser, Steven R.; Koch, Walter J.; Cheung, Joseph Y.; Madesh, Muniswamy; Elrod, John W.
      Cell Reports (2015), 12 (1), 23-34CODEN: CREED8; ISSN:2211-1247. (Cell Press)
      Cardiac contractility is mediated by a variable flux in intracellular calcium (Ca2+), thought to be integrated into mitochondria via the mitochondrial calcium uniporter (MCU) channel to match energetic demand. Here, we examine a conditional, cardiomyocyte-specific, mutant mouse lacking Mcu, the pore-forming subunit of the MCU channel, in adulthood. Mcu-/- mice display no overt baseline phenotype and are protected against mCa2+ overload in an in vivo myocardial ischemia-reperfusion injury model by preventing the activation of the mitochondrial permeability transition pore, decreasing infarct size, and preserving cardiac function. In addn., we find that Mcu-/- mice lack contractile responsiveness to acute β-adrenergic receptor stimulation and in parallel are unable to activate mitochondrial dehydrogenases and display reduced bioenergetic reserve capacity. These results support the hypothesis that MCU may be dispensable for homeostatic cardiac function but required to modulate Ca2+-dependent metab. during acute stress.
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    39. 39
      Kwong, J. Q.; Lu, X.; Correll, R. N.; Schwanekamp, J. A.; Vagnozzi, R. J.; Sargent, M. A.; York, A. J.; Zhang, J.; Bers, D. M.; Molkentin, J. D. The Mitochondrial Calcium Uniporter Selectively Matches Metabolic Output to Acute Contractile Stress in the Heart. Cell Rep. 2015, 12, 15– 22,  DOI: 10.1016/j.celrep.2015.06.002
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      The Mitochondrial Calcium Uniporter Selectively Matches Metabolic Output to Acute Contractile Stress in the Heart
      Kwong, Jennifer Q.; Lu, Xiyuan; Correll, Robert N.; Schwanekamp, Jennifer A.; Vagnozzi, Ronald J.; Sargent, Michelle A.; York, Allen J.; Zhang, Jianyi; Bers, Donald M.; Molkentin, Jeffery D.
      Cell Reports (2015), 12 (1), 15-22CODEN: CREED8; ISSN:2211-1247. (Cell Press)
      In the heart, augmented Ca2+ fluxing drives contractility and ATP generation through mitochondrial Ca2+ loading. Pathol. mitochondrial Ca2+ overload with ischemic injury triggers mitochondrial permeability transition pore (MPTP) opening and cardiomyocyte death. Mitochondrial Ca2+ uptake is primarily mediated by the mitochondrial Ca2+ uniporter (MCU). Here, we generated mice with adult and cardiomyocyte-specific deletion of Mcu, which produced mitochondria refractory to acute Ca2+ uptake, with impaired ATP prodn., and inhibited MPTP opening upon acute Ca2+ challenge. Mice lacking Mcu in the adult heart were also protected from acute ischemia-reperfusion injury. However, resting/basal mitochondrial Ca2+ levels were normal in hearts of Mcu-deleted mice, and mitochondria lacking MCU eventually loaded with Ca2+ after stress stimulation. Indeed, Mcu-deleted mice were unable to immediately sprint on a treadmill unless warmed up for 30 min. Hence, MCU is a dedicated regulator of short-term mitochondrial Ca2+ loading underlying a "fight-or-flight" response that acutely matches cardiac workload with ATP prodn.
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    40. 40
      Medvedeva, Y. V.; Weiss, J. H.; Weiss, J. H.; Medvedeva, Y. V. Intramitochondrial Zn2+ Accumulation via the Ca2+ Uniporter Contributes to Acute Ischemic Neurodegeneration. Neurobiol. Dis. 2014, 68, 137– 144,  DOI: 10.1016/j.nbd.2014.04.011
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      Intramitochondrial Zn2 + accumulation via the Ca2 + uniporter contributes to acute ischemic neurodegeneration
      Medvedeva, Yuliya V.; Weiss, John H.
      Neurobiology of Disease (2014), 68 (), 137-144CODEN: NUDIEM; ISSN:0969-9961. (Elsevier Inc.)
      Ca2 + and Zn2 + have both been implicated in the induction of acute ischemic neurodegeneration. We recently examd. changes in intracellular Zn2 + and Ca2 + in CA1 pyramidal neurons subjected to oxygen glucose deprivation (OGD), and found that Zn2 + rises precede and contribute to the onset of terminal Ca2 + rises ("Ca2 + deregulation"), which are causatively linked to a lethal loss of membrane integrity. The present study seeks to examine the specific role of intramitochondrial Zn2 + accumulation in ischemic injury, using blockers of the mitochondrial Ca2 + uniporter (MCU), through which both Zn2 + and Ca2 + appear able to enter the mitochondrial matrix. In physiol. extracellular Ca2 +, treatment with the MCU blocker, Ruthenium Red (RR), accelerated the Ca2 + deregulation, most likely by disrupting mitochondrial Ca2 + buffering and thus accelerating the lethal cytosolic Ca2 + overload. However, when intracellular Ca2 + overload was slowed, either by adding blockers of major Ca2 + entry channels or by lowering the concn. of Ca2 + in the extracellular buffer, Ca2 + deregulation was delayed, and under these conditions either Zn2 + chelation or MCU blockade resulted in similar further delays of the Ca2 + deregulation. In parallel studies using the reactive oxygen species (ROS) indicator, hydroethidine, lowering Ca2 + surprisingly accelerated OGD induced ROS generation, and in these low Ca2 + conditions, either Zn2 + chelation or MCU block slowed the ROS generation. These studies suggest that, during acute ischemia, Zn2 + entry into mitochondria via the MCU induces mitochondrial dysfunction (including ROS generation) that occurs upstream of, and contributes to the terminal Ca2 + deregulation.
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    41. 41
      Giorgi, C.; Agnoletto, C.; Bononi, A.; Bonora, M.; De Marchi, E.; Marchi, S.; Missiroli, S.; Patergnani, S.; Poletti, F.; Rimessi, A.; Suski, J. M.; Wieckowski, M. R.; Pinton, P. Mitochondrial Calcium Homeostasis as Potential Target for Mitochondrial Medicine. Mitochondrion 2012, 12, 77– 85,  DOI: 10.1016/j.mito.2011.07.004
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      Mitochondrial calcium homeostasis as potential target for mitochondrial medicine
      Giorgi, Carlotta; Agnoletto, Chiara; Bononi, Angela; Bonora, Massimo; De Marchi, Elena; Marchi, Saverio; Missiroli, Sonia; Patergnani, Simone; Poletti, Federica; Rimessi, Alessandro; Suski, Jan M.; Wieckowski, Mariusz R.; Pinton, Paolo
      Mitochondrion (2012), 12 (1), 77-85CODEN: MITOCN; ISSN:1567-7249. (Elsevier B.V.)
      A review. Mitochondria are crucial in different intracellular pathways of signal transduction. Mitochondria are capable of decoding a variety of extracellular stimuli into markedly different intracellular actions, ranging from energy prodn. to cell death. The fine modulation of mitochondrial calcium (Ca2+) homeostasis plays a fundamental role in many of the processes involving this organelle. When mitochondrial Ca2+ homeostasis is compromised, different pathol. conditions can occur, depending on the cell type involved. Recent data have shed light on the mol. identity of the main proteins involved in the handling of mitochondrial Ca2+ traffic, opening fascinating and ambitious new avenues for mitochondria-based pharmacol. strategies.
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    42. 42
      Arduino, D. M.; Wettmarshausen, J.; Vais, H.; Navas-Navarro, P.; Cheng, Y.; Leimpek, A.; Ma, Z.; Delrio-Lorenzo, A.; Giordano, A.; Garcia-Perez, C.; Médard, G.; Kuster, B.; García-Sancho, J.; Mokranjac, D.; Foskett, J. K.; Alonso, M. T.; Perocchi, F. Systematic Identification of MCU Modulators by Orthogonal Interspecies Chemical Screening. Mol. Cell 2017, 67, 711– 723,  DOI: 10.1016/j.molcel.2017.07.019
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      Systematic Identification of MCU Modulators by Orthogonal Interspecies Chemical Screening
      Arduino, Daniela M.; Wettmarshausen, Jennifer; Vais, Horia; Navas-Navarro, Paloma; Cheng, Yiming; Leimpek, Anja; Ma, Zhongming; Delrio-Lorenzo, Alba; Giordano, Andrea; Garcia-Perez, Cecilia; Medard, Guillaume; Kuster, Bernhard; Garcia-Sancho, Javier; Mokranjac, Dejana; Foskett, J. Kevin; Alonso, M. Teresa; Perocchi, Fabiana
      Molecular Cell (2017), 67 (4), 711-723.e7CODEN: MOCEFL; ISSN:1097-2765. (Elsevier Inc.)
      The mitochondrial calcium uniporter complex is essential for calcium (Ca2+) uptake into mitochondria of all mammalian tissues, where it regulates bioenergetics, cell death, and Ca2+ signal transduction. Despite its involvement in several human diseases, we currently lack pharmacol. agents for targeting uniporter activity. Here we introduce a high-throughput assay that selects for human MCU-specific small-mol. modulators in primary drug screens. Using isolated yeast mitochondria, reconstituted with human MCU, its essential regulator EMRE, and aequorin, and exploiting a D-lactate- and mannitol/sucrose-based bioenergetic shunt that greatly minimizes false-pos. hits, we identify mitoxantrone out of more than 600 clin. approved drugs as a direct selective inhibitor of human MCU. We validate mitoxantrone in orthogonal mammalian cell-based assays, demonstrating that our screening approach is an effective and robust tool for MCU-specific drug discovery and, more generally, for the identification of compds. that target mitochondrial functions.
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    43. 43
      Kon, N.; Murakoshi, M.; Isobe, A.; Kagechika, K.; Miyoshi, N.; Nagayama, T. DS16570511 is a Small-Molecule Inhibitor of the Mitochondrial Calcium Uniporter. Cell Death Discov. 2017, 3, 17045,  DOI: 10.1038/cddiscovery.2017.45
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      DS16570511 is a small-molecule inhibitor of the mitochondrial calcium uniporter
      Kon, Naohiro; Murakoshi, Michiko; Isobe, Aya; Kagechika, Katsuji; Miyoshi, Naoki; Nagayama, Takahiro
      Cell Death Discovery (2017), 3 (), 17045CODEN: CDDEB5; ISSN:2058-7716. (Nature Publishing Group)
      In cardiac myocytes, regulation of mitochondrial Ca2+ is important for cellular signaling and cardiac contraction. Ca2+ entry into the mitochondria is mediated by a highly selective Ca2+ channel called the mitochondrial calcium uniporter, which consists of a pore-forming subunit MCU and regulatory subunits such as MICU1. Although pharmacol. regulation of the mitochondrial Ca2+ influx is a promising approach to controlling the cellular functions, a cell-permeable and specific inhibitor of the mitochondrial calcium uniporter has not yet been developed. Here, we identify a novel cell-permeable inhibitor of the uniporter by a high-throughput screening of 120 000 small-mol. compds. In our study, DS16570511 dose-dependently inhibited serum-induced mitochondrial Ca2+ influx in HEK293A cells with an IC50 of 7 μM. DS16570511 inhibited Ca2+ uptake of isolated mitochondria from human cells, rat heart and pig heart. Overexpression of hMCU or hMICU1 in HEK293A cells increased mitochondrial Ca2+ influx, and the increases were completely suppressed by the pretreatment with DS16570511. DS16570511 also blocks mitochondrial Ca2+ overload in a Langendorff perfused beating rat heart. Interestingly, DS16570511 increased cardiac contractility without affecting heart rate in the perfused heart. These results show that DS16570511 is a novel cell-permeable inhibitor of the mitochondrial calcium uniporter and applicable for control of the cardiac functions.
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    44. 44
      Thu, V. T.; Kim, H. K.; Long, L. T.; Lee, S. R.; Hanh, T. M.; Ko, T. H.; Heo, H. J.; Kim, N.; Kim, S. H.; Ko, K. S.; Rhee, B. D.; Han, J. NecroX-5 Prevents Hypoxia/Reoxygenation Injury by Inhibiting the Mitochondrial Calcium Uniporter. Cardiovasc. Res. 2012, 94, 342– 350,  DOI: 10.1093/cvr/cvs122
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      NecroX-5 prevents hypoxia/reoxygenation injury by inhibiting the mitochondrial calcium uniporter
      Thu, Vu Thi; Kim, Hyoung-Kyu; Long, Le Thanh; Lee, Sung-Ryul; Hanh, Tran My; Ko, Tae Hee; Heo, Hye-Jin; Kim, Nari; Kim, Soon Ha; Ko, Kyung Soo; Rhee, Byoung Doo; Han, Jin
      Cardiovascular Research (2012), 94 (2), 342-350CODEN: CVREAU; ISSN:0008-6363. (Oxford University Press)
      Aims: Preservation of mitochondrial function is essential to limit myocardial damage in ischemic heart disease. We examd. the protective effects and mechanism of a new compd., NecroX-5, on rat heart mitochondria in a hypoxia/reoxygenation (HR) model. Methods and results: NecroX-5 reduced mitochondrial oxidative stress, prevented the collapse in mitochondrial membrane potential, improved mitochondrial oxygen consumption, and suppressed mitochondrial Ca2+ overload during reoxygenation in an in vitro rat heart HR model. Furthermore, NecroX-5 reduced the ouabain- or histamine-induced increase in mitochondrial Ca2+. Conclusion: These findings suggest that NecroX-5 may act as a mitochondrial Ca2+ uniporter inhibitor to protect cardiac mitochondria against HR damage.
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    45. 45
      Antonenko, Y. N.; Rokitskaya, T. I.; Cooper, A. J. L.; Krasnikov, B. F. Minocycline Chelates Ca2+, Binds to Membranes, and Depolarizes Mitochondria by Formation of Ca2+-Dependent Ion Channels. J. Bioenerg. Biomembr. 2010, 42, 151– 163,  DOI: 10.1007/s10863-010-9271-1
      There is no corresponding record for this reference.
    46. 46
      Santo-Domingo, J.; Vay, L.; Hernández-Sanmiguel, E.; Lobatón, C. D.; Moreno, A.; Montero, M.; Alvarez, J. The Plasma Membrane Na+/Ca2+ Exchange Inhibitor KB-R7943 is Also a Potent Inhibitor of the Mitochondrial Ca2+ Uniporter. Br. J. Pharmacol. 2007, 151, 647– 654,  DOI: 10.1038/sj.bjp.0707260
      46
      The plasma membrane Na+/Ca2+ exchange inhibitor KB-R7943 is also a potent inhibitor of the mitochondrial Ca2+ uniporter
      Santo-Domingo, J.; Vay, L.; Hernandez-SanMiguel, E.; Lobaton, C. D.; Moreno, A.; Montero, M.; Alvarez, J.
      British Journal of Pharmacology (2007), 151 (5), 647-654CODEN: BJPCBM; ISSN:0007-1188. (Nature Publishing Group)
      Background and purpose: The thiourea deriv. KB-R7943, originally developed as inhibitor of the plasma membrane Na+/Ca2+ exchanger, has been shown to protect against myocardial ischemia-reperfusion injury. We have studied here its effects on mitochondrial Ca2+ fluxes. [Ca2+] in cytosol, mitochondria and endoplasmic reticulum (ER), and mitochondrial membrane potential were monitored using both luminescent (targeted aequorins) and fluorescent (fura-2, tetramethylrhodamine Et ester) probes in HeLa cells. KB-R7943 was also a potent inhibitor of the mitochondrial Ca2+ uniporter (MCU). In permeabilized HeLa cells, KB-R7943 inhibited mitochondrial Ca2+ uptake with a Ki of 5.5±1.3 μM (mean±S.D.). In intact cells, 10μM KB-R7943 reduced by 80% the mitochondrial [Ca2+] peak induced by histamine. KB-R7943 did not modify the mitochondrial membrane potential and had no effect on the mitochondrial Na+/Ca2+ exchanger. KB-R7943 inhibited histamine-induced ER-Ca2+ release in intact cells, but not in cells loaded with a Ca2+-chelator to damp cytosolic [Ca2+] changes. Therefore, inhibition of ER-Ca2+-release by KB-R7943 was probably due to the increased feedback Ca2+-inhibition of inositol 1,4,5-trisphosphate receptors after MCU block. This mechanism also explains why KB-R7943 reversibly blocked histamine-induced cytosolic [Ca2+] oscillations in the same range of concns. required to inhibit MCU. Inhibition of MCU by KB-R7943 may contribute to its cardioprotective activity by preventing mitochondrial Ca2+-overload during ischemia-reperfusion. In addn., the effects of KB-R7943 on Ca2+ homeostasis provide new evidence for the role of mitochondria modulating Ca2+-release and regenerative Ca2+-oscillations. Search for permeable and selective MCU inhibitors may yield useful pharmacol. tools in the future.
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    47. 47
      Kapuscinski, J.; Darzynkiewicz, Z. Interactions of Antitumor Agents Ametantrone and Mitoxantrone (Novatrone) with Double-Stranded DNA. Biochem. Pharmacol. 1985, 34, 4203– 4213,  DOI: 10.1016/0006-2952(85)90275-8
      47
      Interactions of antitumor agents Ametantrone and mitoxantrone (Novatrone) with double-stranded DNA
      Kapuscinski, Jan; Darzynkiewicz, Zbigniew
      Biochemical Pharmacology (1985), 34 (24), 4203-13CODEN: BCPCA6; ISSN:0006-2952.
      Interactions of mitoxantrone and ametantrone with natural and synthetic nucleic acids in aq. medium [0.15 NaCl, 5 mM 4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid (Hepes), pH 7.0. 25%] were studied using computer-aided spectrophotometric techniques. Absorption spectra of the drugs in monomeric and dimeric form and their complexes with DNAs at low drug/phosphate ratios (D/P) were established. The latter were red-shifted and had lower amplitude as compared with the spectra of the free ligand's monomer; the change is consistent with the already well-established intercalative mode of drug-nucleic acid interaction. Drug-DNA equil. were studied using the McGhee-von Hippel model of noncooperative ligand-polymer interaction, with the correction for dimerization of drugs. Although mitoxantrone was 2 orders of magnitude more potent an antitumor drug than Ametantrone, the intrinsic assocn. consts. (Ki) of both drugs were of similar magnitude. Also, no significant DNA-base specificity for either of the drugs (measured as Ki value for various homopolymers) was obsd. Therefore, no correlation was apparent between the intercalative mode of binding to DNA, regardless of base compn., and the pharmacol. activity of these drugs. At higher D/P ratios, a secondary mode of binding was detected by both spectroscopy and light-scattering measurement. Homopolymer-pairs and polymers contg. only dI and dC were esp. susceptible to this secondary type of binding. The possibility that this secondary type of binding may be responsible for the antitumor properties of the drugs is considered.
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    48. 48
      Mazerski, J.; Martelli, S.; Borowski, E. The Geometry of Intercalation Complex of Antitumor Mitoxantrone and Ametantrone with DNA: Molecular Dynamics Simulations. Acta Biochim. Polym. 1998, 45, 1– 11
      48
      The geometry of intercalation complex of antitumor mitoxantrone and ametantrone with DNA: molecular dynamics simulations
      Mazerski, Jan; Martelli, Sante; Borowski, Edward
      Acta Biochimica Polonica (1998), 45 (1), 1-11CODEN: ABPLAF; ISSN:0001-527X. (Polish Biochemical Society)
      Intercalative binding of the antitumor drugs ametantrone and mitoxantrone to the dodecamer duplex d(CGCGAGCTCGCG)2 was studied by applying mol. dynamics in water with the GROMOS 87 force field. A no. of reasonable binding orientations were tested by short pre-simulations. It was shown that in energetically favorable orientation the anthraquinone chromophore is perpendicular to the direction of inter-base hydrogen bonds. Helically shaped side-chains of the drugs fit to the minor groove. The best orientation obtained in pre-simulations was applied in the main simulations. Small but significant differences were found between structures of inter-calation complexes of the two drugs with the dodecamer duplex, the mitoxantrone complex possessing more favorable energy. The mol. nature of interactions responsible for those differences has been discussed.
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    49. 49
      Boland, M. P.; Fitzgerald, K. A.; O’Neill, L. A. J. Topoisomerase II Is Required for Mitoxantrone to Signal Nuclear Factor ΚB Activation in HL60 Cells. J. Biol. Chem. 2000, 275, 25231– 25238,  DOI: 10.1074/jbc.275.33.25231
      There is no corresponding record for this reference.
    50. 50
      Smith, P. J.; Morgan, S. A.; Fox, M. E.; Watson, J. V. Mitoxantrone-DNA Binding and the Induction of Topoisomerase II Associated DNA Damage in Multi-Drug Resistant Small Cell Lung Cancer Cells. Biochem. Pharmacol. 1990, 40, 2069– 2078,  DOI: 10.1016/0006-2952(90)90237-F
      There is no corresponding record for this reference.
    51. 51
      Nägele, H.; Castel, M. A.; Deutsch, O.; Wagner, F. M.; Reichenspurner, H. Heart Transplantation in a Patient with Multiple Sclerosis and Mitoxantrone-Induced Cardiomyopathy. J. Heart Lung Transplant. 2004, 23, 641– 643,  DOI: 10.1016/S1053-2498(03)00307-3
      51
      Heart transplantation in a patient with multiple sclerosis and mitoxantrone-induced cardiomyopathy
      Nagele H; Castel M A; Deutsch O; Wagner F M; Reichenspurner H
      The Journal of heart and lung transplantation : the official publication of the International Society for Heart Transplantation (2004), 23 (5), 641-3 ISSN:1053-2498.
      We describe a 30-year-old man with end-stage heart failure after therapy with mitoxantrone for multiple sclerosis. A successful orthotopic heart transplantation was performed when intensified medical therapy failed to improve the patient's hemodynamics. In spite of the severe underlying disease he did well on dual immunosuppression with methylprednisone and cyclosporine. Neurologic symptoms remained stable throughout the procedure and, after 2 months, he resumed preoperative ambulatory status. Eight years after the operation, the patient is now in New York Heart Association (NYHA) Class I status. Using canes, he is able to walk short distances. Repeated urinary tract infections caused by Escherichia coli became a problem, but have been controlled by long-term oral antibiotic prophylaxis with trimethoprim.
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    52. 52
      Goebel, M.; Kaplan, E. Anthracycline-Induced Cardiotoxicity – A Review. Oncol. Res. Treat. 2004, 15, 198– 204,  DOI: 10.1159/000217360
      There is no corresponding record for this reference.
    53. 53
      Ying, W.-L.; Emerson, J.; Clarke, M. J.; Sanadi, D. R. Inhibition of Mitochondrial Calcium Ion Transport by an Oxo-Bridged Dinuclear Ruthenium Ammine Complex. Biochemistry 1991, 30, 4949– 4952,  DOI: 10.1021/bi00234a016
      53
      Inhibition of mitochondrial calcium ion transport by an oxo-bridged dinuclear ruthenium ammine complex
      Ying, Wen Long; Emerson, Jeffrey; Clarke, Michael J.; Sanadi, D. Rao
      Biochemistry (1991), 30 (20), 4949-52CODEN: BICHAW; ISSN:0006-2960.
      Ruthenium red is a well-known and effective inhibitor of the mitochondria Ca2+ uniporter; however, K.C. Reed and F. Bygrave (1974) tentatively attributed this inhibition to a colorless impurity present in com. samples of ruthenium red (RR). This component has now been isolated and a deriv., (μ-O)[(HCO2)(NH3)4Ru]2Cl3, structurally characterized. The active species in soln. appears to be the sym. oxo-bridged ion, [X(NH3)4Ru-O-Ru(NH3)4X]3+, where X = Cl- or OH-. Its absorption spectrum shows a max. at 360 nm. The dinuclear ruthenium ammine complex inhibits Ca2+-stimulated respiration of rat liver mitochondria with an I50 of 3.5 pmol/mg of protein compared to the value of 60 pmol of RR/mg of protein. The inhibition by the dinuclear compd. is noncompetitive with Ca2+. Respiration-linked swelling of mitochondria induced by Cd2+ also responds similarly to both the dinuclear complex and RR. A close correlation was obsd. between binding to mitochondria as monitored with 103Ru-labeled dinuclear complex and inhibition of Ca2+ transport. A Scatchard plot yielded ests. of max. specific binding and dissocn. const. of 7.5 pmol/mg of protein and 1.3 nM, resp. The inhibitor has the characteristics of a satisfactory affinity ligand for purifn. of the uniporter.
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    54. 54
      Matlib, M. A.; Zhou, Z.; Knight, S.; Ahmed, S.; Choi, K. M.; Krause-Bauer, J.; Phillips, R.; Altschuld, R.; Katsube, Y.; Sperelakis, N.; Bers, D. M. Oxygen-Bridged Dinuclear Ruthenium Amine Complex Specifically Inhibits Ca2+ Uptake into Mitochondria in Vitro and in Situ in Single Cardiac Myocytes. J. Biol. Chem. 1998, 273, 10223– 10231,  DOI: 10.1074/jbc.273.17.10223
      54
      Oxygen-bridged dinuclear ruthenium amine complex specifically inhibits Ca2+ uptake into mitochondria in vitro and in situ in single cardiac myocytes
      Matlib, Mohammed A.; Zhou, Zhuan; Knight, Selena; Ahmed, Saadia; Choi, Kin M.; Krause-Bauer, Jeanette; Phillips, Ronald; Altschuld, Ruth; Katsube, Yasuhiro; Sperelakis, Nicholas; Bers, Donald M.
      Journal of Biological Chemistry (1998), 273 (17), 10223-10231CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
      Ruthenium red is a well known inhibitor of Ca2+ uptake into mitochondria in vitro. However, its utility as an inhibitor of Ca2+ uptake into mitochondria in vivo or in situ in intact cells is limited because of its inhibitory effects on sarcoplasmic reticulum Ca2+ release channel and other cellular processes. We have synthesized a ruthenium deriv. and found it to be an oxygen-bridged dinuclear ruthenium amine complex. It has the same chem. structure as Ru360 reported previously (Emerson, J., Clarke, M. J., Ying, W-L., and Sanadi, D. R. (1993) J. Am. Chem. Soc. 115, 11799-11805). Ru360 has been shown to be a potent inhibitor of Ca2+-stimulated respiration of liver mitochondria in vitro. However, the specificity of Ru360 on Ca2+ uptake into mitochondria in vitro or in intact cells has not been detd. The present study reports in detail the potency, the effectiveness, and the mechanism of inhibition of mitochondrial Ca2+ uptake by Ru360 and its specificity in vitro in isolated mitochondria and in situ in isolated cardiac myocytes. Ru360 was more potent (IC50 = 0.184 nM) than ruthenium red (IC50 = 6.85 nM) in inhibiting Ca2+ uptake into mitochondria. 103Ru360 was found to bind to isolated mitochondria with high affinity (Kd = 0.34 nM, Bmax = 80 fmol/mg of mitochondrial protein). The IC50 of 103Ru360 for the inhibition of Ca2+ uptake into mitochondria was also 0.2 nM, indicating that satn. of a specific binding site is responsible for the inhibition of Ca2+ uptake. Ru360, as high as 10 μM, produced no effect on sarcoplasmic reticulum Ca2+ uptake or release, sarcolemmal Na+/Ca2+ exchange, actomyosin ATPase activity, L-type Ca2+ channel current, cytosolic Ca2+ transients, or cell shortening. 103Ru360 was taken up by isolated myocytes in a time-dependent biphasic manner. Ru360 (10 μM) applied outside intact voltage-clamped ventricular myocytes prevented Ca2+ uptake into mitochondria in situ where the cells were progressively loaded with Ca2+ via sarcolemmal Na+/Ca2+ exchange by depolarization to +110 mV. We conclude that Ru360 specifically blocks Ca2+ uptake into mitochondria and can be used in intact cells.
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    55. 55
      Emerson, J.; Clarke, M. J.; Ying, W. L.; Sanadi, D. R. The Component of “Ruthenium Red” Responsible for Inhibition of Mitochondrial Calcium Ion Transport. Spectra, Electrochemistry, and Aquation Kinetics. Crystal Structure of μ-O-[(HCO)2(NH3)4Ru]2Cl3. J. Am. Chem. Soc. 1993, 115, 11799– 11805,  DOI: 10.1021/ja00078a019
      There is no corresponding record for this reference.
    56. 56
      Nathan, S. R.; Pino, N. W.; Arduino, D. M.; Perocchi, F.; MacMillan, S. N.; Wilson, J. J. Synthetic Methods for the Preparation of a Functional Analogue of Ru360, a Potent Inhibitor of Mitochondrial Calcium Uptake. Inorg. Chem. 2017, 56, 3123– 3126,  DOI: 10.1021/acs.inorgchem.6b03108
      56
      Synthetic Methods for the Preparation of a Functional Analogue of Ru360, a Potent Inhibitor of Mitochondrial Calcium Uptake
      Nathan, Sarah R.; Pino, Nicholas W.; Arduino, Daniela M.; Perocchi, Fabiana; MacMillan, Samantha N.; Wilson, Justin J.
      Inorganic Chemistry (2017), 56 (6), 3123-3126CODEN: INOCAJ; ISSN:0020-1669. (American Chemical Society)
      The mixed-valent oxo-bridged Ru complex [(HCO2)(NH3)4Ru(μ-O)Ru(NH3)4(O2CH)]3+, known as Ru360, is a selective inhibitor of mitochondrial Ca uptake. Although this compd. is useful for studying the role of mitochondrial Ca in biol. processes, its widespread availability is limited because of challenges in purifn. and characterization. Here, the authors describe the authors' studies of three different synthetic methods for the prepn. of a functional analog of this valuable compd. This analog, isolated from the authors' procedures, exhibits potent mitochondrial Ca uptake inhibitory properties in permeabilized HeLa cells and in isolated mitochondria.
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    57. 57
      Nathan, S. R.; Wilson, J. J. Synthesis and Evaluation of a Ruthenium-Based Mitochondrial Calcium Uptake Inhibitor. J. Visualized Exp. 2017, 128, e56527,  DOI: 10.3791/56527
      There is no corresponding record for this reference.
    58. 58
      Cao, C.; Wang, S.; Cui, T.; Su, X.-C.; Chou, J. J. Ion and Inhibitor Binding of the Double-Ring Ion Selectivity Filter of the Mitochondrial Calcium Uniporter. Proc. Natl. Acad. Sci. U. S. A. 2017, 114, E2846– E2851,  DOI: 10.1073/pnas.1620316114
      58
      Ion and inhibitor binding of the double-ring ion selectivity filter of the mitochondrial calcium uniporter
      Cao, Chan; Wang, Shuqing; Cui, Tanxing; Su, Xun-Cheng; Chou, James J.
      Proceedings of the National Academy of Sciences of the United States of America (2017), 114 (14), E2846-E2851CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      The mitochondrial Ca2+ uniporter (MCU) is a holocomplex consisting of the Ca2+-conducting channel and several accessory and regulatory components. A previous electrophysiol. study found that the uniporter had high Ca2+ selectivity and conductance and this depended critically on the conserved amino acid sequence motif, DXXE (Asp-X-X-Glu) of MCU. A recent NMR structure of the MCU channel from Caenorhabditis elegans revealed that the DXXE formed 2 parallel carboxylate rings at the channel entrance that seem to serve as the ion selectivity filter, although direct ion interaction of this structural motif has not been addressed. Here, we used a paramagnetic probe, Mn2+, to investigate ion and inhibitor binding of this putative selectivity filter. The paramagnetic NMR data showed that mutants with a single carboxylate ring, NXXE (Asn-X-X-Glu) and DXXQ (Asp-X-X-Gln), each could bind Mn2+ specifically, whereas in the wild type the 2 rings bound Mn2+ cooperatively, resulting in ∼1000-fold higher apparent affinity. Ca2+ could specifically displace bound Mn2+ at the DXXE site in the channel. Furthermore, titrating the sample with the known channel inhibitor, ruthenium 360 (Ru360), could displace Mn2+ binding from the solvent-accessible Asp site but not the inner Glu site. The NMR titrn. data, together with structural anal. of the DXXE motif and mol. dynamics simulations, indicated that the double carboxylate rings at the apex of the MCU pore constitute the ion selectivity filter and that Ru360 directly blocks ion entry into the filter by binding to the outer carboxylate ring.
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    59. 59
      Gushchin, A. L.; Laricheva, Y. A.; Abramov, P. A.; Sokolov, M. N.; Abramov, P. A. Synthesis and Electrochemical Properties of [RuIV2O(PhCN)4Cl6]. Inorg. Chem. Commun. 2018, 95, 1387– 7003,  DOI: 10.1016/j.inoche.2018.07.033
      There is no corresponding record for this reference.
    60. 60
      Urgiles, J.; Nathan, S. R.; MacMillan, S. N.; Wilson, J. J. Dinuclear Nitrido-Bridged Ruthenium Complexes Bearing Diimine Ligands. Dalton Trans. 2017, 46, 14256– 14263,  DOI: 10.1039/C7DT03085A
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      Dinuclear nitrido-bridged ruthenium complexes bearing diimine ligands
      Urgiles, Julie; Nathan, Sarah R.; MacMillan, Samantha N.; Wilson, Justin J.
      Dalton Transactions (2017), 46 (41), 14256-14263CODEN: DTARAF; ISSN:1477-9226. (Royal Society of Chemistry)
      Reactions of K3[Ru2NCl8(H2O)2] with 2,2'-bipyridine (bpy), 4,4'-dimethyl-2,2'-bipyridine (dmbpy), and 4,4'-dimethoxy-2,2'-bipyridine (dmobpy) yielded the nitrido-bridged dinuclear complexes [Ru2N(L)2Cl5(DMF)] where L = bpy (1), dmbpy (2), and dmobpy (3). The crystal structures of these complexes reveal a linear Ru-N-Ru moiety with each Ru center bearing a bidentate diimine ligand. The complexes were further characterized by NMR, IR, and UV-visible spectroscopic methods and cyclic voltammetry. Because the compds. bear some structural similarities with the mitochondrial Ca uptake inhibitor Ru360, the ability of these complexes to act in this capacity was evaluated. 1-3 All fail to block mitochondrial Ca uptake, revealing new facets of the structure-activity relations for Ru-based mitochondrial Ca uptake inhibitors.
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    61. 61
      Dunitz, J. D.; Orgel, L. E. Application of Molecular-Orbital Theory to Some Binuclear Coordination Compounds. J. Chem. Soc. 1953, 2594– 2596,  DOI: 10.1039/jr9530002594
      There is no corresponding record for this reference.
    62. 62
      Cleare, M. J.; Griffith, W. P. Binuclear Nitrido-Complexes of Ruthenium. Chem. Commun. 1968, 21, 1302,  DOI: 10.1039/c19680001302
      There is no corresponding record for this reference.
    63. 63
      Griffith, W. P.; McManus, N. T.; Skapski, A. C. X-Ray Crystal Structure of [Ru2N(ethylenediamine)5]Cl5· H2O; a Novel Complex Containing Both Nitrido and Ethylenediamine Bridges. J. Chem. Soc., Chem. Commun. 1984, 7, 434,  DOI: 10.1039/c39840000434
      There is no corresponding record for this reference.
    64. 64
      Ng, H.-Y.; Cheung, W.-M.; Kwan Huang, E.; Wong, K.-L.; Sung, H. H.-Y.; Williams, I. D.; Leung, W.-H. Ruthenium Chalcogenonitrosyl and Bridged Nitrido Complexes Containing Chelating Sulfur and Oxygen Ligands. Dalton Trans. 2015, 44, 18459– 18468,  DOI: 10.1039/C5DT02513C
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      Ruthenium chalcogenonitrosyl and bridged nitrido complexes containing chelating sulfur and oxygen ligands
      Ng, Ho-Yuen; Cheung, Wai-Man; Kwan Huang, Enrique; Wong, Kang-Long; Sung, Herman H.-Y.; Williams, Ian D.; Leung, Wa-Hung
      Dalton Transactions (2015), 44 (42), 18459-18468CODEN: DTARAF; ISSN:1477-9226. (Royal Society of Chemistry)
      Ruthenium thio- and seleno-nitrosyl complexes contg. chelating S and O ligands were synthesized and their de-chalcogenation reactions were studied. The reaction of mer-[Ru(N)Cl3(AsPh3)2] with elemental S and Se in THF at reflux afforded the chalcogenonitrosyl complexes mer-[Ru(NX)Cl3(AsPh3)2] [X = S (1), Se (2)]. Treatment of 1 with KN(R2PS)2 afforded trans-[Ru(NS)Cl{N(R2PS)2}2] [R = Ph (3), Pri (4), But (5)]. Alternatively, the thionitrosyl complex 5 was obtained from [Bu4N][Ru(N)Cl4] and KN(But2PS)2, presumably via S atom transfer from [N(But2PS)2]- to the nitride. Reactions of 1 and 2 with NaLOEt (LOEt- = [Co(η5-C5H5){P(O)(LOEt)2}3]-) gave [Ru(NX)LOEtCl2] (X = S (8), Se (9)). Treatment of [Bu4N][Ru(N)Cl4] with KN(R2PS)2 produced RuIV-RuIV μ-nitrido complexes [Ru2(μ-N){N(R2PS)2}4Cl] [R = Ph (6), Pri (7)]. Reactions of 3 and 9 with PPh3 afforded 6 and [Ru(NPPh3)LOEtCl2], resp. The desulfurization of 5 with [Ni(cod)2] (cod = 1,5-cyclooctadiene) gave the mixed valance RuIII-RuIV μ-nitrido complex [Ru2(μ-N){N(But2PS)2}4] (10) that was oxidized by [Cp2Fe](PF6) to give the RuIV-RuIV complex [Ru2(μ-N){N(But2PS)2}4](PF6) ([10]PF6). The crystal structures of 1, 2, 3, 7, 9 and 10 were detd.
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    65. 65
      Ciechanowicz, M.; Skapski, A. C. Crystal Structure of Potassium μ-Nitrido-Bis[Aquotetrachlororuthenate(IV)]. J. Chem. Soc. A 1971, 84, 1792– 1794,  DOI: 10.1039/J19710001792
      There is no corresponding record for this reference.
    66. 66
      Freshney, R. I. Culture of Animal Cells, 5th ed.; John Wiley & Sons, Inc.: Hoboken, NJ, 2005.
      There is no corresponding record for this reference.
    67. 67
      Shanmughapriya, S.; Rajan, S.; Hoffman, N. E.; Zhang, X.; Guo, S.; Kolesar, J. E.; Hines, K. J.; Ragheb, J.; Jog, N. R.; Caricchio, R.; Baba, Y.; Zhou, Y.; Kaufman, B. A.; Cheung, J. Y.; Kurosaki, T.; Gill, D. L.; Madesh, M. Ca2+ Signals Regulate Mitochondrial Metabolism by Stimulating CREB-Mediated Expression of the Mitochondrial Ca2+ Uniporter Gene MCU. Sci. Signaling 2015, 8, ra23,  DOI: 10.1126/scisignal.2005673
      There is no corresponding record for this reference.
    68. 68
      Reers, M.; Smith, T. W.; Chen, L. B. J-aggregate formation of a carbocyanine as a quantitative fluorescent indicator of membrane potential. Biochemistry 1991, 30, 4480– 4486,  DOI: 10.1021/bi00232a015
      68
      J-aggregate formation of a carbocyanine as a quantitative fluorescent indicator of membrane potential
      Reers, Martin; Smith, Thomas W.; Chen, Lan Bo
      Biochemistry (1991), 30 (18), 4480-6CODEN: BICHAW; ISSN:0006-2960.
      The spectral properties of a novel membrane potential sensitive probe (JC-1) were characterized in aq. buffers and in isolated cardiac mitochondria. JC-1 is a carbocyanine with a delocalized pos. charge. It formed under favorable conditions a concn.-dependent fluorescent nematic phase consisting of J-aggregates. When excited at 490 nm, the monomers exhibited an emission max. at 527 nm and J-aggregates at 590 nm. Increasing concns. of JC-1 above a certain concn. caused a linear rise in the J-aggregate fluorescence, while the monomer fluorescence remained const. The membrane potential of energized mitochondria (neg. inside) promoted a directional uptake of JC-1 into the matrix, also with subsequent formation of J-aggregates. The J-aggregate fluorescence was sensitive to transient membrane potential changes induced by ADP and to metabolic inhibitors of oxidative phosphorylation. The J-aggregate fluorescence was pH independent within the physiol. pH range of 7.15-8.0 and could be linearly calibrated with valinomycin-induced K+ diffusion potentials. The advantage of JC-1 over rhodamines and other carbocyanines is that its color altered reversibly from green to red with increasing membrane potentials. This can be exploited for imaging live mitochondria on the stage of a microscope.
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    69. 69
      King, A. P.; Gellineau, H. A.; Ahn, J. E.; MacMillan, S. N.; Wilson, J. J. Bis(Thiosemicarbazone) Complexes of Cobalt(III). Synthesis, Characterization, and Anticancer Potential. Inorg. Chem. 2017, 56, 6609– 6623,  DOI: 10.1021/acs.inorgchem.7b00710
      69
      Bis(thiosemicarbazone) Complexes of Cobalt(III). Synthesis, Characterization, and Anticancer Potential
      King, A. Paden; Gellineau, Hendryck A.; Ahn, Jung-Eun; MacMillan, Samantha N.; Wilson, Justin J.
      Inorganic Chemistry (2017), 56 (11), 6609-6623CODEN: INOCAJ; ISSN:0020-1669. (American Chemical Society)
      Nine bis(thiosemicarbazone) (BTSC) cobalt(III) complexes of the general formula [Co(BTSC)(L)2]NO3 were synthesized, where BTSC = diacetyl bis(thiosemicarbazone) (ATS), pyruvaldehyde bis(thiosemicarbazone) (PTS), or glyoxal bis(thiosemicarbazone) (GTS) and L = ammonia, imidazole (Im), or benzylamine (BnA). These compds. were characterized by multinuclear NMR spectroscopy, mass spectrometry, cyclic voltammetry, and x-ray crystallog. Their stability in phosphate-buffered saline was investigated and is highly dependent on the nature of the axial ligand, L. These studies revealed that complex stability is primarily dictated by the axial ligand following the sequence NH3 > Im > BnA. The cellular uptake and cytotoxicity in cancer cells were also detd. Both the cellular uptake and cytotoxicity were significantly affected by the nature of the equatorial BTSC. Complexes of ATS were taken up much more effectively than those of PTS and GTS. The cytotoxicity of the complexes was correlated to that of the free ligand. Cell uptake and cytotoxicity were also detd. under hypoxic conditions. Only minor differences in the hypoxia activity and uptake were obsd. Treatment of the cancer cells with the copper-depleting agent tetrathiomolybdate decreased the cytotoxic potency of the complexes, indicating that they may operate via a copper-dependent mechanism. These results provide a structure-activity relation for this class of compds., which may be applied for the rational design of new cobalt(III) anticancer agents.
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    70. 70
      Egger, A. E.; Rappel, C.; Jakupec, M. A.; Hartinger, C. G.; Heffeter, P.; Keppler, B. K. Development of an Experimental Protocol for Uptake Studies of Metal Compounds in Adherent Tumor Cells. J. Anal. At. Spectrom. 2009, 24, 51– 61,  DOI: 10.1039/B810481F
      70
      Development of an experimental protocol for uptake studies of metal compounds in adherent tumor cells
      Egger, Alexander E.; Rappel, Christina; Jakupec, Michael A.; Hartinger, Christian G.; Heffeter, Petra; Keppler, Bernhard K.
      Journal of Analytical Atomic Spectrometry (2009), 24 (1), 51-61CODEN: JASPE2; ISSN:0267-9477. (Royal Society of Chemistry)
      Cellular uptake is being widely investigated in the context of diverse biol. activities of metal compds. on the cellular level. However, the applied techniques differ considerably, and a validated methodol. is not at hand. Therefore, we have varied numerous aspects of sample prepn. of the human colon carcinoma cell line SW480 exposed in vitro to the tumor-inhibiting metal complexes cisplatin and indazolium trans-[tetrachlorobis(1H-indazole)ruthenate(III)] (KP1019) prior to anal. with ICP-MS, and the results were found to be tremendously influenced by adsorption to the culture dishes. Adsorption to culture plates increases linearly with the concn. of KP1019, depends on the protein content of the medium, the duration of contact to protein-contg. medium prior to drug addn. and the hydrophilicity/lipophilicity of the compd. For varying degrees of cell confluence, adsorption of Ru hardly differs from cell-free expts. Desorption from the plates contributes to total Ru detected in dependence on the cell harvesting method. Desorption kinetics for lysis in HNO3 and tetramethylammonium hydroxide (TMAH) are comparable, but TMAH is a more potent desorbant. Sample storage conditions prior to anal. influence significantly the recovery of analyte. Protocols using cell lysis in the culture plate without proper corrections run the risk of producing artifacts resulting from metal adsorption/desorption to an extent comparable with the actual cellular content. However, exptl. protocols reported in the literature frequently do not contain information whether adsorption or blank correction were performed and should be regarded with caution, esp. if lysis was performed directly in the culture dishes.
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    71. 71
      Komor, A. C.; Schneider, C. J.; Weidmann, A. G.; Barton, J. K. Cell-Selective Biological Activity of Rhodium Metalloinsertors Correlates with Subcellular Localization. J. Am. Chem. Soc. 2012, 134, 19223– 19233,  DOI: 10.1021/ja3090687
      71
      Cell-Selective Biological Activity of Rhodium Metalloinsertors Correlates with Subcellular Localization
      Komor, Alexis C.; Schneider, Curtis J.; Weidmann, Alyson G.; Barton, Jacqueline K.
      Journal of the American Chemical Society (2012), 134 (46), 19223-19233CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      Deficiencies in the mismatch repair (MMR) pathway are assocd. with several types of cancers, as well as resistance to commonly used chemotherapeutics. Rhodium metalloinsertors have been found to bind DNA mismatches with high affinity and specificity in vitro, and also exhibit cell-selective cytotoxicity, targeting MMR-deficient cells over MMR-proficient cells. Ten distinct metalloinsertors with varying lipophilicities have been synthesized and their mismatch binding affinities and biol. activities detd. Although DNA photocleavage expts. demonstrate that their binding affinities are quite similar, their cell-selective antiproliferative and cytotoxic activities vary significantly. Inductively coupled plasma mass spectrometry (ICP-MS) expts. have uncovered a relation between the subcellular distribution of these metalloinsertors and their biol. activities. Specifically, we find that all of our metalloinsertors localize in the nucleus at sufficient concns. for binding to DNA mismatches. However, the metalloinsertors with high rhodium localization in the mitochondria show toxicity that is not selective for MMR-deficient cells, whereas metalloinsertors with less mitochondrial rhodium show activity that is highly selective for MMR-deficient vs. proficient cells. This work supports the notion that specific targeting of the metalloinsertors to nuclear DNA gives rise to their cell-selective cytotoxic and antiproliferative activities. The selectivity in cellular targeting depends upon binding to mismatches in genomic DNA.
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    72. 72
      Boyle, K. M.; Barton, J. K. A Family of Rhodium Complexes with Selective Toxicity toward Mismatch Repair-Deficient Cancers. J. Am. Chem. Soc. 2018, 140, 5612– 5624,  DOI: 10.1021/jacs.8b02271
      72
      A Family of Rhodium Complexes with Selective Toxicity toward Mismatch Repair-Deficient Cancers
      Boyle, Kelsey M.; Barton, Jacqueline K.
      Journal of the American Chemical Society (2018), 140 (16), 5612-5624CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      Rhodium metalloinsertors are a unique set of metal complexes that bind specifically to DNA base pair mismatches in vitro and kill mismatch repair (MMR)-deficient cells at lower concns. than their MMR-proficient counterparts. A family of metalloinsertors contg. rhodium-oxygen ligand coordination, termed "Rh-O" metalloinsertors, has been prepd. and shown to have a significant increase in both overall potency and selectivity toward MMR-deficient cells regardless of structural changes in the ancillary ligands. Here we describe DNA-binding and cellular studies with the second generation of Rh-O metalloinsertors in which an ancillary ligand is varied in both steric bulk and lipophilicity. These complexes, of the form [Rh(L)(chrysi)(PPO)]2+, all include the O-contg. PPO ligand (PPO = 2-(pyridine-2-yl)propan-2-ol) and the arom. inserting ligand chrysi (5,6-chrysene quinone diimine) but differ in the identity of their ancillary ligand L, where L is a phenanthroline or bipyridyl deriv. The Rh-O metalloinsertors in this family all show micromolar binding affinities for a 29-mer DNA hairpin contg. a single CC mismatch. The complexes display comparable lipophilic tendencies and pKa values of 8.1-9.1 for dissocn. of an imine proton on the chrysi ligand. In cellular proliferation and cytotoxicity assays with MMR-deficient cells (HCT116O) and MMR-proficient cells (HCT116N), the complexes contg. the phenanthroline-derived ligands show highly selective cytotoxic preference for the MMR-deficient cells at nanomolar concns. Using mass spectral analyses, it is shown that the complexes are taken into cells through a passive mechanism and exhibit low accumulation in mitochondria, an off-target organelle that, when targeted by parent metalloinsertors, can lead to nonselective cytotoxicity. Overall, these Rh-O metalloinsertors have distinct and improved behavior compared to previous generations of parent metalloinsertors, making them ideal candidates for further therapeutic assessment.
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    73. 73
      Ahmad, K. A.; Iskandar, K. B.; Hirpara, J. L.; Clement, M. V.; Pervaiz, S. Hydrogen Peroxide-Mediated Cytosolic Acidification is a Signal for Mitochondrial Translocation of Bax during Drug-Induced Apoptosis of Tumor Cells. Cancer Res. 2004, 64, 7867– 7878,  DOI: 10.1158/0008-5472.CAN-04-0648
      There is no corresponding record for this reference.
    74. 74
      Dong, Z.; Shanmughapriya, S.; Tomar, D.; Siddiqui, N.; Lynch, S.; Nemani, N.; Breves, S. L.; Zhang, X.; Tripathi, A.; Palaniappan, P.; Riitano, M. F.; Worth, A. M.; Seelam, A.; Carvalho, E.; Subbiah, R.; Jaña, F.; Soboloff, J.; Peng, Y.; Cheung, J. Y.; Joseph, S. K.; Caplan, J.; Rajan, S.; Stathopulos, P. B.; Madesh, M. Mitochondrial Ca2+ Uniporter is a Mitochondrial Luminal Redox Sensor That Augments MCU Channel Activity. Mol. Cell 2017, 65, 1014– 1028,  DOI: 10.1016/j.molcel.2017.01.032
      74
      Mitochondrial Ca2+ Uniporter Is a Mitochondrial Luminal Redox Sensor that Augments MCU Channel Activity
      Dong, Zhiwei; Shanmughapriya, Santhanam; Tomar, Dhanendra; Siddiqui, Naveed; Lynch, Solomon; Nemani, Neeharika; Breves, Sarah L.; Zhang, Xueqian; Tripathi, Aparna; Palaniappan, Palaniappan; Riitano, Massimo F.; Worth, Alison M.; Seelam, Ajay; Carvalho, Edmund; Subbiah, Ramasamy; Jana, Fabian; Soboloff, Jonathan; Peng, Yizhi; Cheung, Joseph Y.; Joseph, Suresh K.; Caplan, Jeffrey; Rajan, Sudarsan; Stathopulos, Peter B.; Madesh, Muniswamy
      Molecular Cell (2017), 65 (6), 1014-1028.e7CODEN: MOCEFL; ISSN:1097-2765. (Elsevier Inc.)
      Ca2+ dynamics and oxidative signaling are fundamental mechanisms for mitochondrial bioenergetics and cell function. The MCU complex is the major pathway by which these signals are integrated in mitochondria. Whether and how these coactive elements interact with MCU have not been established. As an approach toward understanding the regulation of MCU channel by oxidative milieu, we adapted inflammatory and hypoxia models. We identified the conserved cysteine 97 (Cys-97) to be the only reactive thiol in human MCU that undergoes S-glutathionylation. Furthermore, biochem., structural, and superresoln. imaging anal. revealed that MCU oxidn. promotes MCU higher order oligomer formation. Both oxidn. and mutation of MCU Cys-97 exhibited persistent MCU channel activity with higher [Ca2+]m uptake rate, elevated mROS, and enhanced [Ca2+]m overload-induced cell death. In contrast, these effects were largely independent of MCU interaction with its regulators. These findings reveal a distinct functional role for Cys-97 in ROS sensing and regulation of MCU activity.
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    75. 75
      Zhao, Y.; Araki, S.; Wu, J.; Teramoto, T.; Chang, Y.-F.; Nakano, M.; Abdelfattah, A. S.; Fujiwara, M.; Ishihara, T.; Nagai, T.; Campbell, R. E. An Expanded Palette of Genetically Encoded Ca2+ Indicators. Science 2011, 333, 1888– 1891,  DOI: 10.1126/science.1208592
      75
      An Expanded Palette of Genetically Encoded Ca2+ Indicators
      Zhao, Yongxin; Araki, Satoko; Wu, Jiahui; Teramoto, Takayuki; Chang, Yu-Fen; Nakano, Masahiro; Abdelfattah, Ahmed S.; Fujiwara, Manabi; Ishihara, Takeshi; Nagai, Takeharu; Campbell, Robert E.
      Science (Washington, DC, United States) (2011), 333 (6051), 1888-1891CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
      Engineered fluorescent protein (FP) chimeras that modulate their fluorescence in response to changes in calcium ion (Ca2+) concn. are powerful tools for visualizing intracellular signaling activity. However, despite a decade of availability, the palette of single FP-based Ca2+ indicators has remained limited to a single green hue. The authors have expanded this palette by developing blue, improved green, and red intensiometric indicators, as well as an emission ratiometric indicator with an 11,000% ratio change. This series enables improved single-color Ca2+ imaging in neurons and transgenic Caenorhabditis elegans. In HeLa cells, Ca2+ was imaged in three subcellular compartments, and, in conjunction with a cyan FP-yellow FP-based indicator, Ca2+ and ATP were simultaneously imaged. This palette of indicators paints the way to a colorful new era of Ca2+ imaging.
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    76. 76
      Nakai, J.; Ohkura, M.; Imoto, K. A High Signal-to-Noise Ca2+ Probe Composed of a Single Green Fluorescent Protein. Nat. Biotechnol. 2001, 19, 137– 141,  DOI: 10.1038/84397
      76
      A high signal-to-noise Ca2+ probe composed of a single green fluorescent protein
      Nakai, Junichi; Ohkura, Masamichi; Imoto, Keiji
      Nature Biotechnology (2001), 19 (2), 137-141CODEN: NABIF9; ISSN:1087-0156. (Nature America Inc.)
      Recently, several groups have developed green fluorescent protein (GFP)-based Ca2+ probes. When applied in cells, however, these probes are difficult to use because of a low signal-to-noise ratio. Here we report the development of a high-affinity Ca2+ probe composed of a single GFP (named G-CaMP). G-CaMP showed an apparent Kd for Ca2+ of 235 μM. Assocn. kinetics of Ca2+ binding were faster at higher Ca2+ concns., with time consts. decreasing from 230 ms at 0.2 μM Ca2+ to 2.5 ms at 1 μM Ca2+. Dissocn. kinetics (τ -200 ms) are independent of Ca2+ concns. In HEK-293 cells and mouse myotubes expressing G-CaMP, large fluorescent changes were obsd. in response to application of drugs or elec. stimulations. G-CaMP will be a useful tool for visualizing intracellular Ca2+ in living cells. Mutational anal., together with previous structural information, suggests the residues that may alter the fluorescence of GFP.
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    77. 77
      Chiesi, M.; Schwaller, R.; Eichenberger, K. Structural Dependency of the Inhibitory Action of Benzodiazepines and Related Compounds on the Mitochondrial Na+-Ca2+ Exchanger. Biochem. Pharmacol. 1988, 37, 4399– 4403,  DOI: 10.1016/0006-2952(88)90623-5
      There is no corresponding record for this reference.
    78. 78
      Luongo, T. S.; Lambert, J. P.; Gross, P.; Nwokedi, M.; Lombardi, A. A.; Shanmughapriya, S.; Carpenter, A. C.; Kolmetzky, D.; Gao, E.; van Berlo, J. H.; Tsai, E. J.; Molkentin, J. D.; Chen, X.; Madesh, M.; Houser, S. R.; Elrod, J. W. The Mitochondrial Na+/Ca2+ Exchanger is Essential for Ca2+ Homeostasis and Viability. Nature 2017, 545, 93– 97,  DOI: 10.1038/nature22082
      78
      The mitochondrial Na+/Ca2+ exchanger is essential for Ca2+ homeostasis and viability
      Luongo, Timothy S.; Lambert, Jonathan P.; Gross, Polina; Nwokedi, Mary; Lombardi, Alyssa A.; Shanmughapriya, Santhanam; Carpenter, April C.; Kolmetzky, Devin; Gao, Erhe; van Berlo, Jop H.; Tsai, Emily J.; Molkentin, Jeffery D.; Chen, Xiongwen; Madesh, Muniswamy; Houser, Steven R.; Elrod, John W.
      Nature (London, United Kingdom) (2017), 545 (7652), 93-97CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
      Mitochondrial calcium (mCa2+) has a central role in both metabolic regulation and cell death signaling, however its role in homeostatic function and disease is controversial. Slc8b1 encodes the mitochondrial Na+/Ca2+ exchanger (NCLX), which is proposed to be the primary mechanism for mCa2+ extrusion in excitable cells. Here we show that tamoxifen-induced deletion of Slc8b1 in adult mouse hearts causes sudden death, with less than 13% of affected mice surviving after 14 days. Lethality correlated with severe myocardial dysfunction and fulminant heart failure. Mechanistically, cardiac pathol. was attributed to mCa2+ overload driving increased generation of superoxide and necrotic cell death, which was rescued by genetic inhibition of mitochondrial permeability transition pore activation. Corroborating these findings, overexpression of NCLX in the mouse heart by conditional transgenesis had the beneficial effect of augmenting mCa2+ clearance, preventing permeability transition and protecting against ischemia-induced cardiomyocyte necrosis and heart failure. These results demonstrate the essential nature of mCa2+ efflux in cellular function and suggest that augmenting mCa2+ efflux may be a viable therapeutic strategy in disease.
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    79. 79
      Storey, N. M.; Lambert, D. G. Mitochondrial Pharmacology Turns Its Sights on the Ca2+ Uniporter. Cell Death Discov. 2017, 3, 17064,  DOI: 10.1038/cddiscovery.2017.64
      79
      Mitochondrial pharmacology turns its sights on the Ca(2+) uniporter
      Storey Nina M; Lambert David G
      Cell death discovery (2017), 3 (), 17064 ISSN:2058-7716.
      There is no expanded citation for this reference.
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    80. 80
      Sebag, S. C.; Koval, O. M.; Paschke, J. D.; Winters, C. J.; Comellas, A. P.; Grumbach, I. M. Inhibition of the Mitochondrial Calcium Uniporter Prevents IL-13 and Allergen-Mediated Airway Epithelial Apoptosis and Loss of Barrier Function. Exp. Cell Res. 2018, 362, 400– 411,  DOI: 10.1016/j.yexcr.2017.12.003
      80
      Inhibition of the mitochondrial calcium uniporter prevents IL-13 and allergen-mediated airway epithelial apoptosis and loss of barrier function
      Sebag, Sara C.; Koval, Olha M.; Paschke, John D.; Winters, Christopher J.; Comellas, Alejandro P.; Grumbach, Isabella M.
      Experimental Cell Research (2018), 362 (2), 400-411CODEN: ECREAL; ISSN:0014-4827. (Elsevier B.V.)
      Mitochondria are increasingly recognized as key mediators of acute cellular stress responses in asthma. However, the distinct roles of regulators of mitochondrial physiol. on allergic asthma phenotypes are currently unknown. The mitochondrial Ca2+ uniporter (MCU) resides in the inner mitochondrial membrane and controls mitochondrial Ca2+ uptake into the mitochondrial matrix. To understand the function of MCU in models of allergic asthma, in vitro and in vivo studies were performed using models of functional deficiency or knockout of MCU. In primary human respiratory epithelial cells, MCU inhibition abrogated mitochondrial Ca2+ uptake and reactive oxygen species (ROS) prodn., preserved the mitochondrial membrane potential and protected from apoptosis in response to the pleiotropic Th2 cytokine IL-13. Consequently, epithelial barrier function was maintained with MCU inhibition. Similarly, the endothelial barrier was preserved in respiratory epithelium isolated from MCU-/- mice after exposure to IL-13. In the ovalbumin-model of allergic airway disease, MCU deficiency resulted in decreased apoptosis within the large airway epithelial cells. Concordantly, expression of the tight junction protein ZO-1 was preserved, indicative of maintenance of epithelial barrier function. These data implicate mitochondrial Ca2+ uptake through MCU as a key controller of epithelial cell viability in acute allergic asthma.
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    81. 81
      Xie, N.; Wu, C.; Wang, C.; Cheng, X.; Zhang, L.; Zhang, H.; Lian, Y. Inhibition of the Mitochondrial Calcium Uniporter Inhibits Aβ-Induced Apoptosis by Reducing Reactive Oxygen Species-Mediated Endoplasmic Reticulum Stress in Cultured Microglia. Brain Res. 2017, 1676, 100– 106,  DOI: 10.1016/j.brainres.2017.08.035
      81
      Inhibition of the mitochondrial calcium uniporter inhibits Aβ-induced apoptosis by reducing reactive oxygen species-mediated endoplasmic reticulum stress in cultured microglia
      Xie, Nanchang; Wu, Chuanjie; Wang, Cui; Cheng, Xuan; Zhang, Lu; Zhang, Haifeng; Lian, Yajun
      Brain Research (2017), 1676 (), 100-106CODEN: BRREAP; ISSN:0006-8993. (Elsevier B.V.)
      Amyloid-beta (Aβ) has been shown to induce microglial apoptosis, which is itself sensitive to disturbed mitochondrial calcium (Ca2+) homeostasis. The mitochondrial calcium uniporter (MCU) plays an important regulatory role in mitochondrial Ca2+ homeostasis, but its role in Aβ-induced microglia apoptosis is unknown. In this study, we found increased mitochondrial Ca2+ concn. in Aβ-treated primary microglia and BV-2 cells; also, the MCU inhibitor Ru360 significantly attenuated Aβ-induced microglial apoptosis, whereas the MCU activator spermine augmented it. In addn., Ru360 significantly attenuated Aβ-induced mitochondrial reactive oxygen species (ROS) prodn., as well as endoplasmic reticulum (ER) stress characterized by glucose-regulated protein 78 (GRP78) and C/-EBP homologous protein (CHOP) expression. Spermine, however, exerted the opposite effects on mitochondrial ROS prodn. and ER stress. We also found that mitochondria-targeted antioxidant (Mito-TEMPO) treatment decreased GRP78 and CHOP expression in Aβ-treated microglia. Moreover, blocking endogenous CHOP expression using a CHOP small interfering RNA (siRNA) attenuated Aβ-induced cell death. Altogether, our data suggested that (1) inhibition of MCU exerts a neuroprotective effect on Aβ-induced microglia apoptosis, and (2) that the underlying mechanism may be related to reducing mitochondrial ROS-mediated ER stress.
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    82. 82
      Alessio, E. Thirty Years of the Drug Candidate NAMI-A and the Myths in the Field of Ruthenium Anticancer Compounds: A Personal Perspective. Eur. J. Inorg. Chem. 2017, 2017, 1549– 1560,  DOI: 10.1002/ejic.201600986
      82
      Thirty Years of the Drug Candidate NAMI-A and the Myths in the Field of Ruthenium Anticancer Compounds: A Personal Perspective
      Alessio, Enzo
      European Journal of Inorganic Chemistry (2017), 2017 (12), 1549-1560CODEN: EJICFO; ISSN:1434-1948. (Wiley-VCH Verlag GmbH & Co. KGaA)
      As anticipated in the title, this contribution is basically divided into two, strictly connected, parts. The first is a personal overview of the ruthenium drug candidate NAMI-A, almost 30 years after its synthesis and the discovery of its unprecedented antimetastatic properties in animal models at nontoxic dosages. The sections relating to the chem. and biol. behavior of the complex, and the hypotheses on its mechanism(s) of action, are kept to a min., whereas more space is devoted to discussion of the results of the clin. investigations. The second part deals in detail with a no. of undemonstrated misconceptions (or myths) that, over the years, have thrived around NAMI-A and other ruthenium drug candidates, thus neg. affecting the whole field of Ru anticancer drugs.
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    83. 83
      Wachter, E.; Heidary, D. K.; Howerton, B. S.; Parkin, S.; Glazer, E. C. Light-Activated Ruthenium Complexes Photobind DNA and are Cytotoxic in the Photodynamic Therapy Window. Chem. Commun. 2012, 48, 9649– 9651,  DOI: 10.1039/c2cc33359g
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      Light-activated ruthenium complexes photobind DNA and are cytotoxic in the photodynamic therapy window
      Wachter, Erin; Heidary, David K.; Howerton, Brock S.; Parkin, Sean; Glazer, Edith C.
      Chemical Communications (Cambridge, United Kingdom) (2012), 48 (77), 9649-9651CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)
      Incorporation of biquinoline ligands into Ru(ii) polypyridyl complexes produces light-activated systems that eject a ligand and photobind DNA after irradn. with visible and near-IR light. Structural anal. shows that distortion facilitates the photochem., and gel shift and cytotoxicity studies prove the compds. act as anti-cancer photodynamic therapy (PDT) agents in the tissue penetrant region.
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    84. 84
      Han Ang, W.; Dyson, P. J. Classical and Non-Classical Ruthenium-Based Anticancer Drugs: Towards Targeted Chemotherapy. Eur. J. Inorg. Chem. 2006, 2006, 4003– 4018,  DOI: 10.1002/ejic.200600723
      There is no corresponding record for this reference.
    85. 85
      Süss-Fink, G. Areneruthenium Complexes as Anticancer Agents. Dalton Trans. 2010, 39, 1673– 1688,  DOI: 10.1039/B916860P
      85
      Arene ruthenium complexes as anticancer agents
      Suess-Fink, Georg
      Dalton Transactions (2010), 39 (7), 1673-1688CODEN: DTARAF; ISSN:1477-9226. (Royal Society of Chemistry)
      A review. Neutral or cationic arene ruthenium complexes providing both hydrophilic as well as hydrophobic properties due to the robustness of the ruthenium-arene unit hold a high potential for the development of metal-based anticancer drugs. Mononuclear arene ruthenium complexes contg. P- or N-donor ligands or N,N-, N,O- or O,O-chelating ligands, dinuclear arene ruthenium systems with adjustable org. linkers, trinuclear arene ruthenium clusters contg. an oxo cap, tetranuclear arene ruthenium porphyrin derivs. that are photoactive, as well as hexanuclear ruthenium cages that are either empty or filled with other mols. have been shown to be active against a variety of cancer cells.
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    86. 86
      Wang, F.; Chen, H.; Parsons, S.; Oswald, I. D. H.; Davidson, J. E.; Sadler, P. J. Kinetics of Aquation and Anation of Ruthenium(II) Arene Anticancer Complexes, Acidity and X-Ray Structures of Aqua Adducts. Chem. - Eur. J. 2003, 9, 5810– 5820,  DOI: 10.1002/chem.200304724
      86
      Kinetics of aquation and anation of ruthenium(II) arene anticancer complexes, acidity and X-ray structures of aqua adducts
      Wang, Fuyi; Chen, Haimei; Parsons, Simon; Oswald, Iain D. H.; Davidson, James E.; Sadler, Peter J.
      Chemistry - A European Journal (2003), 9 (23), 5810-5820CODEN: CEUJED; ISSN:0947-6539. (Wiley-VCH Verlag GmbH & Co. KGaA)
      The aqua adducts of the anticancer complexes [(η6-X)Ru(en)Cl] [PF6] (X = biphenyl (Bip) 1, X = 5,8,9,10-tetrahydroanthracene (THA) 2, X = 9,10-dihydroanthracene (DHA) 3; en = ethylenediamime) were sepd. by HPLC and characterized by mass spectrometry as the products of hydrolysis in H2O. The x-ray structures of the aqua complexes [(η6-X)Ru(en)Y] [PF6]n, X = Bip, Y = 0.5H2O/0.5OH, n = 1.5 (4), X = THA, Y = 0.5H2O/0.5OH, n = 1.5 (5A), X = THA, Y = H2O, n = 2 (5B), and X = DHA, Y = H2O, n = 2(6), are reported. In complex 4 there is a large propeller twist of 45° of the pendant Ph ring with respect to the coordinated Ph ring. Although the THA ligand in 5A and 5B is relatively flat, the DHA ring system in 6 is markedly bent (hinge bend ∼35°) as in the chloro complex 3 (41°). The rates of aquation of 1-3 detd. by UV/visible spectroscopy at various ionic strengths and temps. (1.23-2.59 × 10-3s-1 at 298 K, I = 0.1M) are >20× faster than that of cisplatin. The reverse, anation reactions were very rapid on addn. of 100 mM NaCl (a similar concn. to that in blood plasma). The aquation and anation reactions were about two times faster for the DHA and THA complexes compared to the biphenyl complex. The hydrolysis reactions appear to occur by an associative pathway. The pKa values of the aqua adducts were detd. by 1H NMR spectroscopy as 7.71 for 4, 8.01 for 5 and 7.89 for 6. At physiol.-relevant concns. (0.5-5 μM) and temp. (310 K), the complexes will exist in blood plasma as >89% chloro complex, whereas in the cell nucleus significant amts. (45-65%) of the more reactive aqua adducts would be formed together with smaller amts. of the hydroxo complexes (9-25%, pH 7.4, [Cl-] = 4 mM).
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    87. 87
      Hartinger, C. G.; Jakupec, M. A.; Zorbas-Seifried, S.; Groessl, M.; Egger, A.; Berger, W.; Zorbas, H.; Dyson, P. J.; Keppler, B. K. KP1019, A New Redox-Active Anticancer Agent - Preclinical Development and Results of a Clinical Phase I Study in Tumor Patients. Chem. Biodiversity 2008, 5, 2140– 2155,  DOI: 10.1002/cbdv.200890195
      87
      KP1019, a new redox-active anticancer agent - preclinical development and results of a clinical phase I study in tumor patients
      Hartinger, Christian G.; Jakupec, Michael A.; Zorbas-Seifried, Stefanie; Groessl, Michael; Egger, Alexander; Berger, Walter; Zorbas, Haralabos; Dyson, Paul J.; Keppler, Bernhard K.
      Chemistry & Biodiversity (2008), 5 (10), 2140-2155CODEN: CBHIAM; ISSN:1612-1872. (Verlag Helvetica Chimica Acta)
      A review. The promising drug candidate indazolium trans-[tetrachlorobis(1H-indazole)ruthenate(III)] (KP1019) is the second Ru-based anticancer agent to enter clin. trials. In this review, which is an update of a paper from 2006, the exptl. evidence for the proposed mode of action of this coordination compd. is discussed, including transport into the cell via the transferrin cycle and activation by redn. The results of the early clin. development of KP1019 are summarized in which 5 out of 6 evaluated patients experienced disease stabilization with no severe side effects.
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    88. 88
      Peacock, A. F. A.; Habtemariam, A.; Fernández, R.; Walland, V.; Fabbiani, F. P. A.; Parsons, S.; Aird, R. E.; Jodrell, D. I.; Sadler, P. J. Tuning the Reactivity of Osmium(II) and Ruthenium(II) Arene Complexes under Physiological Conditions. J. Am. Chem. Soc. 2006, 128, 1739– 1748,  DOI: 10.1021/ja055886r
      88
      Tuning the Reactivity of Osmium(II) and Ruthenium(II) Arene Complexes under Physiological Conditions
      Peacock, Anna F. A.; Habtemariam, Abraha; Fernandez, Rafael; Walland, Victoria; Fabbiani, Francesca P. A.; Parsons, Simon; Aird, Rhona E.; Jodrell, Duncan I.; Sadler, Peter J.
      Journal of the American Chemical Society (2006), 128 (5), 1739-1748CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      The OsII arene ethylenediamine (en) complexes [(η6-biphenyl)Os(en)Cl][Z], Z = BPh4 (4) and BF4 (5), are inactive toward A2780 ovarian cancer cells despite 4 being isostructural with an active RuII analog, 4R. Hydrolysis of 5 occurred 40 times more slowly than 4R. The aqua adduct 5A has a low pKa (6.3) compared to that of [(η6-biphenyl)Ru(en)(OH2)]2+ (7.7) and is therefore largely in the hydroxo form at physiol. pH. The rate and extent of reaction of 5 with 9-ethylguanine were also less than those of 4R. The authors replaced the neutral en ligand by anionic acetylacetonate (acac). The complexes [(η6-arene)Os(acac)Cl], arene = biphenyl (6), benzene (7), and p-cymene (8), adopt piano-stool structures similar to those of the RuII analogs and form weak dimers through intermol. (arene)C-H···O(acac) H-bonds. Remarkably, these OsII acac complexes undergo rapid hydrolysis to produce not only the aqua adduct, [(η6-arene)Os(acac)(OH2)]+, but also the hydroxo-bridged dimer, [(η6-arene)Os(μ2-OH)3Os(η6-arene)]+. The pKa values for the aqua adducts 6A, 7A, and 8A (7.1, 7.3, and 7.6, resp.) are lower than that for [(η6-p-cymene)Ru(acac)(OH2)]+ (9.4). Complex 8A rapidly forms adducts with 9-ethylguanine and adenosine, but not with cytidine or thymidine. Despite their reactivity toward nucleobases, complexes 6-8 were inactive toward A549 lung cancer cells. This is attributable to rapid hydrolysis and formation of unreactive hydroxo-bridged dimers which, surprisingly, were the only species present in aq. soln. at biol. relevant concns. Hence, the choice of chelating ligand in OsII (and RuII) arene complexes can have a dramatic effect on hydrolysis behavior and nucleobase binding and provides a means of tuning the reactivity and the potential for discovery of anticancer complexes.
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    89. 89
      Mühlgassner, G.; Bartel, C.; Schmid, W. F.; Jakupec, M. A.; Arion, V. B.; Keppler, B. K. Biological Activity of Ruthenium and Osmium Arene Complexes with Modified Paullones in Human Cancer Cells. J. Inorg. Biochem. 2012, 116, 180– 187,  DOI: 10.1016/j.jinorgbio.2012.06.003
      89
      Biological activity of ruthenium and osmium arene complexes with modified paullones in human cancer cells
      Muehlgassner, Gerhard; Bartel, Caroline; Schmid, Wolfgang F.; Jakupec, Michael A.; Arion, Vladimir B.; Keppler, Bernhard K.
      Journal of Inorganic Biochemistry (2012), 116 (), 180-187CODEN: JIBIDJ; ISSN:0162-0134. (Elsevier)
      In an attempt to combine the ability of indolobenzazepines (paullones) to inhibit cyclin-dependent kinases (Cdks) and that of platinum-group metal ions to interact with proteins and DNA, ruthenium(II) and osmium(II) arene complexes with paullones were prepd., expecting synergies and an increase of soly. of paullones. Complexes with the general formula [MIICl(η6-p-cymene)L]Cl, where M = Ru (1, 3) or Os (2, 4), and L = L1 (1, 2) or L2 (3, 4), L1 = N-(9-bromo-7,12-dihydroindolo[3,2-d][1]-benzazepin-6(5H)-yliden-N'-(2-hydroxybenzylidene)azine and L2 = N-(9-bromo-7,12-dihydroindolo[3,2-d][1]benzazepin-6-yl)-N'-[3-hydroxy-5-(hydroxymethyl)-2-methylpyridin-4-yl-methylene]azinium chloride (L2*HCl), were now investigated regarding cytotoxicity and accumulation in cancer cells, impact on the cell cycle, capacity of inhibiting DNA synthesis and inducing apoptosis as well as their ability to inhibit Cdk activity. The MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) assay yielded IC50 values in the nanomolar to low micromolar range. In accordance with cytotoxicity data, the BrdU assay showed that 1 is the most and 4 the least effective of these compds. regarding inhibition of DNA synthesis. Effects on the cell cycle are minor, although concn.-dependent inhibition of Cdk2/cyclin E activity was obsd. in cell-free expts. Induction of apoptosis is most pronounced for complex 1, accompanied by a low fraction of necrotic cells, as obsd. by annexin V-fluorescein isothiocyanate/propidium iodide staining and flow cytometric anal.
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    90. 90
      Lameijer, L. N.; Ernst, D.; Hopkins, S. L.; Meijer, M. S.; Askes, S. H. C.; Le Dévédec, S. E.; Bonnet, S. A Red-Light-Activated Ruthenium-Caged NAMPT Inhibitor Remains Phototoxic in Hypoxic Cancer Cells. Angew. Chem., Int. Ed. 2017, 56, 11549– 11553,  DOI: 10.1002/anie.201703890
      90
      A Red-Light-Activated Ruthenium-Caged NAMPT Inhibitor Remains Phototoxic in Hypoxic Cancer Cells
      Lameijer, Lucien N.; Ernst, Daniel; Hopkins, Samantha L.; Meijer, Michael S.; Askes, Sven H. C.; Le Devedec, Sylvia E.; Bonnet, Sylvestre
      Angewandte Chemie, International Edition (2017), 56 (38), 11549-11553CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
      We describe two water-sol. ruthenium complexes, [1]Cl2 and [2]Cl2, that photodissociate to release a cytotoxic nicotinamide phosphoribosyltransferase (NAMPT) inhibitor with a low dose (21 J cm-2) of red light in an oxygen-independent manner. Using a specific NAMPT activity assay, up to an 18-fold increase in inhibition potency was measured upon red-light activation of [2]Cl2, while [1]Cl2 was thermally unstable. For the first time, the dark and red-light-induced cytotoxicity of these photocaged compds. could be tested under hypoxia (1 % O2). In skin (A431) and lung (A549) cancer cells, a 3- to 4-fold increase in cytotoxicity was found upon red-light irradn. for [2]Cl2, whether the cells were cultured and irradiated with 1 % or 21 % O2. These results demonstrate the potential of photoactivated chemotherapy for hypoxic cancer cells, in which classical photodynamic therapy, which relies on oxygen activation, is poorly efficient.
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    91. 91
      Dutta, B.; Scolaro, C.; Scopelliti, R.; Dyson, P. J.; Severin, K. Importance of the π-Ligand: Remarkable Effect of the Cyclopentadienyl Ring on the Cytotoxicity of Ruthenium PTA Compounds. Organometallics 2008, 27, 1355– 1357,  DOI: 10.1021/om800025a
      91
      Importance of the π-Ligand: Remarkable Effect of the Cyclopentadienyl Ring on the Cytotoxicity of Ruthenium PTA Compounds
      Dutta, Barnali; Scolaro, Claudine; Scopelliti, Rosario; Dyson, Paul J.; Severin, Kay
      Organometallics (2008), 27 (7), 1355-1357CODEN: ORGND7; ISSN:0276-7333. (American Chemical Society)
      The water-sol. complexes [(Cp'OR)RuCl(PTA)2] (Cp'OR = η5-1-alkoxy-2,4-di-tert-butyl-3-neopentylcyclopentadienyl; R = Me, Et; PTA = 1,3,5-triaza-7-phosphaadamantane) are considerably more cytotoxic (∼2 orders of magnitude) than the cyclopentadienyl analog [CpRuCl(PTA)2] (i.e., IC50 = 4-10 vs. >1000 μM, depending on the cell line). The structure of [(Cp'OMe)RuCl(PTA)2] is reported, together with that of the precursor [(Cp'OEt)Ru(μ-Cl)]2.
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    92. 92
      Allardyce, C. S.; Dyson, P. J. Metal-Based Drugs that Break the Rules. Dalton Trans. 2016, 45, 3201– 3209,  DOI: 10.1039/C5DT03919C
      92
      Metal-based drugs that break the rules
      Allardyce, Claire S.; Dyson, Paul J.
      Dalton Transactions (2016), 45 (8), 3201-3209CODEN: DTARAF; ISSN:1477-9226. (Royal Society of Chemistry)
      Cisplatin and other platinum compds. have had a huge impact in the treatment of cancers and are applied in the majority of anticancer chemotherapeutic regimens. The success of these compds. has biased the approaches used to discover new metal-based anticancer drugs. In this perspective we highlight compds. that are apparently incompatible with the more classical (platinum-derived) concepts employed in the development of metal-based anticancer drugs, with respect to both compd. design and the approaches used to validate their utility. Possible design approaches for the future are also suggested.
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    93. 93
      Kilpin, K. J.; Dyson, P. J. Enzyme Inhibition by Metal Complexes: Concepts, Strategies and Applications. Chem. Sci. 2013, 4, 1410– 1419,  DOI: 10.1039/c3sc22349c
      93
      Enzyme inhibition by metal complexes: concepts, strategies and applications
      Kilpin, Kelly J.; Dyson, Paul J.
      Chemical Science (2013), 4 (4), 1410-1419CODEN: CSHCCN; ISSN:2041-6520. (Royal Society of Chemistry)
      A review. Metal complexes are increasingly being used to inhibit enzymes. The reasons for this increased interest arise from the special features that metal complexes offer, e.g. the facile construction of 3D architectures that tightly fill enzyme active sites increasing selectivity and the possibility of facile coordination to protein residues that enhances enzyme inhibition. In this review we classify the main modes of enzyme inhibition by metal-based complexes and correlate the enzyme inhibition activity to macroscopic properties such as anticancer activity.
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    94. 94
      Barry, N. P. E.; Sadler, P. J. Exploration of the Medical Periodic Table: Towards New Targets. Chem. Commun. 2013, 49, 5106– 5131,  DOI: 10.1039/c3cc41143e
      94
      Exploration of the medical periodic table: towards new targets
      Barry, Nicolas P. E.; Sadler, Peter J.
      Chemical Communications (Cambridge, United Kingdom) (2013), 49 (45), 5106-5131CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)
      A review. Metallodrugs offer potential for unique mechanisms of drug action based on the choice of the metal, its oxidn. state, the types and no. of coordinated ligands and the coordination geometry. We discuss recent progress in identifying new target sites and elucidating the mechanisms of action of anti-cancer, anti-bacterial, anti-viral, anti-parasitic, anti-inflammatory, and anti-neurodegenerative agents, as well as in the design of metal-based diagnostic agents. Progress in identifying and defining target sites has been accelerated recently by advances in proteomics, genomics and metal speciation anal. Examples of metal compds. and chelating agents (enzyme inhibitors) currently in clin. use, clin. trials or preclin. development are highlighted.
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    The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acscentsci.8b00773.

    • Experimental details, complex characterization data, cell viability curves, cell uptake, UV–vis spectra, and dose–response data for Ca2+ uptake inhibition (PDF)

    • X-ray crystal data for C-2 and C-3 (CIF)


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