skip to main content

Attention:

The NSF Public Access Repository (NSF-PAR) system and access will be unavailable from 5:00 PM ET until 11:00 PM ET on Friday, June 21 due to maintenance. We apologize for the inconvenience.


Title: A ferrocene-containing analogue of the MCU inhibitor Ru265 with increased cell permeability

An analogue of the mitochondrial calcium uniporter (MCU) inhibitor Ru265 containing axial ferrocenecarboxylate ligands is reported. This new complex exhibits enhanced cellular uptake compared to the parent compound Ru265.

 
more » « less
Award ID(s):
1750295
NSF-PAR ID:
10467008
Author(s) / Creator(s):
; ; ;
Publisher / Repository:
Royal Society of Chemistry
Date Published:
Journal Name:
Inorganic Chemistry Frontiers
Volume:
10
Issue:
2
ISSN:
2052-1553
Page Range / eLocation ID:
591 to 599
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
More Like this
  1. Abstract Background and Purpose

    Excitotoxicity due to mitochondrial calcium (Ca2+) overloading can trigger neuronal cell death in a variety of pathologies. Inhibiting the mitochondrial calcium uniporter (MCU) has been proposed as a therapeutic avenue to prevent calcium overloading. Ru265 (ClRu(NH3)4(μ‐N)Ru(NH3)4Cl]Cl3) is a cell‐permeable inhibitor of the mitochondrial calcium uniporter (MCU) with nanomolar affinity. Ru265 reduces sensorimotor deficits and neuronal death in models of ischemic stroke. However, the therapeutic use of Ru265 is limited by the induction of seizure‐like behaviours.

    Experimental Approach

    We examined the effect of Ru265 on synaptic and neuronal function in acute brain slices and hippocampal neuron cultures derived from mice, in control and where MCU expression was genetically abrogated.

    Key Results

    Ru265 decreased evoked responses from calyx terminals and induced spontaneous action potential firing of both the terminal and postsynaptic principal cell. Recordings of presynaptic Ca2+currents suggested that Ru265 blocks the P/Q type channel, confirmed by the inhibition of currents in cells exogenously expressing the P/Q type channel. Measurements of presynaptic K+currents further revealed that Ru265 blocked a KCNQ current, leading to increased membrane excitability, underlying spontaneous spiking. Ca2+imaging of hippocampal neurons showed that Ru265 increased synchronized, high‐amplitude events, recapitulating seizure‐like activity seenin vivo. Importantly, MCU ablation did not suppress Ru265‐induced increases in neuronal activity and seizures.

    Conclusions and Implications

    Our findings provide a mechanistic explanation for the pro‐convulsant effects of Ru265 and suggest counter screening assays based on the measurement of P/Q and KCNQ channel currents to identify safe MCU inhibitors.

     
    more » « less
  2. Abstract

    The mitochondrial calcium uniporter (MCU) is the ion channel that mediates Ca2+uptake in mitochondria. Inhibitors of the MCU are valuable as potential therapeutic agents and tools to study mitochondrial Ca2+. The best‐known inhibitor of the MCU is the ruthenium compound Ru360. Although this compound is effective in permeabilized cells, it does not work in intact biological systems. We have recently reported the synthesis and characterization of Ru265, a complex that selectively inhibits the MCU in intact cells. Here, the physical and biological properties of Ru265 and Ru360 are described in detail. Using atomic absorption spectroscopy and X‐ray fluorescence imaging, we show that Ru265 is transported by organic cation transporter 3 (OCT3) and taken up more effectively than Ru360. As an explanation for the poor cell uptake of Ru360, we show that Ru360 is deactivated by biological reductants. These data highlight how structural modifications in metal complexes can have profound effects on their biological activities.

     
    more » « less
  3. Abstract

    The mitochondrial calcium uniporter (MCU) is the ion channel that mediates Ca2+uptake in mitochondria. Inhibitors of the MCU are valuable as potential therapeutic agents and tools to study mitochondrial Ca2+. The best‐known inhibitor of the MCU is the ruthenium compound Ru360. Although this compound is effective in permeabilized cells, it does not work in intact biological systems. We have recently reported the synthesis and characterization of Ru265, a complex that selectively inhibits the MCU in intact cells. Here, the physical and biological properties of Ru265 and Ru360 are described in detail. Using atomic absorption spectroscopy and X‐ray fluorescence imaging, we show that Ru265 is transported by organic cation transporter 3 (OCT3) and taken up more effectively than Ru360. As an explanation for the poor cell uptake of Ru360, we show that Ru360 is deactivated by biological reductants. These data highlight how structural modifications in metal complexes can have profound effects on their biological activities.

     
    more » « less
  4. Abstract

    The synthesis and characterization of the15N‐labeled analogue of the mitochondrial calcium uptake inhibitor [Cl(NH3)4Ru(μ‐N)Ru(NH3)4Cl]3+(Ru265) bearing [15N]NH3ligands is reported. Using [1H,15N] HSQC NMR spectroscopy, the rate constants for the axial chlorido ligand aquation of [15N]Ru265 in pH 7.4 buffer at 25 °C were found to bek1=(3.43±0.03)×10−4 s−1andk2=(4.03±0.09)×10−3 s−1. The reactivity of [15N]Ru265 towards biologically relevant small molecules was also assessed via this method, revealing that this complex can form coordination bonds to anionic oxygen and sulfur donors. Time‐based studies on these ligand‐binding reactions reveal this process to be slow relative to the time required for the complex to inhibit mitochondrial calcium uptake, suggesting that hydrogen‐bonding interactions, rather than the formation of coordination bonds, may play a more significant role in mediating the inhibitory properties of this complex.

     
    more » « less
  5. The mitochondrial calcium (Ca2+) uniporter (MCU) mediates high-capacity mitochondrial Ca2+ uptake implicated in ischemic/reperfusion cell death. We have recently shown that inducible MCU ablation in Thy1-expressing neurons renders mice resistant to sensorimotor deficits and forebrain neuron loss in a model of hypoxic/ischemic (HI) brain injury. These findings encouraged us to compare the neuroprotective effects of Ru360 and the recently identified cell permeable MCU inhibitor Ru265. Unlike Ru360, Ru265 (2-10 µM) reached intracellular concentrations in cultured cortical neurons that preserved cell viability, blocked the protease activity of Ca2+-dependent calpains and maintained mitochondrial respiration and glycolysis after a lethal period of oxygen-glucose deprivation (OGD). Intraperitoneal (i.p.) injection of adult male C57Bl/6 mice with Ru265 (3 mg/kg) also suppressed HI-induced sensorimotor deficits and brain injury. However, higher doses of Ru265 (10 and 30 mg/kg, i.p.) produced dose-dependent increases in the frequency of seizure-like behaviours and the duration of clonic convulsions. Ru265 is proposed to promote convulsions by reducing Ca2+ buffering and energy production in highly energetic interneurons that suppress brain seizure activity. These findings support the potential therapeutic utility of MCU inhibition in the acute management of ischemic stroke but also indicate that such clinical translation will require drug delivery strategies which mitigate the pro-convulsant effects of Ru265. 
    more » « less