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The mitochondrial calcium uniporter (MCU) is a transmembrane protein that resides on the inner membrane of the mitochondria and mediates calcium uptake into this organelle. Given the critical role of mitochondrial calcium trafficking in cellular function, inhibitors of this channel have arisen as tools for studying the biological relevance of this process and as potential therapeutic agents. In this study, four new analogues of the previously reported Ru-based MCU inhibitor [ClRu(NH 3 ) 4 (μ-N)Ru(NH 3 ) 4 Cl]Cl 3 (Ru265) are reported. These compounds, which bear axial carboxylate ligands, are of the general formula [(RCO 2 )Ru(NH 3 ) 4 (μ-N)Ru(NH 3 ) 4 (O 2 CR)]X 3 , where X = NO 3 - or CF 3 SO 3 - and R = H ( 1 ), CH 3 ( 2 ), CH 2 CH 3 ( 3 ), and (CH 2 ) 2 CH 3 ( 4 ). These complexes were fully characterized by IR spectroscopy, NMR spectroscopy, and elemental analysis. X-ray crystal structures of 1 and 3 were obtained, revealing the expected presence of both the linear Ru(μ-N)Ru core and axial formate and propionate ligands. The axial carboxylate ligands of complexes 1 - 4 are displaced by water in buffered aqueous solution to give the aquated compound Ru265'. The kinetics of these processes were measured by 1 H NMR spectroscopy, revealing half-lives that span 5.9-9.9 h at 37 °C. Complex 1 to inhibit mitochondrial calcium uptake in permeabilized HEK293T cells were assessed and compared to that of Ru265. Fresh solutions of 1 - 4 to inhibit mitochondrial calcium uptake in permeabilized HEK293T cells were assessed and compared to that of Ru265. Fresh solutions of 1 - 4 are approximately 2-fold less potent than Ru265 with IC 50 values in the range of 14.7-19.1 nM. Preincubating 1 - 4 in aqueous buffers for longer time periods to allow for the aquation reactions to proceed increases their potency of mitochondrial uptake inhibition to match that of Ru265. This result indicates that 1 - 4 were shown to inhibit mitochondrial calcium uptake in intact, nonpermeabilized cells, revealing their value as tools and potential therapeutic agents for mitochondrial calcium-related disorders.1 - 4 were shown to inhibit mitochondrial calcium uptake in intact, nonpermeabilized cells, revealing their value as tools and potential therapeutic agents for mitochondrial calcium-related disorders.

The mitochondrial calcium uniporter (MCU) mediates uptake of calcium ions (Ca2+) into the mitochondria, a process that is vital for maintaining normal cellular function. Inhibitors of the MCU, the most promising of which are dinuclear ruthenium coordination compounds, have found use as both therapeutic agents and tools for studying the importance of this ion channel. In this study, six Co3+ cage compounds with sarcophagine-like ligands were assessed for their abilities to inhibit MCU-mediated mitochondrial Ca2+ uptake. These complexes were synthesized and characterized according to literature procedures and then investigated in cellular systems for their MCU-inhibitory activities. Among these six compounds, [Co(sen)]3+ (3, sen = 5-(4-amino-2-azabutyl)-5-methyl-3,7-diaza-1,9-nonanediamine) was identified to be a potent MCU inhibitor, with IC50 values of inhibition of 160 and 180 nM in permeabilized HeLa and HEK293T cells, respectively. Furthermore, the cellular uptake of compound 3 was determined, revealing moderate accumulation in cells. Most notably, 3 was demonstrated to operate in intact cells as an MCU inhibitor. Collectively, this work presents the viability of using cobalt coordination complexes as MCU inhibitors, providing a new direction for researchers to investigate in future studies.