The mitochondrial calcium uniporter (MCU), a transmembrane protein residing on the mitochondrial inner membrane, traffics calcium ions (Ca2+) into the mitochondrial matrix and is crucial in the maintenance of cellular function. This channel has been linked to pathological conditions that implicate mitochondrial Ca2+ (mCa2+) overload, which has inspired the development of MCU inhibitors as potential therapeutic agents. In this work, four analogues of the previously reported Ru-based MCU inhibitor [ClRu(NH3)4(µ-N)(NH3)4RuCl]3+ (Ru265) are described. These compounds, which are of the general formula [(RCO2)ClRu(NH3)4(µ-N)(NH3)4Ru(O2CR)]3+, where R = H (1), CH3 (2), CH2CH3 (3), or (CH2)2CH3 (4), bear axial carboxylate ligands in place of the chloride ligands in Ru265. These complexes were fully characterized, and crystal structures of 1 and 3 were obtained, revealing the linear Ru(µ-N)Ru core and axial carboxylate ligands. Aquation kinetic studies revealed that these complexes are displaced by water in aqueous buffered solutions to give the aquated compound Ru265¢ with half-lives spanning 5.9–9.9 h at 37 °C. In vitro cytotoxicity and mitochondrial membrane potential measurements performed in HeLa and HEK293T cells revealed that these complexes do not negatively impact cell survival or mitochondrial function. The MCU-mediated Ca2+ uptake of HEK293T cells was examined in the presence of each of these complexes, revealing them to be approximately 2-fold less potent inhibitors than Ru265, with IC50 values ranging from 14.7–19.1 nM. Preincubation of these complexes in aqueous buffered solution over 6 h demonstrated that the aquation reactions proceeded to release Ru265¢, increasing the inhibitory activity of mCa2+ uptake over time. Lastly, each compound was effective in inhibiting mCa2+ uptake in intact, non-permeabilized cells, demonstrating their value as potential as therapeutic agents for mCa2+ related disorders.

Aquation-activated prodrugs of mitochondrial calcium uniporter inhibition

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