👤 Samantha N. MacMillan

The transmembrane protein known as the mitochondrial calcium uniporter (MCU) mediates the influx of calcium ions (Ca2+) into the mitochondrial matrix. An overload of mitochondrial Ca2+ (mCa2+) is directly linked to damaging effects in pathological conditions. Therefore, inhibitors of the MCU are important chemical biology tools and therapeutic agents. Here, two new analogues of previously reported Ru- and Os-based MCU inhibitors Ru265 and Os245, of the general formula [(C10H15CO2)M(NH3)4(μ-N)M(NH3)4(O2CC10H15)](CF3SO3)3, where M = Ru (1) or Os (2), are reported. These analogues bear adamantane functional groups, which were installed to act as guests for the host molecule cucurbit-[7]-uril (CB[7]). These complexes were characterized and analyzed for their efficiency as guests for CB[7]. As shown through a variety of spectroscopic techniques, each adamantane ligand is encapsulated into one CB[7], affording a supramolecular complex of 1 : 2 stoichiometry. The biological effects of these compounds in the presence and absence of two equiv. CB[7] were assessed. Both complexes 1 and 2 exhibit enhanced cellular uptake compared to the parent compounds Ru265 and Os245, and their uptake is increased further in the presence of CB[7]. Compared to Ru265 and Os245, 1 and 2 are less potent as mCa2+ uptake inhibitors in permeabilized cell models. However, in intact cell systems, 1 and 2 inhibit the MCU at concentrations as low as 1 μM, marking an advantage over Ru265 and Os245 which require an order of magnitude higher doses for similar biological effects. The presence of CB[7] did not affect the inhibitory properties of 1 and 2. Experiments in primary cortical neurons showed that 1 and 2 can elicit protective effects against oxygen-glucose deprivation at lower doses than those required for Ru265 or Os245. At low concentrations, the protective effects of 1 were modulated by CB[7], suggesting that supramolecular complex formation can play a role in these biological conditions. The in vivo biocompatibility of 1 was investigated in mice. The intraperitoneal administration of these compounds and their CB[7] complexes led to time-dependent induction of seizures with no protective effects elicited by CB[7]. This work demonstrates the potential for supramolecular interactions in the development of MCU inhibitors. Show less

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(NH₃)₄(μ-N)Ru(NH₃)₄Cl]Cl₃ (Ru265) are reported. These compounds, which bear axial carboxylate ligands, are of the general formula [(RCO₂)Ru(NH₃)₄(μ-N)Ru(NH₃)₄(O₂CR)]X₃, where X = NO₃^- or CF₃SO₃^- and R = H (1), CH₃ (2), CH₂CH₃ (3), and (CH₂)₂CH₃ (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 ¹H NMR spectroscopy, revealing half-lives that span 5.9-9.9 h at 37 °C. Complex 1 with axial formate ligands underwent aquation approximately twice as fast as the other compounds. In vitro cytotoxicity and mitochondrial membrane potential measurements carried out in HeLa and HEK293T cells demonstrated that none of these four complexes negatively affects cell viability or mitochondrial function. The abilities 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 IC₅₀ 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 are aquation-activated prodrugs of Ru265'. Finally, 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. Show less