A Fluorogenic Inhibitor of the Mitochondrial Calcium Uniporter

Abstract

Inhibitors of the mitochondrial calcium uniporter (MCU) are valuable tools for studying the role of mitochondrial Ca²⁺ in various pathophysiological conditions. In this study, a new fluorogenic MCU inhibitor, RuOCou, is presented. This compound is an analogue of the known MCU inhibitor Ru265 that contains fluorescent axial coumarin carboxylate ligands. Upon aquation of RuOCou and release of the axial coumarin ligands, a simultaneous increase in its MCU-inhibitory activity and fluorescence intensity is observed. The fluorescence response of this compound enabled its aquation to be monitored in both HeLa cell lysates and live HeLa cells. This fluorogenic prodrug represents a potential theranostic MCU inhibitor that can be leveraged for the treatment of human diseases related to MCU activity.

Keywords: Bioinorganic Chemistry; Fluorescent Probes; Mitochondrial Calcium Uniporter; Prodrugs; Ruthenium.

Figure 1.

Crystal structure of RuOCou. Thermal ellipsoids are shown at the 50% probability level. Solvents and counterions are omitted for clarity.

Figure 2.

Left: evolution of the emission spectrum of RuOCou (1 μM) in pH 7.4 MOPS-buffered (100 μM) aqueous solution over a 48 h period at 37 °C (excitation wavelength: 402 nm). Right: plot of integrated fluorescence intensity versus time with the best exponential fit.

Figure 3.

Frontier Kohn-Sham molecular orbital diagrams of the coumarin-carboxylate ligand (OCou⁻) and RuOCou with an isovalue of 0.02. Purple arrows: photoexcitation from ground state to the coumarin-based excited state. Blue arrow: electronic transition involved in fluorescence from the coumarin-based excited state. Red wavy arrows: electronic transitions involved in photoinduced electron transfer leading to non-radiative relaxation to the ground state.

Figure 4.

Left: representative traces of extramitochondrial Ca²⁺ clearance after addition of 10 μM Ca²⁺ in permeabilized HEK293T cells treated with either 4 nM Ru265 or RuOCou prepared from stocks that had been pre-incubated at 37 °C (pH 7.4) for different durations. Right: mCa²⁺ uptake rate based on the kinetics of extramitochondrial Ca²⁺ clearance. The mCa²⁺ uptake rate of the untreated cells was normalized to 100.

Figure 5.

Confocal fluorescent microscopy images of HeLa cells treated with 20 μM RuOCou (a) for 24 h and then MitoTracker Red (b) for 30 min. (c) is the overlaid image. Scale bar = 50 μm.

Figure 6.

Confocal fluorescent microscopy images of HeLa cells treated with 75 μM RuOCou for 30 min and subsequently imaged for 40 min. (a): image at 0 min. (b): image at 40 min. (c): mean fluorescence intensity versus time with the initial intensity normalized to 1. Scale bar = 10 μm.

Scheme 1.

Activation pathways of Ru265 and RuOCou.