Enzyme-responsive platforms for mitochondria targeted drug delivery – MitoRedAct

In the last decades, mitochondria has emerged as a therapeutic target of interest. This organelle of eukaryotic cells plays an essential role in the cell's energy production. Mitochondrial disorders have been linked to a wide variety of pathologies such as neurodegenerative diseases, metabolic, cardiovascular and rare mitochondrial diseases. Therefore, since the end of the 1980s, a number of different vectorization strategies have been developed, enabling the transport of active substances into mitochondria. They are based on a non-cleavable covalent linkage between a drug and a specific chemical moiety called mitochondrial vector. The rising number of molecules acting on mitochondrial function that are currently in clinical trials or on the market testifies to the importance of this challenge. In the meantime, the activity of several Red/Ox enzymes has been detected in mitochondria using fluorescence microscopy, thanks to the design and synthesis of enzymatically activatable fluorogenic probes. The MitoRedAct project plans to use these enzymatic activities in order to activate so-called "mito-targeted" pro-drugs. To the best of our knowledge, no similar strategy has been described for the selective release of molecules of therapeutic interest within mitochondria. The aim of this project is to design and then apply strategies for the chemical functionalization of small molecules that will allow their transport and then release selectively within mitochondria. For this purpose, three component molecular systems composed of an enzymatically activatable trigger, a mitochondrial vector, and a fluorogen or a pro-drug will be designed and synthesized. Original "self-immolative" spacers (SISs) incorporating a mito-targeting moiety will be developed. They will make possible the correlation of various enzymatic trigger activation with the release of a fluorophore or a pro-drug in the mitochondrion. The MitoRedAct project is a medicinal chemistry project that is structured around two workpackages. The first one (WP1) aims at the design and synthesis of SISs for the construction of mito-targeted pro-drugs that can be activated by Type 1 nitroreductase, whose presence and activity in the mitochondria has already been demonstrated. They are designed to ensure both the delivery and the release of a wide variety of molecules of therapeutic interest within mitochondria. The selective release of S-adenosyl methionine (SAM) has been chosen as a model for the validation of the "mito-targeted" pro-drug systems that will be prepared. We will study whether this vectorized SAM can compensate for the deficiency of the SLC25A26 carrier (required for SAM delivery into mitochondria), which causes a defective mitochondrial methylation leading to a severe neonatal disease. A second workpackage (WP2) will focus on finding other intra-mitochondrial enzyme activities as an alternative to nitroreductase. This will help broaden the range of mito-targeted pro-drugs. To this aim, mito-targeted fluorogenic probes will be designed, synthesized and tested in vitro and in cellulo. Their fluorogenic response will be induced by the targeted enzyme activity. These probes will be of the "OFF-ON" and/or ratiometric type in order to visualize their activation in cellulo. Confocal microscopy experiments will be conducted to localize the light signal and confirm the intra-mitochondrial nature of the enzymatic activation. The approach proposed is detailed in this project for the detection of azoreductase activity and can easily be extended to other enzymatic activities by modifying the trigger.