Novel fluorescent imaging and theranostic probes targeting mitochondria Marta Domínguez-Prieto, Nicoleta Moisoi and Federico Brucoli De Montfort University, UK
Delocalised lipophilic cations (DLCs) are small-molecules that can selectively accumulate in the mitochondria due to the difference in the negative mitochondrial transmembrane potential of cancer cells compared to normal cells. 1 Mitochondria play key roles in several biological processes, including cellular metabolism, generation of cellular chemical energy and overall cellular homeostasis. Dysfunction, or damage, occurring to these organelles is correlated with pathological conditions such as neurodegenerative disorders, cardiovascular diseases, cancer, obesity, and insulin resistance/type 2 diabetes. 2 There is currently much interest in the development of mitochondria-selective fluorescent probes 3 that can be used to monitor the activity of mitochondria within living cells in real-time and investigate mitochondria-associated dysfunctions in certain diseases. Moreover, one can capitalise on the avidity of cancer cells’ mitochondria for fluorescent DLCs and design fluorescent-dyes that function as mitochondria-targeting anti-cancer and diagnostic (“theranostic”) agents. In this work, we have prepared a focussed library of fluorescent probes, in which a series of aromatic and aliphatic heterocyclic rings were appended to the pyridinium moiety of ( E )-4-(1 H -indol-3-ylvinyl)- N -methylpyridinium iodide (F16), a known fluorescent DLC. 4 The resulting novel probes were characterised for optical properties ( i.e ., absorbance, fluorescence, quantum yield), and screened for anti-proliferative activity against cancer cells, i.e. , human bone osteosarcoma epithelial cells (U2OS), and normal cells, i.e. , human dermal fibroblasts (HDF 6C193). Staining of U2OS cells with the novel fluorescent probes at a concentration of 10 μM in water allowed for the visualisation of mitochondria through confocal microscopy. It was found that one of the probes effectively arrested the growth of the U2OS cells and showed low toxicity in normal fibroblast HDF up to a concentration of 50 µM. On the other hand, F16 was found to have very weak growth inhibition properties in the cancer cell lines used in this study even at the highest concentration, i.e., 100 µM. F16 selectively accumulates in the mitochondria of cancer cells dissipating proton gradient across mitochondrial membrane, but this dye has limited anti-proliferative activity and therapeutic efficacy and can be used as a cargo unit to deliver bioactive compounds to the mitochondria. Here, we have shown that connection of heterocyclic rings to the scaffold of F16 via short alkyl-chain spacers yield derivatives with improved anti-proliferative efficacy, albeit maintaining excellent imaging properties, compared to the parent compound. The probes are currently being evaluated for their ability to bind to predetermined structures within mitochondria with the aim of developing selective, fluorescent anti-cancer agents to track, detect and image these organelles. References 1. T Madak, J., Neamati, N., 2015. Membrane permeable lipophilic cations as mitochondrial directing groups. Current Topics in Medicinal Chemistry , 15 (8), pp.745-766. 2. Wang, H., et al. 2021. Recent advances in chemical biology of mitochondria targeting. Frontiers in Chemistry , 9 , p.683220. 3. Crawford H, et al. 2022. Mitochondrial Targeting and Imaging with Small Organic Conjugated Fluorophores: A Review. Chemistry – A European Journal. 28(72). doi:https://doi.org/10.1002/chem.202202366. 4. Fantin, V.R., et al. 2002. A novel mitochondriotoxic small molecule that selectively inhibits tumor cell growth. Cancer cell , 2 (1), pp.29-42.
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