Deciphering and modelling remyelinating mechanisms induced by clinically-used azole antifungals with exploitable repurposing properties

INTRODUCTION AND OBJECTIVES One of the physiological stimuli driving myelin production is the exposition of oligodendrocytes precursor cells to retinoic acid, which promotes their maturation and differentiation by modulating the expression of several genes. In the nervous system, the concentration of retinoic acid, a derivative of vitamin A, is regulated by the balance between its synthesis and catabolism. Retinoic acid catabolism is mediated by CYP26 enzymes, members of the cytochrome P450 superfamily. Inhibition of catabolic enzymes may then represent a strategy to raise retinoic acid local concentration and favor its pro-myelinating activity. The aim of our research was to identify CYP26 inhibitors, starting from drugs already marketed for other therapeutic indications to accelerate their possible repurposing in multiple sclerosis reparative therapy. Drugs with these characteristics have already been described: one of these is miconazole, widely employed for fungal infections. The choice of evaluating molecules already of clinical use, specifically of the same family of miconazole (azoles), is tied to the abundance of data regarding their short and long term safety. The strategy of employ an already marketed drug with a different therapeutic indication is technically defined as repurposing, and could permit a rapid identification of useful molecules in multiple sclerosis reparative therapy, with strong reductions in research and development costs. RESULTS We studied the properties of eight azoles, using a combination of computational procedures and cell cultures: the former to identify the most promising compounds, the latter to validate our predictions and assess their efficacy. To evaluate compounds ability to increase the local concentration of retinoic acid, we have first built three-dimensional models of CYP26 and studied its interactions with azoles. These interactions were evaluated by a computational technique named molecular docking. For all the tested compounds, we observed that the drug is able to bind the enzyme and impair its catabolic activity. Itraconazole and posaconazole, very active according to our predictions, and fluconazole, taken as a reference, were selected for further experiments on cell cultures. In the first of the assays on oligodendrocytes precursor cells, we evaluated the effects of two different doses of azoles, administered alone or in combination with a known inhibitor of retinoic acid biosynthesis. The number of cells positive for myelin synthesis was always greater for treated cells compared to controls. In a successive assay, more specific for the myelinating activity, based on a co-culture of neurons and oligodendrocytes precursor cells, we obtained the same positive results when testing the same selected azoles. In a third experiment, we incubated cerebral organoids (micromasses) with the most active azoles (itraconazole and fluconazole), that were identified in the previous steps, even after cellular differentiation interruption induced by serotonine. Fluconazole was able to promote cellular differentiation, while itraconazole, at the tested concentrations, had a toxic activity and blocked cellular proliferation and differentiation of micromass cells. CONCLUSIONS Our investigation took into account all the activities indicated in the granted proposal. Our results provide a proof of concept of the myelinating activity of azoles, known antifungals, by inhibition of retinoic acid catabolism. Specifically, our results suggest that these drugs, efficient CYP26 inhibitors, could be used to promote myelin regeneration in people affected by multiple sclerosis. Among the eight evaluated drugs, fluconazole was proven to be effective in all our experiments, including the micromass assay, the most complex among the employed assays. This experiment, in fact, allows to reproduce the three-dimensional tissue organization of the central nervous system in a simplified manner. It’s our intention to continue along this line of research, by individuating other marketed drugs able to promote myelination mechanisms, with the goal to exploit their additive or synergistic properties with azoles. In this way, it will be possible to lower azole doses and reduce the toxicity and the adverse effects of chronic administration of these drugs.