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

Recent evidence demonstrated the ability of some antifungals to induce (re)myelination in speci c experimental models. These compounds, already marketed for di erent pathologies, are interesting candidates for a repurposing strategy in neurodegenerative diseases, like multiple sclerosis (MS), characterized by strong demyelination. Our preliminary results show that the inhibition of enzymes involved in retinoic acid (RA) catabolism (CYP26), by miconazole and other azole antifungals, induces an increase of cellular RA concentration and that this increase may be behind the observed oligodendrocyte precursor cell (OPC) maturation and di erentiation. On this basis, our proposal is aimed at studying the molecular mechanisms of azole antifungals used in human pharmacology, both in silico and in vitro, and at identifying the most promising ones according to their ability to increase Myelin Basic Protein (MBP) expression on OPCs and to promote their di erentiation and myelination. In our investigation, the ability of 7 azoles to inhibit CYP26 isozymes was evaluated through molecular docking: in silico results were the basis for the selection of the drugs for the in vitro experiments. The selected azoles were tested in three di erent cell models, characterized by increasing complexity: i) OPC cultures, a maturation assay for myelin-producing cells; ii) OPC-dorsal root ganglion (DRG) neuron co-cultures, a myelination assay; iii) cerebral micromass cultures, a cell di erentiation assay for CNS. In parallel, the e ects of exogenous RA and of an inhibitor of the synthesis of RA (citral), were evaluated as well. In the next phase of the project, the outcomes from the di erent in vitro models will be statistically correlated with the pharmacological treatments. Our results will be useful: 1) for identifying the most promising azole antifungals with respect to their pro-myelinating activity, 2) for clarifying the molecular mechanisms underlying their reparative e ect and 3) for formalizing mathematical models to identify the most active concentrations for the most potent azoles.