Evaluating the Neuroprotective and Acetylcholinesterase Inhibitory Properties of Four Calcineurin Inhibitor Drugs: Tacrolimus, Pimecrolimus, Cyclosporin A, and Voclosporin.

Neurodegenerative diseases (ND), marked by progressive neuronal degeneration, often involve dysregulation of acetylcholinesterase (AChE), a key enzyme in cholinergic neurotransmission. AChE inhibition is a well-established therapeutic strategy for Alzheimer's disease (AD), the most prevalent ND, as it aims to restore impaired cholinergic function. However, the effects of calcineurin inhibitors (CNIs), primarily used as immunosuppressants, on AChE activity remain largely unexplored. Recent evidence suggests CNIs possess neuroprotective properties, highlighting their potential for ND treatment. This study evaluated the binding affinities of FDA-approved CNIs-Tacrolimus (Tac), Pimecrolimus (Pim), Cyclosporine A (Csa), and Voclosporin (Voc)-to AChE via molecular docking and molecular dynamic simulation. AChE inhibition was assessed in vitro using the Ellman method and in HO-induced degenerative neuron-like SH-SY5Y cells via ELISA and qRT-PCR. Neuroprotection was examined through MTT assays and neurite analysis. Additionally, the antiapoptotic effect was examined by ELISA analysis measuring caspase-3. Docking studies confirmed strong AChE binding for all CNIs, with Voc exhibiting the highest affinity. Voc demonstrated superior in vitro AChE inhibition, surpassing galantamine at low concentrations. Cellular assays showed that CNIs, particularly Voc, significantly inhibited AChE expression at the gene level. Moreover, Voc markedly restored cell viability and reduced neuronal degeneration in HO-treated cells. These findings suggest CNIs, especially Voc, as promising candidates for ND treatment, targeting AChE overactivity and oxidative stress.