The therapeutic landscape for AD has remained relatively static over the past two decades, predominantly relying on second-generation AChE inhibitors such as donepezil, rivastigmine, and galantamine, all of which were approved between 1997 and 2001. These AChEIs function by inhibiting AChE, thereby elevating ACh levels and mitigating the cholinergic deficits associated with AD [48].

Despite their clinical utility, existing AChEIs have limitations, including variable efficacy across patients and adverse side effects such as gastrointestinal disturbances and cardiovascular issues [49]. For instance, tacrine, the first AChEI approved in 1993, was withdrawn due to severe hepatotoxicity, highlighting the need for safer alternatives [50]. Consequently, there is a pressing need for the development of novel AChEIs that could offer enhanced efficacy, improved safety profiles, and better cost-effectiveness. Furthermore, emerging research into novel inhibitors may reveal new mechanisms of action or enhance understanding of cholinergic system modulation, potentially leading to more effective and personalized therapeutic strategies. Continued exploration in this field is essential for overcoming the limitations of current treatments and ultimately improving the management of AD.

The concept of “drug repurposing” involves evaluating existing medications, initially developed for one indication, for their potential efficacy in treating different diseases. This strategy leverages the well-established safety profiles and pharmacokinetic data of these drugs, reducing the time and costs associated with drug development. Repurposing can also reveal novel therapeutic applications that were not originally anticipated, thus accelerating the availability of new treatments [51]. In this context, the current study investigates the neuroprotective and AChEI effects of all FDA-approved CNI drugs (Tac, Pim, Csa, and Voc) as therapeutic agents for AD.

The selection of CNI drugs for this study was guided by their established mechanisms of action and emerging evidence linking calcineurin signaling to neurodegeneration [18, 52,53,54]. CNIs such as Tac, Pim, Csa, and Voc, initially developed for managing organ transplant rejection and autoimmune disorders, exhibit potent modulatory effects on calcium-dependent signaling pathways [55]. Dysregulation of calcium homeostasis is closely associated with the neuropathological features of AD, such as neuronal dysfunction, Aβ aggregation, and tau hyperphosphorylation [57,58,59,60,61]. By inhibiting calcineurin, these drugs can attenuate aberrant calcium signaling, potentially reducing the downstream neurotoxic effects that contribute to AD pathogenesis.

In addition to their impact on calcium homeostasis, CNIs possess anti-inflammatory properties, which are particularly relevant to AD, a condition characterized by chronic neuroinflammation [17]. Microglial activation, driven by pro-inflammatory cytokines, exacerbates neuronal damage in AD [62]. By suppressing calcineurin-dependent pathways, these drugs may reduce microglial overactivation and modulate the release of neuroinflammatory mediators, thereby preserving neuronal integrity [53]. These multifaceted mechanisms—modulating cholinergic dysfunction, calcium dysregulation, and neuroinflammation—highlight the potential of CNIs to offer a comprehensive therapeutic approach for AD. Furthermore, their well-characterized safety profiles and pharmacokinetics, established through decades of clinical use, provide a strong foundation for their repurposing as novel AD therapies.

In our study, the binding potential of Tac, Pim, Csa, and Voc to AChE was investigated by molecular docking. To further evaluate the suitability of these compounds for potential therapeutic use, ADMET analysis was performed. In addition, both in vitro AChE inhibitory effects of these drugs and cellular AChE inhibitory activities in neuron-like SH-SY5Y cells exposed to H2O2-induced oxidative stress were examined at the gene level. In addition, the effects of the drugs on cell viability and neurite lengths were determined in the same cell model. This evaluation aimed to determine the ability of these drugs to modulate AChE activity under oxidative conditions and their neuroprotective effects.

Tac, approved in 1994, is a potent immunosuppressant primarily used in organ transplantation [63]. Beyond its CNI effects, recent studies have highlighted its anti-inflammatory and antioxidative actions, suggesting potential neuroprotective benefits [64,65,66,67,68]. However, it is important to note that Tac is associated with nephrotoxicity and neurotoxicity, which limits its broader therapeutic use [69]. Tac, chemically classified as a macrolide, exerts its effects by binding to the immunophilin FKBP-12 (FK506-binding protein), forming a novel FKBP12-FK506 complex. This complex inhibits peptidyl-prolyl isomerase activity and suppresses calcineurin, a critical phosphatase involved in T-lymphocyte signaling. By inhibiting calcineurin, the FKBP12-FK506 complex disrupts the transcription of interleukin-2 (IL-2), thereby effectively attenuating T-cell activation and immune response [70]. Pim is utilized topically for atopic dermatitis and offers targeted immunosuppression with minimal systemic effects [71]. Emerging research indicates that Pim may also exert neuroprotective effects, potentially due to its ability to modulate inflammatory responses and oxidative stress in neural contexts [23, 72]. Like the Tac used, Pim binds to immunophilins to form a complex, and this complex inhibits the calcineurin phosphatase component. This mechanism suppresses cytokine production in T-lymphocytes and thus inflammatory stability. However, Pim was developed to have local rather than systemic effects and is usually used topically in the treatment of inflammatory skin diseases such as atopic dermatitis [73]. Csa, a foundational drug in transplantation since 1983, functions by binding to cyclophilin and inhibiting calcineurin, thereby suppressing T-cell activation [74]. While primarily known for its nephrotoxicity and hypertension, recent studies have begun to explore its neuroprotective potential, particularly through mechanisms involving modulation of neuroinflammation and protection against neurotoxic insults [75,76,77,78]. Voc, a newer drug approved in 2021, provides enhanced pharmacokinetics and a more favorable side effect profile compared to older CNIs [79, 80]. In addition to its primary role in transplantation, Voc may show promising results for neuroprotection by reducing neuroinflammation and oxidative damage, potentially due to its ability to modulate calcineurin activity more precisely. Collectively, while these drugs are well-established for their CNI roles, their emerging neuroprotective properties warrant further investigation in the context of NDs and cholinergic dysfunction.

The binding affinity of human AChE for its substrates and inhibitors is a key determinant of its physiological and pharmacological roles. The enzyme’s binding affinity is primarily dictated by its active site, which includes a catalytic triad composed of serine, histidine, and glutamate residues [81, 82]. This triad is essential for the hydrolysis of acetylcholine and interacts with the substrate through precise electrostatic and hydrophobic interactions.

Recent studies have elucidated that human AChE exhibits high specificity and affinity for ACh, facilitated by a deep and narrow active site gorge that effectively accommodates and catalyzes the breakdown of the neurotransmitter [83]. This structural feature also has implications for the binding of various inhibitors. Inhibitors that target the active site of the enzyme and can form strong non-covalent interactions with the catalytic triad and other important residues in the active site generally show high binding affinity [84]. In summary, the binding affinity characteristics of human AChE are fundamental to understanding its function and interaction with inhibitors. Continued research into these interactions is essential for advancing the development of more effective and selective therapies for NDs.

In this study, the SH-SY5Y cell line was selected as a model system to investigate the neuroprotective and AChE inhibitory effects of CNI drugs under oxidative stress conditions. SH-SY5Y cells, derived from a human neuroblastoma, are widely utilized in neurodegenerative research due to their ability to differentiate into neuron-like cells with morphological, biochemical, and functional characteristics of dopaminergic and cholinergic neurons [85]. This makes them a robust model for studying neuronal responses to various treatments and stressors. A significant advantage of SH-SY5Y cells is their ease of culture and differentiation, which enables the consistent generation of neuron-like cells for experimental studies [86]. (d)-SH-SY5Y cells express key neuronal markers, including neurofilament proteins and synaptic proteins, and exhibit functional ACh release and AChE activity, making them particularly suitable for exploring cholinergic dysfunction in diseases such as AD [86]. Moreover, SH-SY5Y cells are highly amenable to genetic and pharmacological manipulations, allowing for the evaluation of specific molecular pathways and drug effects in a controlled environment [87]. Their responsiveness to oxidative stress, induced by agents like H2O2, further strengthens their relevance for modeling neurodegenerative processes, including oxidative damage and its impact on neuronal survival and function [88]. Overall, the SH-SY5Y cell line offers a cost-effective, reproducible, and human-relevant model system for studying the cellular and molecular mechanisms underlying NDs and for evaluating the therapeutic potential of candidate drugs, including the immunosuppressive agents investigated in this study.

Our findings demonstrate that these CNI drugs exhibit varying degrees of AChE inhibitory activity, with Voc showing the most promising effects. Molecular docking studies revealed that Voc, in particular, has a high binding affinity to AChE, surpassing the standard AChE inhibitors, Gal. This superior binding interaction is attributed to optimized contact with the active site residues of AChE, which likely enhances its inhibitory potential. Although Tac, Pim, and Csa AChE binding affinities were less pronounced compared to Voc and galantamine, they still demonstrated potential as AChE inhibitors. These results suggest that these drugs, particularly Voc, may offer superior AChE inhibition due to their optimal interactions with the enzyme’s active site.

A comparative analysis of the molecular docking results reveals that Voc, Gal, Tac, Pim, and Csa interact with AChE at both overlapping and distinct binding sites, suggesting different modes of inhibition across these compounds. Gal primarily engages residues within the catalytic active site, including Ser203, His447, and Tyr337, and several residues in the anionic and oxyanion subsites. This is characteristic of classical AChE inhibitors designed to block acetylcholine hydrolysis directly. Gal’s interactions suggest a competitive inhibition mechanism, where it binds to the catalytic region, preventing the enzyme from efficiently hydrolyzing acetylcholine. Voc, in contrast, binds predominantly to residues located at the peripheral anionic site (PAS), such as Tyr72, Thr75, Leu76, Trp286, and His287, without direct interaction with the catalytic triad. This suggests that Voc may exert its inhibitory effect through allosteric modulation, potentially altering the enzyme’s conformation or interfering with substrate access. The broader and more hydrophobic interaction profile of Voc, involving residues like Val340 and Phe346, may provide greater binding stability despite the lack of direct catalytic site engagement. Its cyclic peptide structure, enhanced lipophilicity, and rigid conformation may contribute to more stable interactions at the PAS, reinforcing its potential as a non-competitive inhibitor. The distinct pharmacophoric features of Voc thus support both its binding advantage and therapeutic promise in modulating AChE in ND contexts. This non-competitive or mixed-type inhibition mechanism offers a novel approach to cholinergic modulation, especially in ND contexts. Tac, Pim, and Csa also interact with AChE, but their binding profiles differ significantly from those of Gal and Voc. Tac binds at several locations, including residues ASN233, GLY234, PRO235, TRP236, THR238, VAL239, and others, with no direct interaction with the catalytic triad. The broad range of interactions and inclusion of hydrophobic residues like Val303 and Phe346 may indicate an allosteric inhibition mechanism, similar to Voc. Pim shares a similar binding profile with Tac, with interactions at residues such as ASN233, GLY234, and TRP236, as well as additional residues like PRO290 and GLY305. The overlap with Tac in binding regions suggests that Pim may also act through an allosteric modulation mechanism, influencing AChE activity indirectly without direct catalytic site engagement. Csa, while also binding to AChE, interacts with a more distinct set of residues, including GLN71, TYR72, VAL73, THR75, LEU76, and others. These interactions are similar to those observed in Voc at the PAS, but Csa’s binding profile is more limited, suggesting that its inhibitory mechanism may be less potent or less specific compared to the other compounds discussed here. In summary, while Gal acts as a direct competitive inhibitor through interactions with the catalytic site, Voc, Tac, and Pim are more likely to exert their effects through allosteric inhibition, with a broader range of binding sites. Csa also shows an allosteric inhibition profile but with a more limited binding interaction. These differences in binding mechanisms suggest that each molecule may offer unique therapeutic potential for modulating AChE activity in ND, with non-competitive inhibitors like Voc potentially providing novel avenues for cholinergic modulation.

In addition to the molecular docking studies, which revealed favorable binding affinities of Tac, Pim, Csa, and Voc to AChE, MD simulations provided deeper insight into the conformational stability and flexibility of these ligand–AChE complexes. RMSF analysis demonstrated that the AChE–Voc complex exhibited the lowest fluctuation values (1.0–1.4 Å), indicating a highly stable and rigid binding conformation. This observation supports the previously obtained docking results, where Voc showed the strongest binding affinity (− 11.6 kcal/mol), likely due to its extensive interaction network with the catalytic residues. Pim also exhibited low RMSF values (1.2–1.7 Å) around the active site, suggesting a robust and stable binding interface consistent with its high docking score and biological performance. In contrast, the AChE–Csa complex showed the greatest fluctuations (1.8–2.5 Å), suggesting a more dynamic binding interaction despite favorable docking energies. Tac displayed moderate stability, with RMSF values ranging from 1.6 to 2.0 Å, correlating with its intermediate docking performance. These MDS findings reinforce the docking predictions and experimental AChE inhibition data, particularly for Pim and Voc, which exhibited both high structural stability and functional potency. Thus, the integration of MD simulations strengthens the reliability of in silico predictions and further supports the potential of these CNIs as repurposed candidates for modulating cholinergic dysfunction in neurodegenerative diseases.

Voc formed the most extensive and geometrically favorable hydrogen bond network with AChE, including a strong short-range bond (1.99 Å) between its ligand H68 and the side-chain carboxyl group of GLU313. Several other interactions involved key residues such as ASN317 and GLY240, suggesting that Voc effectively stabilizes the binding pocket. This dense and well-oriented interaction pattern aligns with Voc’s superior performance in AChE inhibition assays and its pronounced neuroprotective effects observed in cellular experiments. Pim displayed multiple moderate interactions with the catalytic residue SER203, a critical residue within the oxyanion hole of AChE, with hydrogen bond distances between 2.90 and 3.14 Å. While these interactions support a stable binding, the observed bond angles and longer distances compared to Voc may account for its relatively lower AChE inhibition, despite showing potent anti-apoptotic properties. These findings reinforce the notion that Pim may exert neuroprotection through pathways beyond cholinergic modulation, such as inhibition of caspase activation, as confirmed by our caspase-3 data and supported by previous studies. Csa formed high-quality hydrogen bonds with GLU285 and LEU289, residues that lie close to the peripheral anionic site (PAS) of AChE. Although these bonds are strong and well-oriented (distances < 2.0 Å and DHA angles > 160°), their positioning outside the catalytic gorge may explain Csa’s modest inhibitory effect despite good binding affinity. Such peripheral binding could modulate enzyme conformation or allosteric dynamics without directly blocking the active site. Tac formed two weak hydrogen bonds (3.18 and 2.80 Å) with relatively poor angular geometry (DHA < 65°), suggesting a less stable and possibly transient interaction with the AChE binding site. This may underlie its lower efficacy in both inhibition and neuroprotection observed at the tested concentrations. Finally, the reference compound Gal exhibited two stable and geometrically favorable hydrogen bonds with LEU289 and GLN291 (2.38–2.47 Å, DHA > 140°), consistent with its established role as a potent reversible AChE inhibitor.

The ADMET results for Tac, Pim, Csa, and Voc provide valuable insights into their pharmacokinetic and toxicological profiles, which are critical for assessing their therapeutic potential in AD. The high molecular weights of these drugs, particularly Csa and Voc, may limit their oral bioavailability according to Lipinski’s Rule of Five, yet their calculated LogP values (ranging from 4.35 to 6.08) suggest strong Lipophilicity, which supports their ability to cross the BBB. The hydrogen bond donor and acceptor counts, alongside the TPSA values, indicate moderate permeability, balancing their ability to cross cellular membranes while maintaining solubility. Acute toxicity assessments reveal favorable profiles, as all compounds exhibit no signs of acute toxicity or mutagenicity. However, Pim displayed indications of genotoxic and non-genotoxic carcinogenicity, highlighting a potential safety concern that warrants further investigation. Interestingly, the PPB values, which exceed 80% for all drugs, suggest significant binding to circulating proteins. While this may reduce the free drug concentration available for immediate action, it could prolong their half-lives and therapeutic effects. The Vd values varied among the compounds, with Pim exhibiting the highest Vd (1.762), indicative of its extensive tissue distribution, which may influence its overall efficacy and safety. These ADMET profiles highlight the distinct pharmacokinetic and safety properties of each compound, while highlighting that all four drugs show potential for repurposing as AD therapies.

Although Pim exhibits a logP value exceeding 6.0—indicative of high lipophilicity and, by extension, greater membrane permeability and intracellular accumulation—this pharmacokinetic advantage did not translate into superior neuroprotective efficacy at the 0.1 μM concentration, as observed with other CNIs tested at the same dose. Notably, in our updated findings, Pim at 1 μM provided significantly greater protection against H2O2-induced cytotoxicity in SH-SY5Y cells compared to its 0.1 μM concentration. This result aligns with ADMET predictions, which suggest that higher lipophilicity enhances intracellular drug accumulation and may augment biological activity [89, 90]. However, this relationship appears to be both compound- and mechanism-specific. While Pim displayed superior cell viability at 1 μM in the MTT assay, it was less effective than Voc at inhibiting AChE activity at the same concentration. These findings suggest that the neuroprotective mechanism of Pim may be primarily anti-apoptotic rather than cholinergic, a conclusion supported by our recent study demonstrating the modulation of key apoptotic signaling pathways by 1 μM Pim in H2O2-exposed, neuron-like differentiated SH-SY5Y cells [22]. Collectively, these observations underscore that drug efficacy is a multifactorial outcome, influenced not only by physicochemical properties such as lipophilicity but also by compound-specific mechanisms of action and the cellular context in which the drug is evaluated.

The in vitro AChE inhibition results further underscore the potential of these CNI drugs as therapeutic agents for NDs. Voc demonstrated exceptional inhibitory activity at low concentrations, surpassing Gal at 0.01 µM and 0.1 µM. This finding suggests that Voc’s strong binding affinity, as revealed by molecular docking, translates effectively into functional inhibition of AChE activity. Voc is a CNI characterized by an improved calcineurin-binding profile and a reduced burden of drug and metabolites [91]. This unique pharmacokinetic-pharmacodynamic relationship allows for a high AChE inhibition profile at a dose associated with relatively low calcineurin inhibition. The higher concentrations of Voc (1 µM and 10 µM) exhibited a plateau or reduction in inhibitory efficacy, likely reflecting potential saturation effects or off-target interactions. In contrast, Tac, Pim, and Csa exhibited weaker in vitro inhibitory effects across all tested concentrations. While these drugs may not provide the same level of AChE inhibition as Voc, their moderate effects highlight the potential for their use in combination therapies or in targeting other pathways relevant to neuroprotection. The observed concentration-dependent effects also suggest that optimal dosing is critical for maximizing the therapeutic potential of these drugs while minimizing potential side effects.

Gal was included as a positive control in the in vitro AChE inhibition assays but was not assessed in the cellular neuroprotection experiments. However, this decision was based on the well-documented safety profile of Gal at concentrations up to 10 μM in SH-SY5Y cells. Previous studies have demonstrated that Gal exhibits negligible cytotoxicity at therapeutically relevant concentrations. For instance, Gal induced only modest toxicity (17% reduction in viability) at a concentration of 1.25 mg/mL, with a predicted IC50 of 3.53 mg/mL (12.2 mM) [92]. In a follow-up study, no toxic effects were observed at 0.1 μM, and significant reductions in viability occurred only at concentrations ≥ 100 μM [93]. Kola et al. [94] further confirmed that concentrations up to 50 μM had no cytotoxic effect on differentiated SH-SY5Y cells. Similarly, Girgin et al. [95] reported that Gal-induced cytotoxicity was concentration-dependent, but cell viability remained comparable to controls at concentrations ≤ 10 μM. In support of these findings, Wojtunik-Kulesza et al. [96] reported a CC50 value for Gal close to 5 mM, indicating a wide safety margin. These results collectively validate the concentration range (0–10 μM) used in our AChE assays and support the role of Gal as a reliable reference compound in neuropharmacological research.

The IC50 values derived from enzyme kinetics assays provide a quantitative evaluation of the AChE inhibitory potency of CNIs. Among the tested compounds, Voc exhibited a remarkably low IC50 value of 0.004 µM, indicating a strong inhibitory effect on AChE activity. In comparison, Tac (4.243 µM), Pim (2.148 µM), and CsA (2.335 µM) demonstrated weaker inhibition. Notably, Gal—a clinically approved AChEI—displayed an IC50 value of 0.212 µM in our study, which aligns closely with previously reported values using the Ellman method, typically ranging from 0.2 to 0.5 µM depending on experimental conditions and enzyme source [92, 97, 98]. This consistency not only validates the reliability of our assay but also reinforces the significance of Voc’s much lower IC50. The fact that Voc outperformed Gal in AChE inhibition suggests its high binding affinity and efficacy, possibly due to distinct structural interactions with the active site of the enzyme. Considering its additional neuroprotective effects observed in our cellular models, Voc emerges as a highly promising candidate for further investigation in the context of ND. These findings support the potential of CNIs, particularly Voc, as dual-function agents that may address both cholinergic deficits and oxidative stress–related neurotoxicity in diseases such as AD.

In addition to the effects observed with CNIs, it is important to consider established neuroprotective agents such as Gal for comparison. In addition to its well-known AChE inhibitory effects, Gal has been shown to exert direct neuroprotective actions in oxidative stress models. Zhang et al. [99] demonstrated that N-aryl galantamine analogues protected SH-SY5Y cells from H2O2-induced cytotoxicity. Also, Gal acts as an allosteric potentiating ligand of nicotinic acetylcholine receptors (nAChRs), especially the α7 and α3β4 subtypes, thereby enhancing cholinergic neurotransmission and promoting neuronal survival [100, 101]. Its neuroprotective effects involve activation of the α7 nAChR–phosphatidylinositol 3-kinase (PI3K)–protein kinase B (Akt) signaling pathway, stimulation of Aβ phagocytosis by microglia, and suppression of neuroinflammation through reduced production of pro-inflammatory cytokines (such as IL-6, IL-1β, and TNF-α) and inhibition of nuclear factor kappa B (NF-κB) signaling [102,103,104]. Gal also exhibits antioxidant properties by decreasing ROS levels and attenuating caspase-3 activation, which contributes to its anti-apoptotic activity. Additionally, it supports neuronal survival by upregulating neurotrophic factors such as nerve growth factor (NGF) and insulin-like growth factor 2 (IGF2) [105, 106]. Taken together, these diverse molecular actions distinguish Gal as a multifaceted neuroprotective agent. While our study focused on CNIs, which may act through distinct or partially overlapping mechanisms, future research directly comparing CNIs with Gal in oxidative stress models may help elucidate their relative therapeutic potential.

Building on this comparative framework, we next analyzed the dose-dependent cytotoxic and neuroprotective properties of CNIs under oxidative stress conditions in SH-SY5Y cells. The cytotoxicity and neuroprotective activity of the tested CNIs were further elucidated through a detailed analysis of their dose-dependent effects on SH-SY5Y cells. In the absence of oxidative stress (Fig. 3a), Tac and Voc at 1 and 10 μM concentrations significantly reduced cell viability, indicating potential cytotoxic effects at higher doses. In contrast, Pim and Csa did not show significant cytotoxicity at the tested concentrations, suggesting a more favorable safety profile under basal conditions. Our findings indicate that in (d)-SH-SY5Y cells subjected to oxidative stress via H2O2 treatment, cell viability, neurite length, and the number of neurites per cell were significantly reduced compared to untreated cells. This observation aligns with morphological and functional degeneration commonly reported in oxidative stress-related neurodegenerative conditions. These results are consistent with previous literature findings [23, 25, 107,108,109]. However, under oxidative stress conditions (Fig. 3b), simultaneous treatment with H2O2 and CNIs at 0.1 μM significantly attenuated H2O2-induced cytotoxicity. Interestingly, 1 μM Pim provided even greater protection than its 0.1 μM counterpart, an effect not observed with other CNIs. This may be attributed to the higher lipophilicity of Pim.

In contrast, 10 μM concentrations of Tac, Voc, and Csa when co-administered with H2O2 significantly reduced cell viability compared to H2O2 alone. This suggests that beyond a certain threshold, these compounds may exert synergistic toxicity with oxidative stress, potentially through mechanisms such as mitochondrial dysfunction, calcium overload, or increased ROS production.

Our findings underscore the differential effects of CNIs on oxidative stress-induced apoptosis in neuronal cells (Fig. 3d). The marked increase in caspase-3 levels following H2O2 exposure confirms the pro-apoptotic nature of oxidative damage. Notably, co-treatment with Pim and Voc significantly attenuated caspase-3 activation, indicating their potential to counteract H2O2-induced apoptotic signaling. While no prior studies have directly reported antioxidant properti