In Silico and In Vitro Evaluation of Novel Fiscalin Derivatives as P-Glycoprotein Modulators for Alzheimer’s Disease

Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by amyloid-beta (Aβ) accumulation in the brain. Central nervous system homeostasis is maintained, in part, by the blood-brain barrier (BBB), where the luminal efflux transporter P-glycoprotein (P-gp/ABCB1) plays a key role in clearing Aβ into the systemic circulation. In AD, BBB impairment and the loss of P-gp function disrupt this mechanism, accelerating Aβ deposition, neuroinflammation, and cognitive decline [1]. Fiscalins are valine-derived alkaloids bearing an indolyl moiety and a tricyclic anthranilic acid scaffold, previously reported to modulate P-gp activity and exert neuroprotective effects [2]. This work thus aimed to elucidate, in silico, the P-gp binding sites of 10 new synthetic fiscalin derivatives (FDs), using Molecular Operating Environment (MOE) and AutoDock Vina software, and to evaluate, in vitro, their cytotoxicity (0-50 µM; neutral red uptake and MTT reduction assays; 24 h exposure) and their effects on P-gp activity (rhodamine 123 [Rho123] accumulation assay) in differentiated SH-SY5Y cells. Docking analysis revealed that all FDs bound to P-gp at three binding sites: the drug-binding pocket (DBP) and nucleotide-binding domains (NBDs) 1 and 2. Within the DBP - a typical substrate-binding site - FDs displayed lower (more negative) binding energies than known P-gp substrates, indicating higher affinity. They preferentially occupied the modulator (M) site, except FD5.1, whose lowest binding energy occurred at the Rho123 site. In vitro, FD2-FD6.1 were non-cytotoxic at concentrations up to 25 µM, FD6.2 and FD7 were safe up to 10 µM, and FD1 only at 5 µM. Notably, FD1 and FD4 significantly enhanced P-gp activity, suggesting an increased capacity to efflux substrates across the BBB. Given the role of P-gp in Aβ clearance, these findings identify FDs as a novel chemical scaffold for restoring BBB efflux function, opening a promising avenue for disease-modifying strategies in AD.