Multi-functional activities of citrus flavonoid narirutin in Alzheimer's disease therapeutics: An integrated screening approach and in vitro validation.

Alzheimer's disease (AD) is a complex disorder and the disease mechanism is yet to be properly characterized. Over the years, "amyloid cascade" emerges as principal pathogenic event in AD. β-Secretase 1 (BACE1) controls the rate-limiting step in amyloid beta (Aβ) generation and Aβ rapidly aggregates to form neurotoxic amyloid fibrils. Oxidative stress is one of the principal mediators of the observed neurotoxicity of amyloid fibril. The disease pathogenesis involves induction of multiple signaling cascades and the cross-talk therein. Thus inhibiting multiple targets could be an attractive therapeutic strategy. Here, a multi-target virtual screening protocol has been devised and used to screen an in house developed phytochemical library. Narirutin comes out as a multi-potent phytochemical. Steady-state and time-resolved fluorescence along with molecular modelling studies demonstrate its binding to the BACE1 active site that induces a conformational transition of the protein from open to closed form which precludes substrate recognition. Narirutin possesses strong Aβ aggregation inhibitory potential, evident from ANS and Thioflavin-T binding assay and confirmed by AFM study. ABTS·+ scavenging assay shows moderate antioxidant activity for narirutin. The applicability of a multi-target screening strategy in AD therapeutics is thus demonstrated and the identified hit, narirutin, shows strong multi-potent activity.