Conjugation of Dietary Carnosine to Phe-Leu Dipeptide Affords a Potent Inhibitor of Multifaceted Aβ-Induced Neurotoxicity.

Alzheimer's disease (AD) is a chronic neurological disorder manifested by cognitive decline, amyloid-β (Aβ) fibrillation, synaptic dysfunction, and neuronal loss. Furthermore, metal ion dysregulation is implicated in Aβ aggregation, oxidative stress, and neuronal toxicity, thereby exacerbating neurodegeneration. Thus, highly versatile chemical entities with a synergistic effect targeting Aβ aggregation, disintegrate Aβ fibrils, and impede Aβ-Cu2+ interactions hold considerable therapeutic potential against AD. With this aim, a novel tetrapeptide CarFL (β-Ala-His-Phe-Leu) was rationally designed in this work by conjugating a natural dietary endogenous metal chelator, Car, with an Aβ fibrillation inhibitor (FL). Remarkably, CarFL prevented Aβ42 aggregation and disaggregated the preformed Aβ42 fibrils by 75.7 ± 3.8%. Notably, CarFL displayed significant inhibitory activity against Cu2+-induced Aβ42 aggregation (83.6 ± 0.8%) and disaggregated preformed Aβ42/Cu2+ fibrils by 85.6 ± 0.7%. CarFL binds with Cu2+ ions in 1:1 stoichiometry and sequesters Cu2+ from the Aβ42/Cu2+ complex with a binding constant (Ka) of 2.9 × 106 M-1, which, in turn, blocked the Cu2+-catalyzed reactive oxygen species (ROS) production. Importantly, CarFL protected the PC12 cells from Aβ42-mediated toxicity and has a sufficient safety profile as determined by cell viability assay. Furthermore, molecular dynamics (MD) simulations highlighted high-affinity binding (ΔGfit = -11.94 ± 0.42 kcal/mol) of CarFL to the aggregation-prone regions [central hydrophobic core (CHC) and C-terminal] of Aβ42 monomer (Aβ42M) (PDB: 1IYT) with a noteworthy contribution of van der Waals interactions. Importantly, MD simulations depicted that CarFL blocked the conformational transitions in Aβ42M to the aggregation-competent β-sheet structures, consistent with various biophysical studies depicting CarFL-mediated inhibition of Aβ42 fibrillation and disaggregation of preformed fibrillar species. Interestingly, CarFL-mediated noteworthy rise in the helical content from 37.0 ± 1.8 to 54.5 ± 0.6% in Aβ42M-Cu2+ depicts the prevention of Cu2+-induced Aβ42 misfolding. To our knowledge, the present study for the first time depicts the synergistic potential of carnosine conjugated to FL as an inhibitor of multifaceted Aβ-induced cytotoxicity in AD.