Mutations in the Aβ₄₂ Recognition Sequence KLVFF Afford New Pentapeptide Inhibitors of Aβ₄₂ Fibrillation:...

Alzheimer's disease (AD) is the most prevalent neurodegenerative disorder, marked by the accumulation of amyloid-β (Aβ) plaques in the brain, excessive tau protein phosphorylation, and cholinergic neuron degeneration. In this work, a library of computationally designed peptides based on the Aβ₄₂ recognition sequence KLVFF has been generated, which is further evaluated for its ability to effectively attenuate Aβ₄₂ fibrillation. Notably, molecular mechanics Poisson-Boltzmann surface area analysis identified three new peptides, RPPWF (ΔG_binding = -58.08 ± 9.02 kcal/mol), RPPWY (ΔG_binding = -46.13 ± 3.62 kcal/mol), and KPPWW (ΔG_binding = -44.27 ± 4.08 kcal/mol), displaying significantly higher binding affinity to the Aβ₄₂ monomer (Aβ₄₂m) compared to KLVFF (ΔG_binding = -38.70 ± 17.17 kcal/mol). Importantly, among the designed peptides, RPPWF induces the helix conformation in Aβ₄₂m to the maximum extent and prevents the conformational transitions in Aβ₄₂m to aggregation-competent β-sheet structures. Furthermore, the thioflavin T (ThT) fluorescence assay depicted no self-fibrillation of RPPWF and a maximum inhibitory effect among the synthesized peptides (IC₅₀ = 8.90 ± 0.98 μM) against Aβ₄₂ fibrillation, consistent with the computational results. Notably, DLS and TEM analyses confirmed the RPPWF-induced modulation in Aβ₄₂ fibrillation. RPPWF efficiently rescued PC12 cells from Aβ₄₂ aggregate-induced neurotoxicity by notably enhancing cell viability to 89.6% as compared to 42.2% (Aβ₄₂ alone). Remarkably, this study highlights the synergistic effect of multiple substitutions (K → R, L → P, V → P, and F → W) in the amyloidogenic KLVFF sequence of Aβ₄₂ and depicts the importance of arginine, proline, and tryptophan residues in the RPPWF sequence in affording an efficient new potent inhibitor, RPPWF, of Aβ₄₂ fibrillation. Furthermore, RPPWF has the potential for conjugation or further modifications to afford more effective and clinically relevant multifunctional agents against Aβ₄₂ fibrillation in AD.