Synergistic Effect of Mutations in the Amyloidogenic Core of Human Amylin (hAM) Affords a New Potent Inhibitor of hAM Fibrillation: Computational Design and Experimental Validation

The human amylin, hAM, also known as the human islet amyloid polypeptide (hIAPP), fibrils are identified as a substantial risk element in type 2 diabetes (T2D). Thus, targeting hAM fibrillation could significantly influence the prevention and treatment of T2D. In this work, a library of computationally designed peptides based on the hAM amyloidogenic sequence NFGAILSS, an effective inhibitor of hAM fibrillation, has been generated, which is further evaluated for its ability to effectively attenuate hAM fibrillation. Notably, MM-PBSA analysis identified two new peptides, QYGPILSS (Δ<i>G</i>_binding = -37.66 ± 3.96 kcal/mol) and NWGPILSS (Δ<i>G</i>_binding = -33.10 ± 6.19 kcal/mol), displaying significantly higher binding affinity to hAM as compared to NFGAILSS (Δ<i>G</i>_binding = -21.03 ± 6.56 kcal/mol). Importantly, among the designed peptides, QYGPILSS preserves the helix conformation of hAM to the maximum extent and prevents the conformational transformation of hAM to an aggregation-competent β-sheet conformation, which is further corroborated using CD studies. Furthermore, the thioflavin T (ThT) fluorescence assay depicted no self-fibrillation of QYGPILSS and a maximum inhibitory effect among the synthesized peptides (QYGPILSS inhibition efficiency = 77%, IC₅₀ = 5.51 ± 0.81 μM) against hAM fibrillation, consistent with the computational findings. Notably, DLS and TEM analyses confirmed the modulation in hAM fibrillation upon the incorporation of QYGPILSS. Remarkably, QYGPILSS efficiently rescued human embryonic kidney (HEK293) cells against hAM-induced cytotoxicity by enhancing cell viability in the range of 47.5-81.6% as compared to 35.8% (hAM alone). Remarkably, this study highlights the synergistic effect of three key mutations (N → Q, F → Y, and A → P) in the amyloidogenic fragment of hAM (NFGAILSS) in yielding a new potent inhibitor (QYGPILSS) of hAM fibrillation.