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Title: Computational Design and Evaluation of Novel Peptide Inhibitors Against Amyloid Aggregation in Type 2 Diabetes Mellitus and Alzheimer’s Disease
Authors: Kaur, Apneet
Supervisor: Goyal, Bhupesh
Keywords: Protein aggregation;Alzheimer's disease;Type 2 diabetes;Peptide inhibitor;Molecular dynamics
Issue Date: 19-Jun-2024
Abstract: The self-aggregation of normally dissolvable proteins into soluble oligomers and insoluble amyloid filaments characterizes amyloidosis. These aggregates known as amyloid are made up of misfolded proteins with β–sheet structure, have an impact on normal tissue function, and are associated with prevalent amyloidosis diseases such as Alzheimer's disease (AD), Parkinson's disease, and type 2 diabetes (T2D). The human islet amyloid polypeptide (hIAPP, commonly known as amylin) linked to T2D and amyloid−β (Aβ) peptide linked to AD are classic examples of intrinsically disordered proteins (IDPs) that self-assemble to form amyloid fibrils. The hIAPP (37 amino acid residues hormone) is one of the main secretory components of pancreatic islet β−cells. The self-aggregation of hIAPP into cytotoxic aggregates results in islet β–cells apoptosis and finally leads to T2D. Unfortunately, the underlying molecular mechanism by which hIAPP undergoes structural transition and aggregates into transient oligomers and finally to insoluble amyloid plaques causing T2D symptoms remains unknown. Alzheimer's disease (AD) is a multifactorial neurodegenerative disease mainly characterized by extracellular accumulation of amyloid-β (Aβ) peptide. The self-assembly of amyloid-β (Aβ) peptide generated by the proteolysis of amyloid precursor protein (APP) in the amyloidogenic pathway into toxic oligomers and fibrils is the major event in AD pathogenesis A complete understanding of the underlying process of conformational conversion of functional proteins to toxic oligomers and amyloid fibrils may support the development of medical applications and innovative therapies. Thus, inhibiting the formation of hIAPP and Aβ aggregates has therapeutic benefits for the treatment of T2D, and AD, respectively. Among various inhibitors, short peptides derived from the amyloidogenic regions of hIAPP have been employed as hIAPP aggregation inhibitors due to their low immunogenicity, biocompatibility, and high chemical diversity. Recently, hexapeptide ANFLVH was identified as a potent inhibitor of IAPP aggregation among various synthesized peptides derived from the hIAPP sequence. ANFLVH inhibited IAPP aggregation in vitro and in human islet cultures, which significantly enhanced the islet cell viability. However, the molecular mechanism of inhibition of hIAPP aggregation in the presence of ANFLVH remains unclear. MD simulation analysis highlighted that ANFLVH prevented the conformational transition of hIAPP by stabilizing the native helical conformation. The binding free energy analysis by the MM-PBSA method highlighted favourable binding of ANFLVH with hIAPP and depicted a significant contribution of the van der Waals interaction term in the stability of the hIAPP-ANFLVH complex. The per-residue binding free energy highlighted that ANFLVH strongly interacted with His18 of hIAPP, which has been reported as a key residue in mediating the hIAPP self-assembly process. The hIAPP fragment HSSNN18-22 was identified as an amyloidogenic sequence and displayed higher antiproliferative activity to RIN-5F cells. Notably, various inhibitors have been designed by chemical modifications of the highly amyloidogenic sequence (NFGAIL) of hIAPP and evaluated for their efficacy against hIAPP aggregation. As HSSNN has been identified as a highly amyloidogenic sequence, thus it can be employed as a lead domain for designing new hIAPP aggregation inhibitors. Hence, a library of pentapeptides based on fragment HSSNN18-22 was designed and assessed for their efficacy in blocking hIAPP aggregation using an integrated computational screening approach. The binding free energy calculations by molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) method identified HSSQN and HSSNQ that bind to hIAPP monomer with a binding affinity of –21.25 ± 4.90 and –19.73 ± 3.10 kcal/mol, respectively, which is notably higher as compared to HSSNN (–11.90 ± 4.12 kcal/mol). The sampling of the non aggregation-prone helical conformation was notably increased from 23.5 ± 3.0 in hIAPP monomer to 38.1 ± 3.6, and 33.8 ± 3.0% on the incorporation of HSSQN, and HSSNQ, respectively, which indicate reduced aggregation propensity of hIAPP monomer. The computationally designed peptides, HSSQN and HSSNQ, emerged as new, simple, and efficient inhibitors of hIAPP aggregation. Previous studies reported pentapeptide RIIGL as an effective inhibitor of Aβ aggregation and neurotoxicity induced by Aβ aggregates. A library of 912 pentapeptides based on RIIGL has been designed and assessed for their efficacy in inhibiting Aβ42 aggregation using computational techniques. The MD simulations highlighted that the incorporation of proline and arginine in pentapeptides contributed to their strong binding with Aβ42 monomer. Furthermore, RVVPI and RIAPA prevented conformational conversion of Aβ42 monomer to aggregation-prone structures, which, in turn, resulted in a lower aggregation tendency of Aβ42 monomer. Additionally, the disruptive ability of RIIGL, RVVPI, and RIAPA on Aβ42 protofibril was examined using MD simulations and in vitro studies. Notably, RVVPI displays a more pronounced destabilization effect than other peptides due to higher conformational fluctuations, and disruption of K28-A42 salt bridges in Aβ42 protofibril. Among the synthesized peptides, RVVPI exhibited the highest inhibitory activity (Inhibition= 66.2%, IC50= 5.57 ± 0.83 µM) against Aβ42 aggregation consistent with the computational results. Remarkably, RVVPI displayed ~4.5 fold lower IC50 value as compared to the RIIGL. The ThT and TEM studies highlighted the enhanced efficiency of RVVPI (62.4%) in the disassembly of pre-formed Aβ42 fibrils than RIIGL and RIAPA. The combined in silico and in vitro studies in this work identified a new peptide, RVVPI, as an efficient modulator of Aβ42 aggregation and disassembly of pre-formed Aβ42 aggregates. The work presented in the thesis illuminates the inhibitory mechanism of the peptide inhibitors against hIAPP and Aβ42 aggregation as well as sheds light on the Aβ42 protofibril destabilization on the incorporation of peptides. The computational design methodology of the natural peptides against hIAPP and Aβ42 aggregation provides an opportunity for the design of more potent inhibitors against T2D and AD. The results of the present studies will help in the structure-based design of more effective and potent novel inhibitors against hIAPP and Aβ aggregation, which will prevent or slow down T2D and AD pathogenesis. In addition, the pentapeptides (RVVPI and RIAPA) identified as potential inhibitors against Aβ42 aggregation in this work can be further conjugated with various metal chelating peptides to yield more efficacious and clinically relevant multifunctional modulators possessing abilities to chelate the copper ion and reduce Cu2+-mediated formation of reactive oxygen species. Furthermore, peptides could be used as a template and their affinity, stability, and bioavailability could be improved by the addition of linker molecules/conjugates or could be altered into peptidomimetics with improved stability and cellular delivery.
Appears in Collections:Doctoral Theses@SCBC

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