Antagonistic Activity of Naphthoquinone-Based Hybrids toward Amyloids Associated with Alzheimer’s Disease and Type-2 Diabetes

Abstract

Protein misfolding and amyloid formation are associated with various human diseases including Alzheimer's disease (AD), Parkinson's disease (PD), and Type-2 Diabetes mellitus (T2DM). No disease-modifying therapeutics are available for them. Despite the lack of sequence homology between the corresponding proteins, aromatic residues are recognized as common key motifs in the formation and stabilization of amyloid structures via π−π stacking. Thus, targeting aromatic recognition interfaces could be a useful approach for inhibiting amyloid formation as well as disrupting the preformed amyloid fibrils. Combining experimental and computational approaches, we demonstrated the anti-amyloidogenic effect of naphthoquinone-tryptophan-based hybrid molecules toward PHF6 (τ-derived aggregative peptide), Amyloid β (Aβ42), and human islet amyloid polypeptide (hIAPP) implicated in AD and T2DM, respectively. These hybrid molecules significantly inhibited the aggregation and disrupted their preformed fibrillar aggregates in vitro, in a dose-dependent manner as evident from Thioflavin T/S binding assay, CD spectroscopy, and electron microscopy. Dye leakage assay from LUVs and cell-based experiments indicated that the hybrid molecules inhibit membrane disruption and cytotoxicity induced by these amyloids. Furthermore, in silico studies provided probable mechanistic insights into the interaction of these molecules with the amyloidogenic proteins in their monomeric or aggregated forms, including the role of hydrophobic interaction, hydrogen bond formation, and packing during inhibition of aggregation and fibril disassembly. Our findings may help in designing novel therapeutics toward AD, T2DM, and other proteinopathies based on the naphthoquinone derived hybrid molecules.

Tryptophan-galactosylamine conjugates inhibit and disaggregate amyloid fibrils of Aβ42 and hIAPP peptides while reducing their toxicity

“…In ThT kinetics, hIAPP was found to aggregate rapidly and reached plateau within~6 h (Fig. 2d), indicating completion of amyloid fibrils formation in agreement with previous report 44 . Upon co-incubation of hIAPP with the hybrid molecules, a dose-response inhibition of hIAPP fibril formation was observed ( Fig.…”

“…This effect was more pronounced at 5-fold molar excess of WGal, under which condition, the CD intensity was reduced markedly and the peak shifted from 218 nm to 211 nm ( Fig. 2c), indicating a substantial reduction of β-sheet content as observed for other amyloid inhibitors of Aβ42 44,65,66 . A similar effect on the secondary structure of Aβ42 was observed in the presence of WGalNH 2 and WGalNAc ( Fig.…”

“…4, bottom panel and Supplementary Fig. 7) as reported 33,44,67 . When hIAPP was incubated with 5-fold molar excess of the hybrid molecules, no typical fibrillar assemblies were observed, rather some amorphous aggregates of different sizes were detected ( Fig.…”

“…CD spectroscopy in the absence or presence of increasing doses of the hybrid molecules was carried out for Aβ42 and hIAPP to evaluate the effect of the conjugates on their conformation 44,61 (Fig. 2c, f and Supplementary Figs.…”

“…Combining the aromatic elements of amino acids and small molecules has also been attempted for amyloid inhibition. For example, a naphthoquinone-tryptophan hybrid was shown to be effective towards various amyloids (including Aβ42, IAPP, Tau, α-Syn) in vitro as well as in vivo [42][43][44][45][46] . However, a major limitation in the drugability of such molecules is their poor solubility in aqueous media [47][48][49] : Attaching a hydrophilic moiety for increasing solubility may enhance the overall therapeutic capacity of a drug 48 .…”