Peptide-Based Strategies: Combating Alzheimer's Amyloid ß Aggregation through Ergonomic Design and Fibril Disruption.

Amyloidosis of amyloid-ß (Aß) triggers a cascade of events, leading to oxidative damage and neuronal death. Therefore, inhibiting Aß amyloidosis or disrupting the matured fibrils is the primary target to combat progressive Alzheimer's disease (AD) pathogenesis. Here, we undertake optimization strategies to improve the antiamyloid efficiency of our previously reported NF11 (NAVRWSLMRPF) peptide. Among the series of peptides tested, nontoxic and serum-stable peptide 1 or P1 containing an anthranilic acid residue shows immense potential in not only inhibiting the Aß42 amyloid formation but also disrupting the mature Aß42 fibrils into nontoxic small molecular weight soluble species. Our studies provide high-resolution characterization of the peptide's mechanism of action. With a binding affinity within the micromolar range for both the monomer and aggregated Aß42, this α/ß hybrid peptide can efficiently modulate Aß amyloidosis while facilitating the clearance of toxic aggregates and enforcing protection from apoptosis. Thus, our studies highlight that incorporating a ß-amino acid not only imparts protection from proteolytic degradation and improved stability but also functions effectively as a ß breaker, redirecting the aggregation kinetics toward off-pathway fibrillation.

High-resolution structure of a partially folded insulin aggregation intermediate.

Insulin has long been served as a model for protein aggregation, both due to the importance of aggregation in the manufacture of insulin and because the structural biology of insulin has been extensively characterized. Despite intensive study, details about the initial triggers for aggregation have remained elusive at the molecular level. We show here that at acidic pH, the aggregation of insulin is likely initiated by a partially folded monomeric intermediate. High-resolution structures of the partially folded intermediate show that it is coarsely similar to the initial monomeric structure but differs in subtle details-the A chain helices on the receptor interface are more disordered and the B chain helix is displaced from the C-terminal A chain helix when compared to the stable monomer. The result of these movements is the creation of a hydrophobic cavity in the center of the protein that may serve as nucleation site for oligomer formation. Knowledge of this transition may aid in the engineering of insulin variants that retain the favorable pharamacokinetic properties of monomeric insulin but are more resistant to aggregation.

Enhancing amyloid beta inhibition and disintegration by natural compounds: A study utilizing spectroscopy, microscopy and cell biology.

Amyloid proteins and peptides play a pivotal role in the etiology of various neurodegenerative diseases, including Alzheimer's disease (AD). Synthetically designed small molecules/ peptides/ peptidomimetics show promise towards inhibition of various kinds of amyloidosis. However, exploration of compounds isolated from natural extracts having such potential is lacking. Herein, we have investigated the repurposing of a traditional Indian medicine Lasunadya Ghrita (LG) in AD. LG is traditionally used to treat gut dysregulation and mental illnesses. Various extracts of LG were obtained, characterized, and analyzed for inhibition of Aß aggregation. Biophysical studies show that the water extract of LG (LGWE) is more potent in inhibiting Aß peptide aggregation and defibrillation of Aß40/Aß42 aggregates. NMR studies showed that LGWE binds to the central hydrophobic area and C-terminal residues of Aß40/Aß42, thereby modulating the aggregation, and reducing cell membrane damage. Additionally, LGWE rescues Aß toxicity in neuronal SH-SY5Y cells evident from decreases in ROS generation, membrane leakage, cellular apoptosis, and calcium dyshomeostasis. Notably, LGWE is non-toxic to neuronal cells and mouse models. Our study thus delves into the mechanistic insights of a repurposed drug LGWE with the potential to ameliorate Aß induced neuroinflammation.

Raman Spectroscopic Insights of Phase-Separated Insulin Aggregates.

Phase-separated protein accumulation through the formation of several aggregate species is linked to the pathology of several human disorders and diseases. Our current investigation envisaged detailed Raman signature and structural intricacy of bovine insulin in its various forms of aggregates produced in situ at an elevated temperature (60 °C). The amide I band in the Raman spectrum of the protein in its native-like conformation appeared at 1655 cm-1 and indicated the presence of a high content of α-helical structure as prepared freshly in acidic pH. The disorder content (turn and coils) also was predominately present in both the monomeric and oligomeric states and was confirmed by the presence shoulder amide I maker band at ∼1680 cm-1. However, the band shifted to ∼1671 cm-1 upon the transformation of the protein solution into fibrillar aggregates as produced for a longer time of incubation. The protein, however, maintained most of its helical conformation in the oligomeric phase; the low-frequency backbone α-helical conformation signal at ∼935 cm-1 was similar to that of freshly prepared aqueous protein solution enriched in helical conformation. The peak intensity was significantly weak in the fibrillar aggregates, and it appeared as a good Raman signature to follow the phase separation and the aggregation behavior of insulin and similar other proteins. Tyrosine phenoxy moieties in the protein may maintained its H-bond donor-acceptor integrity throughout the course of fibril formation; however, it entered in more hydrophobic environment in its journey of fibril formation. In addition, it was noticed that oligomeric bovine insulin maintained the orientation/conformation of the disulfide bonds. However, in the fibrillar state, the disulfide linkages became more strained and preferred to maintain a single conformation state.

Coomassie brilliant blue G-250 acts as a potential chemical chaperone to stabilize therapeutic insulin.

Our studies show Coomassie Brilliant Blue G-250 as a promising chemical chaperone that stabilises the α-helical native human insulin conformers, disrupting their aggregation. Furthermore, it also increases the insulin secretion. This multipolar effect coupled with its non-toxic nature could be useful for developing highly bioactive, targeted and biostable therapeutic insulin.

An amphiphilic small molecule drives insulin aggregation inhibition and amyloid disintegration.

The aggregation of proteins into ordered fibrillar structures called amyloids, and their disintegration represent major unsolved problems that limit the therapeutic applications of several proteins. For example, insulin, commonly used for the treatment of diabetes, is susceptible to amyloid formation upon exposure to non-physiological conditions, resulting in a loss of its biological activity. Here, we report a novel amphiphilic molecule called PAD-S, which acts as a chemical chaperone and completely inhibits fibrillation of insulin and its biosimilars. Mechanistic investigations and molecular docking lead to the conclusion that PAD-S binds to key hydrophobic regions of native insulin, thereby preventing its self-assembly. PAD-S treated insulin was biologically active as indicated by its ability to phosphorylate Akt, a protein in the insulin signalling pathway. PAD-S is non-toxic and protects cells from insulin amyloid induced cytotoxicity. The high aqueous solubility and easy synthetic accessibility of PAD-S facilitates its potential use in commercial insulin formulations. Notably, PAD-S successfully disintegrated preformed insulin fibrils to non-toxic smaller fragments. Since the structural and mechanistic features of amyloids are common to several human pathologies, the understanding of the amyloid disaggregation activity of PAD-S will inform the development of small molecule disaggregators for other amyloids.

Zinc oxide nanoparticle interface moderation with tyrosine and tryptophan reverses the pro-amyloidogenic property of the particle.

Zinc oxide nanoparticle with negative surface potential (ZnONP) enhances bovine insulin fibrillation. Here, we are exploring ZnONP with positive surface potential (ZnONPUnc) and surface functionalized with tyrosine and tryptophan amino acids to observe the effects of surface potential and surface functional groups on the fibrillation. ZnONPUnc, despite of inversed surface potential, enhances the insulin fibrillation with increase in the interface concentration at physiological pH. Whereas, the interface moderation with the amino acids mitigates the surface-mediated insulin fibrillation propensity. Additionally, the study indicates that the change in interfacial functional groups at ZnONPUnc significantly reverses the interface-mediated destabilization of insulin conformation. The functional groups from the amino acids, like CO, N-H and aromatic functional groups, are anticipated to further stabilize the insulin conformation by forming hydrogen bond and van der Waals interactions with the key amyloidogenic sequences of insulin, A13-A20 from A-chain and B9-B20 from B-chain. Hence, the altered interaction profile, with change in interfacial functional groups, mitigates the interface-mediated insulin fibrillation and the ZnONPUnc-/fibril-mediated cytotoxicity.

Targeted inhibition of amyloidogenesis using a non-toxic, serum stable strategically designed cyclic peptide with therapeutic implications.

Amyloidogenic disorders are currently rising as a global health issue, prompting more and more studies dedicated to the development of effective targeted therapeutics. The innate affinity of these amyloidogenic proteins towards the biomembranes adds further complexities to the systems. Our previous studies have shown that biologically active peptides can effectively target amyloidogenesis serving as an efficient therapeutic alternative in several amyloidogenic disorders. The structural uniqueness of the PWWP motif in the de novo designed heptapeptide, KR7 (KPWWPRR-NH2) was demonstrated to target insulin fiber elongation specifically. By working on insulin, an important model system in amyloidogenic studies, we gained several mechanistic insights into the inhibitory actions at the protein-peptide interface. Here, we report a second-generation non-toxic and serum stable cyclic peptide, based primarily on the PWWP motif that resulted in complete inhibition of insulin fibrillation both in the presence and absence of the model membranes. Using both low- and high-resolution spectroscopic techniques, we could delineate the specific mechanism of inhibition, at atomistic resolution. Our studies put forward an effective therapeutic intervention that redirects the default aggregation kinetics towards off-pathway fibrillation. Based on the promising results, this novel cyclic peptide can be considered an excellent lead to design pharmaceutical molecules against amyloidogenesis.