Structural Features and Toxicity of α-Synuclein Oligomers Grown in the Presence of DOPAC.

The interplay between α-synuclein and dopamine derivatives is associated with oxidative stress-dependent neurodegeneration in Parkinson's disease (PD). The formation in the dopaminergic neurons of intraneuronal inclusions containing aggregates of α-synuclein is a typical hallmark of PD. Even though the biochemical events underlying the aberrant aggregation of α-synuclein are not completely understood, strong evidence correlates this process with the levels of dopamine metabolites. In vitro, 3,4-dihydroxyphenylacetaldehyde (DOPAL) and the other two metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC) and 3,4-dihydroxyphenylethanol (DOPET), share the property to inhibit the growth of mature amyloid fibrils of α-synuclein. Although this effect occurs with the formation of differently toxic products, the molecular basis of this inhibition is still unclear. Here, we provide information on the effect of DOPAC on the aggregation properties of α-synuclein and its ability to interact with membranes. DOPAC inhibits α-synuclein aggregation, stabilizing monomer and inducing the formation of dimers and trimers. DOPAC-induced oligomers did not undergo conformational transition in the presence of membranes, and penetrated the cell, where they triggered autophagic processes. Cellular assays showed that DOPAC reduced cytotoxicity and ROS production induced by α-synuclein aggregates. Our findings show that the early radicals resulting from DOPAC autoxidation produced covalent modifications of the protein, which were not by themselves a primary cause of either fibrillation or membrane binding inhibition. These findings are discussed in the light of the potential mechanism of DOPAC protection against the toxicity of α-synuclein aggregates to better understand protein and catecholamine biology and to eventually suggest a scaffold that can help in the design of candidate molecules able to interfere in α-synuclein aggregation.

Redesigning the kinetics of lysozyme amyloid aggregation by cephalosporin molecules.

In lysozyme amyloidosis, fibrillar aggregates of lysozyme are associated with severe renal, hepatic, and gastrointestinal manifestations, with no definite therapy. Current drugs are now being tested in amyloidosis clinical trials as aggregation inhibitors to mitigate disease progression. The tetracycline group among antimicrobials in use is in phase II of clinical trials, whereas some macrolides and cephalosporins have shown neuroprotection. In the present study, two cephalosporins, ceftazidime (CZD) and cefotaxime (CXM), and a glycopeptide, vancomycin (VNC), are evaluated for inhibition of amyloid aggregation of hen egg white lysozyme (HEWL) under two conditions (i) 4 M guanidine hydrochloride (GuHCl) at pH 6.5 and 37° C, (ii) At pH 1.5 and 65 °C. Fluorescence quench titration and molecular docking methods report that CZD, CXM, and VNC interact more strongly with the partially folded intermediates (PFI) in comparison to the protein's natural state (N). However, only CZD and CXM proficiently inhibit the aggregation. Transmission electron microscopy, tinctorial assessments, and aggregation kinetics all support oligomer-level inhibition. Transition structures in CZD-HEWL and CXM-HEWL aggregation are shown by circular dichroism (CD). On the other hand, kinetic variables and soluble fraction assays point to a localized association of monomers. Intrinsic fluorescence (IF),1-Anilino 8-naphthalene sulphonic acid, and CD demonstrate structural and conformational modifications redesigning the PFI. GuHCl-induced unfolding and differential scanning fluorimetry suggested that the PFI monomers bound to CZD and CXM exhibited partial stability. Our results present two mechanisms that function in both solution conditions, creating a novel avenue for the screening of putative inhibitors for drug repurposing. We extend our proposed mechanisms in the designing of physical inhibitors of amyloid aggregation considering shorter time frames and foolproof methods.Communicated by Ramaswamy H. Sarma.

Drug repurposing has overcome failures in drug discovery and has reduced the overall time and cost of drug discovery and development.We examined the effect of screened antibiotics, ceftazidime (CZD), cefotaxime (CXM), and vancomycin (VNC) on lysozyme aggregation under two solution conditions.These antibiotics inhibit/modulate the aggregation reactions by strongly interacting with aggregation-prone intermediate and modulation of conformation and stability.Our study puts forward with caution two cephalosporins for aggregation inhibition studies.

Hydrophobic C-Terminal Peptide Analog Aß31-41 Protects the Neurons from Aß-Induced Toxicity.

Spontaneous aggregation of amyloid beta (Aß) leads to the formation of neurotoxic senile plaque considered as the most crucial event in Alzheimer's disease (AD) progression. Inhibition or disruption of this deadly aggregate formation is one of the most efficient strategies for the development of potential therapeutics, and extensive research is in progress by various research groups. In this direction, the development of a peptide analogous to that of the native Aß peptide is an attractive strategy. Based on this rationale, ß-sheet breakers were developed from the Aß central hydrophobic core. These peptide derivatives will bind to the full length of the parent Aß and interfere in self-recognition, thereby preventing the folding of the Aß peptide into cross ß-sheet neurotoxic aggregates. However, this approach is effective in the inhibition of fibrillar aggregation, but this strategy is ineffective in the Aß neurotoxic oligomer formation. Therefore, an alternative and efficient approach is to use the Aß peptide analogous to the C-terminal region, which arbitrates fibrillation and oligomerization. Herein, we developed the Aß C-terminal fragment (ACT-1 to ACT-7) for inhibition of oligomerization as well as fibrillar aggregation. Screening of these seven peptides resulted in an efficient anti-Aß peptide aggregative agent (ACT-7), which was evaluated by the ThT assay peptide. The ThT assay reveals complete inhibition and showed significant neuroprotection of PC-12-derived neurons from Aß-induced toxicity and reduced cell apoptosis. Further, analysis using CD and FTIR spectroscopy reveals that the ACT-7 peptide efficiently inhibits the formation of the ß-sheet secondary structure content. HR-TEM microscopic analysis confirmed the inhibition of formation. Therefore, the inhibition of ß-sheet Aß fibrillary aggregation by the protease-stable ACT-7 peptide may provide a beneficial effect on AD treatment to control the Aß aggregates. Finally, we anticipate that our newly designed ACT peptides may also assist as a template molecular scaffold for designing potential anti-AD therapeutics.

Chaperone Activity and Protective Effect against Aß-Induced Cytotoxicity of Artocarpus camansi Blanco and Amaranthus dubius Mart. ex Thell Seed Protein Extracts.

Alzheimer's disease (AD) is the most common type of dementia and is listed as the sixth-leading cause of death in the United States. Recent findings have linked AD to the aggregation of amyloid beta peptides (Aß), a proteolytic fragment of 39-43 amino acid residues derived from the amyloid precursor protein. AD has no cure; thus, new therapies to stop the progression of this deadly disease are constantly being searched for. In recent years, chaperone-based medications from medicinal plants have gained significant interest as an anti-AD therapy. Chaperones are responsible for maintaining the three-dimensional shape of proteins and play an important role against neurotoxicity induced by the aggregation of misfolded proteins. Therefore, we hypothesized that proteins extracted from the seeds of Artocarpus camansi Blanco (A. camansi) and Amaranthus dubius Mart. ex Thell (A. dubius) could possess chaperone activity and consequently may exhibit a protective effect against Aß1-40-induced cytotoxicity. To test this hypothesis, the chaperone activity of these protein extracts was measured using the enzymatic reaction of citrate synthase (CS) under stress conditions. Then, their ability to inhibit the aggregation of Aß1-40 using a thioflavin T (ThT) fluorescence assay and DLS measurements was determined. Finally, the neuroprotective effect against Aß1-40 in SH-SY5Y neuroblastoma cells was evaluated. Our results demonstrated that A. camansi and A. dubius protein extracts exhibited chaperone activity and inhibited Aß1-40 fibril formation, with A. dubius showing the highest chaperone activity and inhibition at the concentration assessed. Additionally, both protein extracts showed neuroprotective effects against Aß1-40-induced toxicity. Overall, our data demonstrated that the plant-based proteins studied in this research work can effectively overcome one of the most important characteristics of AD.

Antioxidant Polymers with Enhanced Neuroprotection Against Insulin Fibrillation.

Lipoic acid (LA) and dihydrolipoic acid (DHLA) are well established antioxidants to scavenge reactive oxygen species (ROS). However, they are carboxylates with ≈4.7 pKa making them negatively charged at physiological pH (7.4) reducing their passive diffusion through cell membranes. LA is known to be capable of reducing protein fibrillation. Incorporation of LA and especially DHLA in polymer side chains are scarce. Herein, the first examples of the anti-amyloidogenic effect of LA and DHLA incorporated into the side-chain of a block copolymer with a water-soluble poly(polyethylene glycol methyl ether methacrylate) (PPEGMA) segment are presented. The resultant polymers show improved ROS scavenging activity and improved ability to reduce insulin fibrillation compared to free LA and DHLA. Furthermore, the resultant polymers are also capable of disintegrating preformed insulin firbrils. Interestingly, polymers with dihydro-lipoate moieties showed 93% free radical scavenging activity with 91% anti-fibrillating efficacies for insulin protein confirmed by 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay and Thioflavin T (ThT) dye binding study, respectively. Further, the antioxidant polymers increase the cell viability against fibrillar insulin aggregates that may be involved in the etiology of several diseases. Overall, this work reveals that antioxidant polymer-based therapeutic agents can serve as a powerful modulation strategy for developing novel drugs in future against amyloid-related disorders.

Side-Chain Proline-Based Polymers as Effective Inhibitors for In Vitro Aggregation of Insulin.

Insulin fibril formation is considered as the hallmark of several debilitating pathological conditions. To develop effective therapeutics that are able to control the amyloidogenesis process and inhibit fibril formation, herein we have designed a side-chain proline (Pro)-based homopolymer and block copolymers through the reversible addition-fragmentation chain transfer (RAFT) polymerization technique and further explored their obligatory role in the in vitro insulin fibrillation process. Using a variety of biophysical tools, including turbidity measurements, thioflavin T (ThT) fluorescence kinetics, tyrosine (Tyr) fluorescence study, Nile red (NR) fluorescence assay, dynamic light scattering (DLS) study, circular dichroism (CD) measurements, and isothermal titration calorimetry (ITC) techniques, we demonstrated that Pro-based polymers can significantly inhibit the insulin fibrillation process. Among them, the Pro-based homopolymer acts as the most potent inhibitor of insulin fibrillation as confirmed by ThT assay, CD study, and transmission electron microscopic (TEM) analysis. Tyrosine fluorescence measurements and NR fluorescence assay revealed that hydrophobic interactions are the crucial factor that mainly controls the inhibition process. Apart from hydrophobic interactions, polar interactions may also be responsible for the inhibition process as evaluated by ITC study.

Green Tea Extracts EGCG and EGC Display Distinct Mechanisms in Disrupting Aß42 Protofibril.

The amyloid beta (Aß) fibrillar aggregate is the hallmark of Alzheimer's disease (AD). Disassembling preformed fibril or inhibiting Aß aggregation is considered as a therapeutic strategy for AD. Increasing evidence shows that green tea extracts, epigallocatechin-3-gallate (EGCG, containing an extra gallic acid ester group compared to EGC) and epigallocatechin (EGC), can disassociate Aß fibrils and attenuate Aß toxicity. However, the underlying molecular mechanism is poorly understood. Herein, we performed microsecond all-atom molecular dynamics (MD) simulations to investigate the influences of EGCG/EGC on the newly cryo-EM resolved LS-shaped Aß42 protofibrils and their detailed interactions. MD simulations demonstrate that both EGCG and EGC can disrupt Aß42 protofibril and EGCG displays a higher disruptive capacity than EGC. EGCG alters the L-shape of Aß42 protofibril by breaking the hydrogen bond between H6 and E11 through π-π interactions with residues H14/Y10 and hydrogen-bonding interactions with E11, while EGC remodels the L-shape by inserting into the hydrophobic core formed by A2, F4, L34, and V36 and via aromatics interaction with H6/Y10. EGCG disrupts the salt bridges between the K28 side chain and A42 COO- through hydrogen-bonding interaction with A42 and cation-π interaction between its gallic acid ester group and K28, while EGC damages the salt bridges through hydrophobic interactions with V39 and I41 as well as with I32, M35, and V40 located in the C-terminal hydrophobic core. This study demonstrates the pivotal role of the gallic acid ester group of EGCG in disrupting Aß42 protofibril and provides atomic-level insights into the distinct mechanism by which EGCG and EGC disrupt Aß protofibril, which could be useful for designing amyloid inhibitors.