PRIMA-1 inhibits Y220C p53 amyloid aggregation and synergizes with cisplatin in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related deaths worldwide. Although many therapeutic options are available, several factors, including the presence of p53 mutations, impact tumor development and therapeutic resistance. TP53 is the second most frequently mutated gene in HCC, comprising more than 30% of cases. Mutations in p53 result in the formation of amyloid aggregates that promote tumor progression. The use of PRIMA-1, a small molecule capable of restoring p53, is a therapeutic strategy to pharmacologically target the amyloid state mutant p53. In this study, we characterize an HCC mutant p53 model for the study of p53 amyloid aggregation in HCC cell lines, from in silico analysis of p53 mutants to a 3D-cell culture model and demonstrate the unprecedented inhibition of Y220C mutant p53 aggregation by PRIMA-1. In addition, our data show beneficial effects of PRIMA-1 in several "gain of function" properties of mutant-p53 cancer cells, including migration, adhesion, proliferation, and drug resistance. We also demonstrate that the combination of PRIMA-1 and cisplatin is a promising approach for HCC therapy. Taken together, our data support the premise that targeting the amyloid-state of mutant p53 may be an attractive therapeutic approach for HCC, and highlight PRIMA-1 as a new candidate for combination therapy with cisplatin.

Biological Evaluation of Valeriana Extracts from Argentina with Potent Cholinesterase Inhibition for the Treatment of Neurodegenerative Disorders and Their Comorbidities-The Case of Valeriana carnosa Sm. (Caprifoliaceae) Studied in Mice.

Alzheimer's disease (AD) is a neurodegenerative disorder whose pathophysiology includes the abnormal accumulation of proteins (e.g., ß-amyloid), oxidative stress, and alterations in neurotransmitter levels, mainly acetylcholine. Here we present a comparative study of the effect of extracts obtained from endemic Argentinian species of valerians, namely V. carnosa Sm., V. clarionifolia Phil. and V. macrorhiza Poepp. ex DC from Patagonia and V. ferax (Griseb.) Höck and V. effusa Griseb., on different AD-related biological targets. Of these anxiolytic, sedative and sleep-inducing valerians, V. carnosa proved the most promising and was assayed in vivo. All valerians inhibited acetylcholinesterase (IC50 between 1.08-12.69 mg/mL) and butyrylcholinesterase (IC50 between 0.0019-1.46 mg/mL). They also inhibited the aggregation of ß-amyloid peptide, were able to chelate Fe2+ ions, and exhibited a direct relationship between antioxidant capacity and phenolic content. Moreover, V. carnosa was able to inhibit human monoamine oxidase A (IC50: 0.286 mg/mL (0.213-0.384)). A daily intake of aqueous V. carnosa extract by male Swiss mice (50 and 150 mg/kg/day) resulted in anxiolytic and antidepressant-like behavior and improved spatial memory. In addition, decreased AChE activity and oxidative stress markers were observed in treated mouse brains. Our studies contribute to the development of indigenous herbal medicines as therapeutic agents for AD.

Modulation of beta-amyloid aggregation using ascorbic acid.

Studies have shown that the level of ascorbic acid (AA) is reduced in the brain of Alzheimer's disease (AD) patients. However, its effect on amyloid-ß 1-42 (Aß42) aggregation has not yet been elucidated. Here we investigated for the first time the effect of AA on Aß42 aggregation using fluorescence assay, circular dichroism, atomic force microscopy, isothermal titration calorimetry, ligand docking, and molecular dynamics. Our results showed that the fibril content decreases in the growth phase when the peptides are co-incubated with AA. AA molecules bind to Aß42 peptides with high binding affinity and a binding site for AA between the ß-strands of Aß42 oligomers prevents the stack of adjacent strands. We demonstrate the inhibitory effect of AA on the aggregation of Aß42 and its molecular interactions, which can contribute to the development of an accessible therapy for AD and also to the design of novel drugs for other amyloidogenic diseases.

Octovespin, a peptide bioinspired by wasp venom, prevents cognitive deficits induced by amyloid-ß in Alzheimer's disease mouse model.

Approximately 46.8 million people have been diagnosed worldwide with dementia, of which the most common type is Alzheimer's disease (AD). Since the current AD treatment is incipient and limited, it is essential to develop new drugs to prevent AD. Considering that evolutionary pressure selected animal venom compounds that are very specific for a unique target, those can be a potential drug against AD. Octovespin was modified from occidentalin-1202, which is a peptide isolated from Polybia occidentalis wasp venom. In this context, this study evaluated the effect of treatment with octovespin against Amyloid-ß (Aß)-induced toxicity, which is postulated to be one of the main causes of AD, in both in vitro and in vivo tests. In vitro, octovespin was able to prevent Aß aggregation in a ThT assay. In vivo, octovespin (0.15 nmol/animal) reverses memory impairment that is due to Aß toxicity, in the Morris Water Maze and Novel Object Recognition Test. Our results suggested that octovespin is a potential drug for the treatment of AD, due to its ability to avoid Aß aggregation in vitro and to prevent Aß -induced memory deficit in mice.

From Hybrids to New Scaffolds: The Latest Medicinal Chemistry Goals in Multi-target Directed Ligands for Alzheimer's Disease.

Alzheimer's disease (AD) is a chronic, progressive, and fatal neurodegenerative disorder affecting cognition, behavior, and function, being one of the most common causes of mental deterioration in elderly people. Once thought as being just developed because of ß amyloid depositions or neurofibrillary Tau tangles, during the last decades, numerous AD-related targets have been established, the multifactorial nature of AD became evident. In this context, the one drug-one target paradigm has resulted in being inefficient in facing AD and other disorders with complex etiology, opening the field for the emergence of the multitarget approach. In this review, we highlight the recent advances within this area, emphasizing in hybridization tools of well-known chemical scaffolds endowed with pharmacological properties concerning AD, such as curcumin-, resveratrol-, chromone- and indole-. We focus mainly on well established and incipient AD therapeutic targets, AChE, BuChE, MAOs, ß-amyloid deposition, 5-HT4 and Serotonin transporter, with the aim to shed light about new insights in the AD multitarget therapy.

Inhibition of ß-amyloid Aggregation of Ugni molinae Extracts.

The ß-amyloid peptide (1-42) is a molecule capable of aggregating into neurotoxic structures that have been implicated as potential etiological factors of Alzheimer's Disease. The aim of this study was to evaluate the inhibition of ß-amyloid aggregation of ethyl acetate and ethanolic extracts obtained from Ugni molinae leaves on neurotoxic actions of ß-amyloid aggregates. Chemical analyses were carried out with the extracts in order to determine their phenolic profile and its quantification. Both extracts showed a tendency to reduce neuronal deaths caused by ß-amyloid. This tendency was inversely proportional to the evaluated concentrations. Moreover, the effect of EAE and ETE on ß-amyloid aggregation was studied by fluorimetric T Thioflavin assay and transmission electronic microscopy (TEM); the extracts showed a modulation in the aggregation process. Partly, it is believed that these effects can be attributed to the polyphenolic compounds present in the extracts.

Green Tea Polyphenol Microparticles Based on the Oxidative Coupling of EGCG Inhibit Amyloid Aggregation/Cytotoxicity and Serve as a Platform for Drug Delivery.

The accumulation of cross-ß-sheet amyloid fibrils is a hallmark of all human amyloid diseases. The compound epigallocatechin-3-gallate (EGCG), the main polyphenol present in green tea, has been described to have beneficial effects in several pathologies, including amyloidogenic diseases. This polyphenol blocks amyloidogenesis and disaggregates a broad range of amyloidogenic peptides comprising amyloid fibrils in vitro. The mechanism by which EGCG acts in the context of amyloid aggregation is not clear. Most of the biological effects of EGCG are attributable to its antioxidant activity. However, EGCG-oxidized products appear to be sufficient for the majority of EGCG amyloid remodeling observed against some polypeptides. If controlled, EGCG oxidation can afford homogenous microparticles (MPs) and can serve as drug delivery agents. Herein, we produced EGCG MPs by oxidative coupling and analyzed their activity during the aggregation of the protein α-synuclein (α-syn), the main protein related to Parkinson's disease. The MPs modestly remodeled mature amyloid fibrils and efficiently inhibited the amyloidogenic aggregation of α-syn. The MPs showed low cytotoxicity against both dopaminergic cells and microglial cells. The MPs reduced the cytotoxic effects of α-syn oligomers. Interestingly, the MPs were loaded with another antiamyloidogenic compound, increasing their activity against amyloid aggregation. We propose the use of EGCG MPs as a bifunctional strategy, blocking amyloid aggregation directly and carrying a molecule that can act synergistically to alleviate the symptoms caused by the amyloidogenic pathway.

Carotenoids as Novel Therapeutic Molecules Against Neurodegenerative Disorders: Chemistry and Molecular Docking Analysis.

Alzheimer's disease (AD) is the most devastating neurodegenerative disorder that affects the aging population worldwide. Endogenous and exogenous factors are involved in triggering this complex and multifactorial disease, whose hallmark is Amyloid-ß (Aß), formed by cleavage of amyloid precursor protein by ß- and γ-secretase. While there is no definitive cure for AD to date, many neuroprotective natural products, such as polyphenol and carotenoid compounds, have shown promising preventive activity, as well as helping in slowing down disease progression. In this article, we focus on the chemistry as well as structure of carotenoid compounds and their neuroprotective activity against Aß aggregation using molecular docking analysis. In addition to examining the most prevalent anti-amyloidogenic carotenoid lutein, we studied cryptocapsin, astaxanthin, fucoxanthin, and the apocarotenoid bixin. Our computational structure-based drug design analysis and molecular docking simulation revealed important interactions between carotenoids and Aß via hydrogen bonding and van der Waals interactions, and shows that carotenoids are powerful anti-amyloidogenic molecules with a potential role in preventing AD, especially since most of them can cross the blood-brain barrier and are considered nutraceutical compounds. Our studies thus illuminate mechanistic insights on how carotenoids inhibit Aß aggregation. The potential role of carotenoids as novel therapeutic molecules in treating AD and other neurodegenerative disorders are discussed.

Resveratrol prevents p53 aggregation in vitro and in breast cancer cells.

One potential target for cancer therapeutics is the tumor suppressor p53, which is mutated in more than 50% of malignant tumors. Loss of function (LoF), dominant negative (DN) and gain of function (GoF) mutations in p53 are associated with amyloid aggregation. We tested the potential of resveratrol, a naturally occurring polyphenol, to interact and prevent the aggregation of wild-type and mutant p53 in vitro using fluorescence spectroscopy techniques and in human breast cancer cells (MDA-MB-231, HCC-70 and MCF-7) using immunofluorescence co-localization assays. Based on our data, an interaction occurs between resveratrol and the wild-type p53 core domain (p53C). In addition, resveratrol and its derivatives pterostilbene and piceatannol inhibit mutant p53C aggregation in vitro. Additionally, resveratrol reduces mutant p53 protein aggregation in MDA-MB-231 and HCC-70 cells but not in the wild-type p53 cell line MCF-7. To verify the effects of resveratrol on tumorigenicity, cell proliferation and cell migration assays were performed using MDA-MB-231 cells. Resveratrol significantly reduced the proliferative and migratory capabilities of these cells. Our study provides evidence that resveratrol directly modulates p53, enhancing our understanding of the mechanisms involved in p53 aggregation and its potential as a therapeutic strategy for cancer treatment.

Exploiting the therapeutic potential of ready-to-use drugs: Repurposing antibiotics against amyloid aggregation in neurodegenerative diseases.

Neurodegenerative diseases are chronic and progressive disorders that affect specific regions of the brain, causing gradual disability and suffering that results in a complete inability of patients to perform daily functions. Amyloid aggregation of specific proteins is the most common biological event that is responsible for neuronal death and neurodegeneration in various neurodegenerative diseases. Therapeutic agents capable of interfering with the abnormal aggregation are required, but traditional drug discovery has fallen short. The exploration of new uses for approved drugs provides a useful alternative to fill the gap between the increasing incidence of neurodegenerative diseases and the long-term assessment of classical drug discovery technologies. Drug re-profiling is currently the quickest possible transition from bench to bedside. In this way, experimental evidence shows that some antibiotic compounds exert neuroprotective action through anti-aggregating activity on disease-associated proteins. The finding that many antibiotics can cross the blood-brain barrier and have been used for several decades without serious toxic effects makes them excellent candidates for therapeutic switching towards neurological disorders. The present review is, to our knowledge, the first extensive evaluation and analysis of the anti-amyloidogenic effect of different antibiotics on well-known disease-associated proteins. In addition, we propose a common structural signature derived from the antiaggregant antibiotic molecules that could be relevant to rational drug discovery.

An ortho-Iminoquinone Compound Reacts with Lysine Inhibiting Aggregation while Remodeling Mature Amyloid Fibrils.

Protein aggregation is a hallmark of several neurodegenerative diseases, including Alzheimer's and Parkinson's diseases. It has been shown that lysine residues play a key role in the formation of these aggregates. Thus, the ability to disrupt aggregate formation by covalently modifying lysine residues could lead to the discovery of therapeutically relevant antiamyloidogenesis compounds. Herein, we demonstrate that an ortho-iminoquinone (IQ) can be utilized to inhibit amyloid aggregation. Using alpha-synuclein and Aß1-40 as model amyloidogenic proteins, we observed that IQ was able to react with lysine residues and reduce amyloid aggregation. We also observed that IQ reacted with free amines within the amyloid fibrils preventing their dissociation and seeding capacity.

Intervening in the ß-barrel structure of lipid binding proteins: consequences on folding, ligand-binding and aggregation propensity.

Natural ß-folds manage to fold up successfully. By contrast, attempts to dissect fragments or peptides from well folded ß-sheet proteins have met with insurmountable difficulties. Here we briefly review selected successful cases of intervention on the well-known scaffold of intestinal fatty acid binding protein (IFABP). Lessons from these examples might set guidelines along the design of proteins belonging to this class. Impact of modifications on topology, binding and aggregation is highlighted. With the aid of abridged variants of IFABP we focus on key structural features responsible for the assembly into oligomeric forms or aggregates.

Decrease of ERK/MAPK overactivation in prefrontal cortex reverses early memory deficit in a mouse model of Alzheimer's disease.

Alzheimer's disease (AD) can be considered as a disease of memory in its initial clinical stages. Amyloid-ß (Aß) peptide accumulation is central to the disease initiation leading later to intracellular neurofibrillary tangles (NFTs) of cytoskeletal tau protein formation. It is under discussion whether different Aß levels of aggregation, concentration, brain area, and/or time of exposure might be critical to the disease progression, as well as which intracellular pathways it activates. The aim of the present work was to study memory-related early molecular and behavioral alterations in a mouse model of AD, in which a subtle deregulation of the physiologic function of Aß can be inferred. For this purpose we used triple-transgenic (3xTg) mice, which develop Aß and tau pathology resembling the disease progression in humans. Memory impairment in novel object recognition task was evident by 5 months of age in 3xTg mice. Hippocampus and prefrontal cortex extra-nuclear protein extracts developed differential patterns of Aß aggregation. ERK1/MAPK showed higher levels of cytosolic activity at 3 months and higher levels of nuclear activity at 6 months in the prefrontal cortex. No significant differences were found in JNK and NF-κB activity and in calcineurin protein levels. Finally, intra-PFC administration of a MEK inhibitor in 6-month-old 3xTg mice was able to reverse memory impairment, suggesting that ERK pathway alterations might at least partially explain memory deficits observed in this model, likely as a consequence of memory trace disruption.