ROF-2 as an Aggregation-Induced Emission (AIE) Probe for Multi-Target Amyloid Detection and Screening of Amyloid Inhibitors.

Misfolding and aggregation of amyloid peptides into ß-structure-rich fibrils represent pivotal pathological features in various neurodegenerative diseases, including Alzheimer's disease (AD), type II diabetes (T2D), and medullary thyroid carcinoma (MTC). The development of effective amyloid detectors and inhibitors for probing and preventing amyloid aggregation is crucial for diagnosing and treating debilitating diseases, yet it poses significant challenges. Here, an aggregation-induced emission (AIE) molecule of ROF2 with multifaceted functionalities as an amyloid probe and a screening tool for amyloid inhibitors using different biophysical, cellular, and worm assays, are reported. As an amyloid probe, ROF2 outperformed ThT, demonstrating its superior sensing capability in monitoring, detecting, and distinguishing amyloid aggregates of different sequences (Amyloid-ß, human islet amyloid polypeptide, or human calcitonin) and sizes (monomers, oligomers, or fibrils). More importantly, the utilization of ROF2 as a screening molecule to identify and repurpose cardiovascular drugs as amyloid inhibitors is introduced. These drugs exhibit potent amyloid inhibition properties, effectively preventing amyloid aggregation and reducing amyloid-induced cytotoxicity both in cells and nematode. The findings present a novel strategy to discovery AIE-based amyloid probes and to be used to repurpose amyloid inhibitors, expanding diagnostic and therapeutic options for neurodegenerative diseases while addressing vascular congestion and amyloid aggregation risks.

Role of Amyloidogenic and Non-Amyloidogenic Protein Spaces in Neurodegenerative Diseases and their Mitigation Using Theranostic Agents.

Neurodegenerative diseases (NDDs) refer to a complex heterogeneous group of diseases which are associated with the accumulation of amyloid fibrils or plaques in the brain leading to progressive loss of neuronal functions. Alzheimer's disease is one of the major NDD responsible for 60-80 % of all dementia cases. Currently, there are no curative or disease-reversing/modifying molecules for many of the NDDs except a few such as donepezil, rivastigmine, galantamine, carbidopa and levodopa which treat the disease-associated symptoms. Similarly, there are very few FDA-approved tracers such as flortaucipir (Tauvid) for tau fibril imaging and florbetaben (Neuraceq), flutemetamol (Vizamyl), and florbetapir (Amyvid) for amyloid imaging available for diagnosis. Recent advances in the cryogenic electron microscopy reported distinctly different microstructures for tau fibrils associated with different tauopathies highlighting the possibility to develop tauopathy-specific imaging agents and therapeutics. In addition, it is important to identify the proteins that are associated with disease development and progression to know about their 3D structure to develop various diagnostics, therapeutics and theranostic agents. The current article discusses in detail the disease-associated amyloid and non-amyloid proteins along with their structural insights. We comprehensively discussed various novel proteins associated with NDDs and their implications in disease pathology. In addition, we document various emerging chemical compounds developed for diagnosis and therapy of different NDDs with special emphasis on theranostic agents for better management of NDDs.

A Peptide Strategy for Inhibiting Different Protein Aggregation Pathways.

Protein aggregation correlates with many human diseases. Protein aggregates differ in structure and shape. Strategies to develop effective aggregation inhibitors that reach the clinic failed so far. Here, we developed a family of peptides targeting early aggregation stages for both amorphous and fibrillar aggregates of proteins unrelated in sequence and structure. They act on dynamic precursors before mechanistic differentiation takes place. Using peptide arrays, we first identified peptides inhibiting the amorphous aggregation of a molten globular, aggregation-prone mutant of the Axin tumor suppressor. Optimization revealed that the peptides activity did not depend on their sequences but rather on their molecular determinants: a composition of 20-30 % flexible, 30-40 % aliphatic and 20-30 % aromatic residues, a hydrophobicity/hydrophilicity ratio close to 1, and an even distribution of residues of different nature throughout the sequence. The peptides also suppressed fibrillation of Tau, a disordered protein that forms amyloids in Alzheimer's disease, and slowed down that of Huntingtin Exon1, an amyloidogenic protein in Huntington's disease, both entirely unrelated to Axin. Our compounds thus target early stages of different aggregation mechanisms, inhibiting both amorphous and amyloid aggregation. Such cross-mechanistic, multi-targeting aggregation inhibitors may be lead compounds for developing drug candidates against various protein aggregation diseases.

η6-Arene Ru(II) Complexes as Modulators of Amyloid Aggregation.

Inorganic medicinal compounds represent a unique and versatile source of potential therapeutics in many diseases and, more recently, in neurodegeneration. Herein we investigated the effects of two η6-arene Ru(II) complexes on the self-aggregation processes of several amyloidogenic peptides endowed with different kinetics and primary sequences. The Ru(II) complexes exhibit, around the metal ion, two chlorides, one NHC = N-heterocyclic carbene, with a glucosyl and a methyl substituent and separately a hexamethylbenzene, which is named Ru1, and one benzene, named Ru2. Both complexes were demonstrated to bind monomeric amyloids suppressing aggregation as evidenced in thioflavin T (ThT) binding assays and autofluorescence experiments. Electrospray ionization mass spectrometry (ESI-MS) indicated the formation of direct adducts between amyloid and metal complexes, which determined the marked conformational variation of peptides and a rescue of cellular viability in SH-SY5Y cells. The complex Ru2 was demonstrated to be a more potent inhibitor of amyloid aggregation compared to Ru1 likely because of the less hindrance of the arene moiety. The presented data strongly support the in vitro ability of η6-arene Ru(II) complexes to suppress amyloid aggregation, providing insights into their potential application as novel therapeutics in neurodegenerative diseases.

Inhibitory effects of extracts from Eucalyptus gunnii on α-synuclein amyloid fibrils.

Amyloid fibril formation is associated with various amyloidoses, including neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Despite the numerous studies on the inhibition of amyloid formation, the prevention and treatment of a majority of amyloid-related disorders are still challenging. In this study, we investigated the effects of various plant extracts on amyloid formation of α-synuclein. We found that the extracts from Eucalyptus gunnii are able to inhibit amyloid formation, and to disaggregate preformed fibrils, in vitro. The extract itself did not lead to cell damage. In the extract, miquelianin, which is a glycosylated form of quercetin and has been detected in the plasma and the brain, was identified and assessed to have a moderate inhibitory activity, compared to the effects of ellagic acid and quercetin, which are strong inhibitors for amyloid formation. The properties of miquelianin provide insights into the mechanisms controlling the assembly of α-synuclein in the brain.

Thiacalixarene Carboxylic Acid Derivatives as Inhibitors of Lysozyme Fibrillation.

Amyloid fibroproliferation leads to organ damage and is associated with a number of neurodegenerative diseases affecting populations worldwide. There are several ways to protect against fibril formation, including inhibition. A variety of organic compounds based on molecular recognition of amino acids within the protein have been proposed for the design of such inhibitors. However, the role of macrocyclic compounds, i.e., thiacalix[4]arenes, in inhibiting fibrillation is still almost unknown. In the present work, the use of water-soluble thiacalix[4]arene derivatives for the inhibition of hen egg-white lysozyme (HEWL) amyloid fibrillation is proposed for the first time. The binding of HEWL by the synthesized thiacalix[4]arenes (logKa = 5.05-5.13, 1:1 stoichiometry) leads to the formation of stable supramolecular systems capable of stabilizing the protein structure and protecting against fibrillation by 29-45%. The macrocycle conformation has little effect on protein binding strength, and the native HEWL secondary structure does not change via interaction. The synthesized compounds are non-toxic to the A549 cell line in the range of 0.5-250 µg/mL. The results obtained may be useful for further investigation of the anti-amyloidogenic role of thiacalix[4]arenes, and also open up future prospects for the creation of new ways to prevent neurodegenerative diseases.

A novel bio-inspired strategy to prevent amyloidogenesis and synaptic damage in Alzheimer's disease.

Alzheimer's disease (AD) is an irreversible neurodegenerative disorder that affects millions of people worldwide. AD pathogenesis is intricate. It primarily involves two main molecular players-amyloid-ß (Aß) and tau-which actually have an intrinsic trend to generate molecular assemblies that are toxic to neurons. Incomplete knowledge of the molecular mechanisms inducing the onset and sustaining the progression of the disease, as well as the lack of valid models to fully recapitulate the pathogenesis of human disease, have until now hampered the development of a successful therapy for AD. The overall experience with clinical trials with a number of potential drugs-including the recent outcomes of studies with monoclonal antibodies against Aß-seems to indicate that Aß-targeting is not effective if it is not accompanied by an efficient challenge of Aß neurotoxic properties. We took advantage from the discovery of a naturally-occurring variant of Aß (AßA2V) that has anti-amyloidogenic properties, and designed a novel bio-inspired strategy for AD based on the intranasal delivery of a six-mer peptide (Aß1-6A2V) retaining the anti-amyloidogenic abilities of the full-length AßA2V variant. This approach turned out to be effective in preventing the aggregation of wild type Aß and averting the synaptic damage associated with amyloidogenesis in a mouse model of AD. The results of our preclinical studies inspired by a protective model already existing in nature, that is the human heterozygous AßA2V carriers which seem to be protected from AD, open the way to an unprecedented and promising approach for the prevention of the disease in humans.

Rational affinity maturation of anti-amyloid antibodies with high conformational and sequence specificity.

The aggregation of amyloidogenic polypeptides is strongly linked to several neurodegenerative disorders, including Alzheimer's and Parkinson's diseases. Conformational antibodies that selectively recognize protein aggregates are leading therapeutic agents for selectively neutralizing toxic aggregates, diagnostic and imaging agents for detecting disease, and biomedical reagents for elucidating disease mechanisms. Despite their importance, it is challenging to generate high-quality conformational antibodies in a systematic and site-specific manner due to the properties of protein aggregates (hydrophobic, multivalent, and heterogeneous) and limitations of immunization (uncontrolled antigen presentation and immunodominant epitopes). Toward addressing these challenges, we have developed a systematic directed evolution procedure for affinity maturing antibodies against Alzheimer's Aß fibrils and selecting variants with strict conformational and sequence specificity. We first designed a library based on a lead conformational antibody by sampling combinations of amino acids in the antigen-binding site predicted to mediate high antibody specificity. Next, we displayed this library on the surface of yeast, sorted it against Aß42 aggregates, and identified promising clones using deep sequencing. The resulting antibodies displayed similar or higher affinities than clinical-stage Aß antibodies (aducanumab and crenezumab). Moreover, the affinity-matured antibodies retained high conformational specificity for Aß aggregates, as observed for aducanumab and unlike crenezumab. Notably, the affinity-maturated antibodies displayed extremely low levels of nonspecific interactions, as observed for crenezumab and unlike aducanumab. We expect that our systematic methods for generating antibodies with unique combinations of desirable properties will improve the generation of high-quality conformational antibodies specific for diverse types of aggregated conformers.

An α-synuclein decoy peptide prevents cytotoxic α-synuclein aggregation caused by fatty acid binding protein 3.

α-synuclein (αSyn) is a protein known to form intracellular aggregates during the manifestation of Parkinson's disease. Previously, it was shown that αSyn aggregation was strongly suppressed in the midbrain region of mice that did not possess the gene encoding the lipid transport protein fatty acid binding protein 3 (FABP3). An interaction between these two proteins was detected in vitro, suggesting that FABP3 may play a role in the aggregation and deposition of αSyn in neurons. To characterize the molecular mechanisms that underlie the interactions between FABP3 and αSyn that modulate the cellular accumulation of the latter, in this report, we used in vitro fluorescence assays combined with fluorescence microscopy, transmission electron microscopy, and quartz crystal microbalance assays to characterize in detail the process and consequences of FABP3-αSyn interaction. We demonstrated that binding of FABP3 to αSyn results in changes in the aggregation mechanism of the latter; specifically, a suppression of fibrillar forms of αSyn and also the production of aggregates with an enhanced cytotoxicity toward mice neuro2A cells. Because this interaction involved the C-terminal sequence region of αSyn, we tested a peptide derived from this region of αSyn (αSynP130-140) as a decoy to prevent the FABP3-αSyn interaction. We observed that the peptide competitively inhibited binding of αSyn to FABP3 in vitro and in cultured cells. We propose that administration of αSynP130-140 might be used to prevent the accumulation of toxic FABP3-αSyn oligomers in cells, thereby preventing the progression of Parkinson's disease.

Nonphosphorylated tau slows down Aß1-42 aggregation, binds to Aß1-42 oligomers, and reduces Aß1-42 toxicity.

The formation of neurofibrillary tangles and amyloid plaques accompanies the progression of Alzheimer's disease. Tangles are made of fibrillar aggregates formed by the microtubule-associated protein tau, whereas plaques comprise fibrillar forms of amyloid-beta (Aß). Both form toxic oligomers during aggregation and are thought to interact synergistically to each promote the accumulation of the other. Recent in vitro studies have suggested that the monomeric nonphosphorylated full-length tau protein hinders the aggregation of Aß1-40 peptide, but whether the same is true for the more aggregation-prone Aß1-42 was not determined. We used in vitro and in vivo techniques to explore this question. We have monitored the aggregation kinetics of Aß1-42 by thioflavine T fluorescence in the presence or the absence of different concentrations of nonphosphorylated tau. We observed that elongation of Aß1-42 fibrils was inhibited by tau in a dose-dependent manner. Interestingly, the fibrils were structurally different in the presence of tau but did not incorporate tau. Surface plasmon resonance indicated that tau monomers bound to Aß1-42 oligomers (but not monomers) and hindered their interaction with the anti-Aß antibody 4G8, suggesting that tau binds to the hydrophobic central core of Aß recognized by 4G8. Tau monomers also antagonized the toxic effects of Aß oligomers in Caenorhabditis elegans. This suggests that nonphosphorylated tau might have a neuroprotective effect by binding Aß1-42 oligomers formed during the aggregation and shielding their hydrophobic patches.

Compound screening identified gossypetin and isoquercitrin as novel inhibitors for amyloid fibril formations of Vλ6 proteins associated with AL amyloidosis.

AL amyloidosis is a life-threatening disease characterized by the deposition of amyloidogenic immunoglobulin light chain secreted from clonal plasma cells. Here we established an in-vitro screening system of amyloid inhibition of a variable domain in λ6 light chain mutant (Vλ6), Wil, and screened a food-additive compound library to identify compounds inhibiting the fibril formation. We found gossypetin and isoquercitrin as novel inhibitors. NMR analysis showed that both compounds directly interacted with natively-folded Wil, and proteolysis experiments demonstrated that these compounds conferred proteolytic resistance, suggesting that the compounds enhance the kinetic stability of Wil. Since gossypetin and isoquercitrin specifically interacted with the protein at micromolar concentrations, these compounds could be used as lead to further develop inhibitors against AL amyloidosis.

Targeting transthyretin - Mechanism-based treatment approaches and future perspectives in hereditary amyloidosis.

The liver-derived, circulating transport protein transthyretin (TTR) is the cause of systemic hereditary (ATTRv) and wild-type (ATTRwt) amyloidosis. TTR stabilization and knockdown are approved therapies to mitigate the otherwise lethal disease course. To date, the variety in phenotypic penetrance is not fully understood. This systematic review summarizes the current literature on TTR pathophysiology with its therapeutic implications. Tetramer dissociation is the rate-limiting step of amyloidogenesis. Besides destabilizing TTR mutations, other genetic (RBP4, APCS, AR, ATX2, C1q, C3) and external (extracellular matrix, Schwann cell interaction) factors influence the type of onset and organ tropism. The approved small molecule tafamidis stabilizes the tetramer and significantly decelerates the clinical course. By sequence-specific mRNA knockdown, the approved small interfering RNA (siRNA) patisiran and antisense oligonucleotide (ASO) inotersen both significantly reduce plasma TTR levels and improve neuropathy and quality of life compared to placebo. With enhanced hepatic targeting capabilities, GalNac-conjugated siRNA and ASOs have recently entered phase III clinical trials. Bivalent TTR stabilizers occupy both binding groves in vitro, but have not been tested in trials so far. Tolcapone is another stabilizer with the potential to cross the blood-brain barrier, but its half-life is short and liver failure a potential side effect. Amyloid-directed antibodies and substances like doxycycline aim at reducing the amyloid load, however, none of the yet developed antibodies has successfully passed clinical trials. ATTR-amyloidosis has become a model disease for pathophysiology-based treatment. Further understanding of disease mechanisms will help to overcome the remaining limitations, including application burden, side effects, and blood-brain barrier permeability.

Structural Insights into Curli CsgA Cross-ß Fibril Architecture Inspire Repurposing of Anti-amyloid Compounds as Anti-biofilm Agents.

Curli amyloid fibrils secreted by Enterobacteriaceae mediate host cell adhesion and contribute to biofilm formation, thereby promoting bacterial resistance to environmental stressors. Here, we present crystal structures of amyloid-forming segments from the major curli subunit, CsgA, revealing steric zipper fibrils of tightly mated ß-sheets, demonstrating a structural link between curli and human pathological amyloids. D-enantiomeric peptides, originally developed to interfere with Alzheimer's disease-associated amyloid-ß, inhibited CsgA fibrillation and reduced biofilm formation in Salmonella typhimurium. Moreover, as previously shown, CsgA fibrils cross-seeded fibrillation of amyloid-ß, providing support for the proposed structural resemblance and potential for cross-species amyloid interactions. The presented findings provide structural insights into amyloidogenic regions important for curli formation, suggest a novel strategy for disrupting amyloid-structured biofilms, and hypothesize on the formation of self-propagating prion-like species originating from a microbial source that could influence neurodegenerative diseases.

Ferulic acid amide derivatives with varying inhibition of amyloid-ß oligomerization and fibrillization.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized, in part, by the misfolding, oligomerization and fibrillization of amyloid-ß (Aß). Evidence suggests that the mechanisms underpinning Aß oligomerization and subsequent fibrillization are distinct, and may therefore require equally distinct therapeutic approaches. Prior studies have suggested that amide derivatives of ferulic acid, a natural polyphenol, may combat multiple AD pathologies, though its impact on Aß aggregation is controversial. We designed and synthesized a systematic library of amide derivatives of ferulic acid and evaluated their anti-oligomeric and anti-fibrillary capacities independently. Azetidine tethered, triphenyl derivatives were the most potent anti-oligomeric agents (compound 2i: IC50 = 1.8 µM ± 0.73 µM); notably these were only modest anti-fibrillary agents (20.57% inhibition of fibrillization), and exemplify the poor correlation between anti-oligomeric/fibrillary activities. These data were subsequently codified in an in silico QSAR model, which yielded a strong predictive model of anti-Aß oligomeric activity (κ = 0.919 for test set; κ = 0.737 for validation set).

Natural spirocyclic alkaloids and polyphenols as multi target dementia leads.

The U rhynchophylla, U tomentosa, Isatis indigotica Fortune, Voacanga Africana, herbal constituents, fungal extracts from Aspergillus duricaulis culture media, include spirooxindoles, polyphenols or bridged spirocyclic alkaloids. Their constituents exhibit specific and synergistic multiple neuroprotective properties including inhibiting of Aß fibril induced cytotoxicity, NMDA receptor inhibition in mice models of Alzheimer's disease (AD). The pioneering research from Woodward to Waldmann has advanced the synthesis of spirocyclic alkaloids. Furthermore, the elucidation of the genetic analysis, biochemical pathways that links strictosidine to the alkaloids akuammicine, stemmadenine, tabersonine, catharanthine, will now enable the biotechnological generation, also stimulate synthesis of related bridged spirocyclic alkaloids for medicinal investigations. From the value of spirocyclic structures as multi target dementia leads, we hypothesise that simpler Lipinski-like natural/synthetic alkaloid analogues may likewise be discovered that provide neurocognitive enhancing activities against dementia and AD.

Blended polar/nonpolar peptide conjugate interferes with human insulin amyloid-mediated cytotoxicity.

Insulin, a peptide hormone and a key regulator of blood glucose level, is routinely administered to type-I diabetic patients to achieve the required glycemic control. Insulin aggregation and ensuing amyloidosis has been observed at repeated insulin injection sites and in injectable formulations. The latter occurs due to insulin agglomeration during shipping and storage. Such insulin amyloid leads to enhanced immunogenicity and allow potential attachment to cell membranes leading to cell permeability and apoptosis. Small molecule inhibitors provide useful interruption of this process and inhibit protein misfolding as well as amyloid formation. In this context, we report the propensity of a palmitoylated peptide conjugate to inhibit insulin aggregation and amyloid-mediated cytotoxicity, via designed interference with polypeptide interfacial interactions.

Microwave-assisted rapid synthesis of spirooxindole-pyrrolizidine analogues and their activity as anti-amyloidogenic agents.

A library of Sox-pyrrolizidines was rapidly prepared by microwave-assisted, one-pot, three-component, 1,3-dipolar cycloaddition of azomethine ylides from l-proline and isatin, with various ß-nitrostyrenes. Nitro-Sox compounds, 4b, 4d and 4e inhibit HEWL amyloid fibril formation by ThT studies with percentages of fluorescence intensity of 55.4, 42.9 and 40.3%, respectively. Further studies with MTT assay, Raman spectroscopy, TEM and molecular docking supported these promising candidates for activity against amyloid misfolding, a phenomenon leading to Alzheimer's disease pathology.

Interest of novel N-alkylpyridinium-indolizine hybrids in the field of Alzheimer's disease: Synthesis, characterization and evaluation of antioxidant activity, cholinesterase inhibition, and amyloid fibrillation interference.

A small library of molecules combining indolizine and N-alkyl pyridinium was synthesized and evaluated in a multi-target-directed-ligand strategy for Alzheimer's disease (AD) treatment. The new compounds were classified in three series depending on the number of methylene residues linking the two heterocycles (Ind-PyCx with x = 0, 2 or 3). The molecules were synthesized from the corresponding bis-pyridines by two-step formation of the indolizine core including mono-alkylation of pyridine and 1,3-dipolar cycloaddition with an alkylpropiolate. Their activities against AD's key-targets were evaluated in vitro: acetyl- and butyrylcholinesterase (AChE and BChE) inhibition, antioxidant properties and inhibition of amyloid fibril formation. None of the three series showed significant activities against all the targets. The Ind-PyC2 and Ind-PyC3 series are active on eeAChE and hAChE (µM IC50 values). Most of the positively charged molecules from these two series also appeared active against eqBChE, however they lost their activity on hBChE. Comparative molecular modeling of 13 and 15 docked in hAChE and hBChE highlighted the importance of the substituent (p-methoxybenzoyl or methyloxycarbonyl, respectively) located on the indolizine C-3 for the binding. The larger molecule 13 fits more tightly at the active site of the two enzymes than 15 that shows a larger degree of freedom. The Ind-PyC2 and Ind-PyC3 hybrids displayed some antioxidant activity when tested at 750 µg/mL (up to 95% inhibition of DPPH radical scavenging for 10). In both series, most hybrids were also able to interact with amyloid fibers, even if the inhibitory effect was observed at a high 100 µM concentration. The Ind-PyC0 molecules stand out completely due to their spectroscopic properties which prevent their evaluation by Ellman's and ThT assays. However, these molecules showed interesting features in the presence of preformed fibers. In particular, the strong increase in fluorescence of 3 in the presence of amyloid fibers is very promising for its use as a fibrillation fluorescent reporter dye.

The Amyloid Inhibitor CLR01 Relieves Autophagy and Ameliorates Neuropathology in a Severe Lysosomal Storage Disease.

Lysosomal storage diseases (LSDs) are inherited disorders caused by lysosomal deficiencies and characterized by dysfunction of the autophagy-lysosomal pathway (ALP) often associated with neurodegeneration. No cure is currently available to treat neuropathology in LSDs. By studying a mouse model of mucopolysaccharidosis (MPS) type IIIA, one of the most common and severe forms of LSDs, we found that multiple amyloid proteins including α-synuclein, prion protein (PrP), Tau, and amyloid ß progressively aggregate in the brain. The amyloid deposits mostly build up in neuronal cell bodies concomitantly with neurodegeneration. Treating MPS-IIIA mice with CLR01, a "molecular tweezer" that acts as a broad-spectrum inhibitor of amyloid protein self-assembly reduced lysosomal enlargement and re-activates autophagy flux. Restoration of the ALP was associated with reduced neuroinflammation and amelioration of memory deficits. Together, these data provide evidence that brain deposition of amyloid proteins plays a gain of neurotoxic function in a severe LSD by affecting the ALP and identify CLR01 as new potent drug candidate for MPS-IIIA and likely for other LSDs.