Discovery of a Highly Selective Glycogen Synthase Kinase‐3 Inhibitor (PF‐04802367) That Modulates Tau Phosphorylation in the Brain: Translation for PET Neuroimaging

Glycogen synthase kinase‐3 (GSK‐3) regulates multiple cellular processes in diabetes, oncology, and neurology. N‐(3‐(1H‐1,2,4‐triazol‐1‐yl)propyl)‐5‐(3‐chloro‐4‐methoxyphenyl)oxazole‐4‐carboxamide (PF‐04802367 or PF‐367) has been identified as a highly potent inhibitor, which is among the most selective antagonists of GSK‐3 to date. Its efficacy was demonstrated in modulation of tau phosphorylation in vitro and in vivo. Whereas the kinetics of PF‐367 binding in brain tissues are too fast for an effective therapeutic agent, the pharmacokinetic profile of PF‐367 is ideal for discovery of radiopharmaceuticals for GSK‐3 in the central nervous system. A ¹¹C‐isotopologue of PF‐367 was synthesized and preliminary PET imaging studies in non‐human primates confirmed that we have overcome the two major obstacles for imaging GSK‐3, namely, reasonable brain permeability and displaceable binding.     

Rhizolutin,a Novel 7/10/6-Tricyclic Dilactone,Dissociates Misfolded Protein Aggregates and Reduces Apoptosis/Inflammation Associated with Alzheimer's Disease

Rhizolutin ( 1 ) was discovered as a natural product of ginseng-rhizospheric Streptomyces sp. WON17. Its structure features an unprecedented 7/10/6-tricyclic dilactone carbon skeleton composed of dimethylcyclodecatriene flanked by a 7-membered and a 6-membered lactone ring based on spectroscopic analysis. During an unbiased screening of natural product libraries, this novel compound was found to dissociate amyloid-β (Aβ) plaques and tau tangles, which are key pathological hallmarks of Alzheimer's disease (AD). Rhizolutin treatment of APP/PS1 double transgenic mice with AD significantly dissociated hippocampal plaques. In vitro, rhizolutin substantially decreased Aβ-induced apoptosis and inflammation in neuronal and glial cells. Our findings introduce a unique chemical entity that targets Aβ and tau concurrently by mimicking misfolded protein clearance mechanisms of immunotherapy, which is prominently investigated in clinical trials.     

Development of Tau Aggregation Inhibitors for Alzheimer's Disease

Small molecules against Alzheimer's : The pathological aggregation of the tau protein is a major hallmark of neurodegenerative diseases such as Alzheimer's disease. The inhibition or reversal of tau aggregation is a potential therapeutic strategy that is currently undergoing clinical trials. The image shows pathological fibers assembled from tau protein, which are the main components of the neurofibrillary tangles of Alzheimer's disease.

     

Acetylcholinesterase Sensor Based on Polyelectrolyte Complexes with DNA Inclusion for the Determination of Reversible Inhibitors

The acetylcholinesterase (AChE) biosensor has been developed for the determination of reversible inhibitors applied in the Alzheimer's disease therapy, i. e., Huperzine A (HupA) and galantamine (Gal). For this purpose, glassy carbon electrode (GCE) was first modified with carbon black (CB) and Co phthalocyanine and then polyelectrolyte complex was self‐assembled on its surface by drop casting of reactants and washing. To extend the stability and improve biosensor performance, it was proposed for the first time to use DNA as polyanion in the complex assembly. The DNA showed higher charge density than conventional polyelectrolytes and stabilized the surface coating by adsorption of higher enzyme amount and prevention of its leaching during the biosensor operation. Complex formation and the influence of structural factors were monitored with surface plasmon resonance. Kinetic study showed mixed inhibition of the enzyme within micro‐ and nanomolar range of inhibitor concentrations. The AChE biosensor showed limit of detection of HupA equal to 0.9 and that of Gal to 70 nM. The sensitivity of drug determination was found to be close or better than that of the AChE biosensors previously reported in the literature. The biosensor was tested on the sample of artificial urine and showed 102 % recovery of the drugs determination.     

A Phosphorylation‐Induced Turn Defines the Alzheimer’s Disease AT8 Antibody Epitope on the Tau Protein

Post mortem biochemical staging of Alzheimer’s disease is currently based on immunochemical analysis of brain slices with the AT8 antibody. The epitope of AT8 is described around the pSer₂₀₂/pThr₂₀₅ region of the hyperphosphorylated form of the neuronal protein tau. In this study, NMR spectroscopy was used to precisely map the AT8 epitope on phosphorylated tau, and derive its defining structural features by a combination of NMR analyses and molecular dynamics. A particular turn conformation is stabilized by a hydrogen bond of the phosphorylated Thr₂₀₅ residue to the amide proton of Gly₂₀₇, and is further stabilized by the two Arg residues opposing the pSer₂₀₂/pThr₂₀₅.     

Advances in Detection Methods of β-Amyloid Protein

Alzheimer's disease (AD) is a neurodegenerative disease with unknown etiology. β-amyloid protein (Aβ) is one of the specific biomarkers of AD, and many clinical studies suggest that abnormal levels of Aβ in blood, cerebrospinal fluid and brain tissue are closely related to the progression of AD. The analysis and evaluation of Aβ are important for early detection, tracking, prevention, and treatment of AD. In this paper, the present situations of the commonly used detection methods of Aβ at home and abroad were summarized and compared. Specifically, the latest application of new electrochemical biosensor in Aβ detection was mainly described, and the summary of its future directions and the potential applications was given.     

3‐O‐Sulfation of Heparan Sulfate Enhances Tau Interaction and Cellular Uptake

Prion‐like transcellular spreading of tau in Alzheimer's Disease (AD) is mediated by tau binding to cell surface heparan sulfate (HS). However, the structural determinants for tau–HS interaction are not well understood. Microarray and SPR assays of structurally defined HS oligosaccharides show that a rare 3‐O‐sulfation (3‐O‐S) of HS significantly enhances tau binding. In Hs3st1^?/? (HS 3‐O‐sulfotransferase‐1 knockout) cells, reduced 3‐O‐S levels of HS diminished both cell surface binding and internalization of tau. In a cell culture, the addition of a 3‐O‐S HS 12‐mer reduced both tau cell surface binding and cellular uptake. NMR titrations mapped 3‐O‐S binding sites to the microtubule binding repeat 2 (R2) and proline‐rich region 2 (PRR2) of tau. Tau is only the seventh protein currently known to recognize HS 3‐O‐sulfation. Our work demonstrates that this rare 3‐O‐sulfation enhances tau–HS binding and likely the transcellular spread of tau, providing a novel target for disease‐modifying treatment of AD and other tauopathies.     

Folding of the Tau Protein on Microtubules

Microtubules are regulated by microtubule‐associated proteins. However, little is known about the structure of microtubule‐associated proteins in complex with microtubules. Herein we show that the microtubule‐associated protein Tau, which is intrinsically disordered in solution, locally folds into a stable structure upon binding to microtubules. While Tau is highly flexible in solution and adopts a β‐sheet structure in amyloid fibrils, in complex with microtubules the conserved hexapeptides at the beginning of the Tau repeats two and three convert into a hairpin conformation. Thus, binding to microtubules stabilizes a unique conformation in Tau.     

Proteophenes – Amino Acid Functionalized Thiophene-based Fluorescent Ligands for Visualization of Protein Deposits in Tissue Sections with Alzheimer's Disease Pathology

Protein deposits composed of specific proteins or peptides are associated with several neurodegenerative diseases and fluorescent ligands able to detect these pathological hallmarks are vital. Here, we report the synthesis of a class of thiophene-based ligands, denoted proteophenes, with different amino acid side-chain functionalities along the conjugated backbone, which display selectivity towards specific disease-associated protein aggregates in tissue sections with Alzheimer's disease (AD) pathology. The selectivity of the ligands towards AD associated pathological hallmarks, such as aggregates of the amyloid-β (Aβ) peptide or tau filamentous inclusions, was highly dependent on the chemical nature of the amino acid functionality, as well as on the location of the functionality along the pentameric thiophene backbone. Finally, the concept of synthesizing donor-acceptor-donor proteophenes with distinct photophysical properties was shown. Our findings provide the structural and functional basis for the development of new thiophene-based ligands that can be utilized for optical assignment of different aggregated proteinaceous species in tissue sections.     

Direct Observation of Aggregation‐Induced Backbone Conformational Changes in Tau Peptides

In tau proteins, the hexapeptides in the R2 and R3 repeats are known to initiate tau fibril formation, which causes a class of neurodegenerative diseases called the taupathies. We show that in R3, in addition to the presence of the hexapeptides, the correct turn conformation upstream to it is also essential for producing prion‐like fibrils that are capable of propagation. A time‐dependent NMR aggregation assay of a slow fibril forming R3‐S316P peptide revealed a trans to cis equilibrium shift in the peptide‐bond conformation preceding P316 during the growth phase of the aggregation process. S316 was identified as the key residue in the turn that confers templating capacity on R3 fibrils to accelerate the aggregation of the R3‐S316P peptide. These results on the specific interactions and conformational changes responsible for tau aggregation could prove useful for developing an efficient therapeutic intervention in Alzheimer's disease.     

A Crossed Immunoelectrophoretic Technique for the Detection of the Down's Syndrome Protein

Abstract

The sera of seventy-one women and twenty-six men of various ages were examined by a two dimensional (crossed) immunoelectrophoresis technique which was modified to detect a protein (the Down's Syndrome protein) originally found in the sera of mothers of trisomy 21 children. The sensitivity of the method is reported to be 0.1 to 1.0 mg/L. Modifications included use of a Tris-Tricine buffer, high voltage in the second dimension, and relatively low antibody concentration to allow easy detection of the Down's Syndrome (DS) protein.

Of twenty-eight mothers and twelve fathers of trisomy 21 children, eighteen mothers and five fathers were positive, while of forty-three women and fourteen men having unaffected or no children, only seven women and two men were positive for the DS protein.

Since the presence of the Down's Syndrome protein may indicate a propensity for giving birth to a trisomy 21 affected child, this crossed immunoelectrophoretic technique may be used as a screening procedure for the DS protein and, therefore, as a tool in genetic counseling.     

A reversed‐phase compatible thin‐layer chromatography autography for the detection of acetylcholinesterase inhibitors

A dual readout autographic assay to detect acetylcholinesterase inhibitors present in complex matrices adsorbed on reversed‐phase or normal‐phase thin‐layer chromatography plates is described. Enzyme gel entrapment with an amphiphilic copolymer was used for assay development. The effects of substrate and enzyme concentrations, pH, incubation time, and incubation temperature on the sensitivity and the detection limit of the assay were evaluated. Experimental design and response surface methodology were used to optimize conditions with a minimum number of experiments. The assay allowed the detection of 0.01% w/w of physostigmine in both a spiked Sonchus oleraceus L. extract chromatographed on normal phase and a spiked Pimenta racemosa (Mill.) J.W. Moore leaf essential oil chromatographed on reversed phase. Finally, the reversed‐phase thin‐layer chromatography assay was applied to reveal the presence of an inhibitor in the Cymbopogon citratus (DC.) Stapf essential oil. The developed assay is able to detect acetylcholinesterase inhibitors present in complex matrixes that were chromatographed in normal phase or reversed‐phase thin‐layer chromatography. The detection limit for physostigmine on both normal and reversed phase was of 1×10^?4 μg. The results can be read by a change in color and/or a change in fluorescence.     

Peptide‐Based Molecular Strategies To Interfere with Protein Misfolding,Aggregation, and Cell Degeneration

Protein misfolding into amyloid fibrils is linked to more than 40 as yet incurable cell‐ and neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and type 2 diabetes. So far, however, only one of the numerous anti‐amyloid molecules has reached patients. This Minireview gives an overview of molecular strategies and peptide chemistry “tools” to design, develop, and discover peptide‐based molecules as anti‐amyloid drug candidates. We focus on two major inhibitor rational design strategies: 1) the oldest and most common strategy, based on molecular recognition elements of amyloid self‐assembly, and 2) a more recent approach, based on cross‐amyloid interactions. We discuss why peptide‐based amyloid inhibitors, in particular their advanced generations, can be promising leads or candidates for anti‐amyloid drugs as well as valuable tools for deciphering amyloid‐mediated cell damage and its link to disease pathogenesis.     

Single Mutations in Tau Modulate the Populations of Fibril Conformers through Seed Selection

Seeded conversion of tau monomers into fibrils is a central step in the progression of tau pathology in Alzheimer’s disease and other neurodegenerative disorders. Self‐assembly is mediated by the microtubule binding repeats in tau. There are either three or four repeats present depending on the protein isoform. Here, double electron‐electron resonance spectroscopy was used to investigate the conformational ensemble of four‐repeat tau fibrils. Single point mutations at key positions in the protein (ΔK280, P301S, P312I, D314I) markedly change the distribution of fibril conformers after template‐assisted growth, whereas other mutations in the protein (I308M, S320F, G323I, G326I, Q336R) do not. These findings provide unprecedented insights into the seed selection of tau disease mutants and establish conformational compatibility as an important driving force in tau fibril propagation.     

An Electrochemiluminescence Biosensor for the Detection of Alzheimer’s Tau Protein Based on Gold Nanostar Decorated Carbon Nitride Nanosheets

Human Tau protein is the most reliable biomarker for the prediction of Alzheimer’s disease (AD). However, the assay to detect low concentrations of tau protein in serum is a great challenge for the early diagnosis of AD. This paper reports an electrochemiluminescence (ECL) immunosensor for Tau protein in serum samples. Gold nanostars (AuNSs) decorated on carbon nitride nanosheets (AuNS@g-CN nanostructure) show highly strong and stable ECL activity compared to pristine CN nanosheets due to the electrocatalytic and surface plasmon effects of AuNSs. As a result of the strong electromagnetic field at branches, AuNSs showed a better ECL enhancement effect than their spherical counterpart. For the fabrication of a specific immunosensor, immobilized AuNSs were functionalized with a monoclonal antibody specific for Tau protein. In the presence of Tau protein, the ECL intensity of the immunosensor decreased considerably. Under the optimal conditions, this ECL based immunosensor exhibits a dynamic linear range from 0.1 to 100 ng mL⁻¹ with a low limit of detection of 0.034 ng mL⁻¹. The LOD is less than the Tau level in human serum; thus, this study provides a useful method for the determination of Tau. The fabricated ECL immunosensor was successfully applied to the detection of Tau, the biomarker in serum samples. Therefore, the present approach is very promising for application in diagnosing AD within the early stages of the disease.     

A metabolomic study of the urine of rats with Alzheimer's disease and the efficacy of Ding‐Zhi‐Xiao‐Wan on the afflicted rats

As a well‐known traditional Chinese medicine formula, Ding‐Zhi‐Xiao‐Wan has long been used for the routine treatment of Alzheimer's disease. However, the mechanism of Ding‐Zhi‐Xiao‐Wan in treating Alzheimer's disease is unclear. Therefore, a nontargeted metabolomics method based on ultrahigh performance liquid chromatography with quadrupole time‐of‐flight mass spectrometry has been established to explore the metabolic variations in the urine of Alzheimer's disease rats and investigate the therapeutic mechanism of Ding‐Zhi‐Xiao‐Wan on Alzheimer's disease. To develop a better rat model of Alzheimer's disease, amyloid β25‐35 was injected into the bilateral hippocampus of Sprague–Dawley rats. Multivariate analysis approaches were applied to differentiate the urine components between the four groups. Thereafter, a targeted metabolomics method was used to verify the identified endogenous metabolites and determine the mechanism of action of Ding‐Zhi‐Xiao‐Wan. Altogether, 26 potential biomarkers were found, of which 15 biomarkers (10 of which are potential biomarkers found in nontargeted metabolomics) were identified. The results show that Ding‐Zhi‐Xiao‐Wan mainly affects the pathways of taurine and hypotaurine metabolism, tryptophan metabolism, and phenylalanine metabolism. Ding‐Zhi‐Xiao‐Wan might play a role in the treatment of Alzheimer's disease by mediating antioxidative stress, regulation of energy metabolism, improvement of intestinal microbes, and protection of nerve cells.     

Simultaneous Detection of Tyrosine and Structure-Specific Intrinsic Fluorescence in the Fibrillation of Alzheimer's Associated Peptides

Understanding of the structural changes during their aggregation and interaction is a prerequisite for establishing the precise clinical relevance of human islet amyloid polypeptide (hIAPP) (involved in Type-II Diabetes Mellitus) in the treatment of Alzheimer's disease stemmed from beta-amyloid (Aβ). Herein, we show that the steady-state emission spectra obtained from photoluminescence (PL) simultaneously capture both the tyrosine derivative (tyrosinate) and the structure-specific intrinsic fluorescence during the aggregation of Aβ and hIAPP. We observe multiple peaks in the emission spectra which exist for structure-specific intrinsic fluorescence, and use the second derivative UV-Vis spectra and the shift in the tyrosine peak as a quantitative measure of the dissimilitude in the electronic states and the fibril growth. We further applied these techniques to detect the static electric field (0, 40, 120, 200 V/cm) induced promotion and inhibition of fibrillation in Aβ, hIAPP and their electric field dependent role in the fibrillation of Aβ : hIAPP(1 : 1). The results were corroborated by field-emission scanning electron microscopy (FESEM), and the determinations of secondary structures by Fourier transform infrared spectroscopy (FTIR). The results indicate that the emission spectrum can be used as a sensor to detect the presence of fibrils; hence for screening potential inhibitors of amyloid fibrillation.