Mapping the binding site topology of amyloid protein aggregates using multivalent ligands

A key process in the development of neurodegenerative diseases such as Alzheimer''s and Parkinson''s diseases is the aggregation of proteins to produce fibrillary aggregates with a cross β-sheet structure, amyloid. The development of reagents that can bind these aggregates with high affinity and selectivity has potential for early disease diagnosis. By linking two benzothiazole aniline (BTA) head groups with different length polyethylene glycol (PEG) spacers, fluorescent probes that bind amyloid fibrils with low nanomolar affinity have been obtained. Dissociation constants measured for interaction with Aβ, α-synuclein and tau fibrils show that the length of the linker determines binding affinity and selectivity. These compounds were successfully used to image α-synuclein aggregates in vitro and in the post-mortem brain tissue of patients with Parkinson''s disease. The results demonstrate that multivalent ligands offer a powerful approach to obtain high affinity, selective reagents to bind the fibrillary aggregates that form in neurodegenerative disease.

Multivalent ligands offer a powerful approach to obtain high affinity reagents to bind the aggregates that form in neurodegenerative disease. Selectivity for different proteins was achieved by using different linkers to connect the head groups.     

An explicitly designed paratope of amyloid-β prevents neuronal apoptosis in vitro and hippocampal damage in rat brain

Synthetic antibodies hold great promise in combating diseases, diagnosis, and a wide range of biomedical applications. However, designing a therapeutically amenable, synthetic antibody that can arrest the aggregation of amyloid-β (Aβ) remains challenging. Here, we report a flexible, hairpin-like synthetic paratope (SP1, ∼2 kDa), which prevents the aggregation of Aβ monomers and reverses the preformed amyloid fibril to a non-toxic species. Structural and biophysical studies further allowed dissecting the mode and affinity of molecular recognition events between SP1 and Aβ. Subsequently, SP1 reduces Aβ-induced neurotoxicity, neuronal apoptosis, and ROS-mediated oxidative damage in human neuroblastoma cells (SH-SY5Y). The non-toxic nature of SP1 and its ability to ameliorate hippocampal neurodegeneration in a rat model of AD demonstrate its therapeutic potential. This paratope engineering module could readily implement discoveries of cost-effective molecular probes to nurture the basic principles of protein misfolding, thus combating related diseases.

Herein, the therapeutic potentials of an explicitly designed peptide probe are systematically illuminated in vitro and in vivo against Aβ aggregation. The probe demonstrates remarkable potency for attenuating neurotoxicity and hippocampal damage.     

3D-visualization of amyloid-β oligomer interactions with lipid membranes by cryo-electron tomography

Amyloid-β (Aβ) assemblies have been shown to bind to lipid bilayers. This can disrupt membrane integrity and cause a loss of cellular homeostasis, that triggers a cascade of events leading to Alzheimer''s disease. However, molecular mechanisms of Aβ cytotoxicity and how the different assembly forms interact with the membrane remain enigmatic. Here we use cryo-electron tomography (cryoET) to obtain three-dimensional nano-scale images of various Aβ assembly types and their interaction with liposomes. Aβ oligomers and curvilinear protofibrils bind extensively to the lipid vesicles, inserting and carpeting the upper-leaflet of the bilayer. Aβ oligomers concentrate at the interface of vesicles and form a network of Aβ-linked liposomes, while crucially, monomeric and fibrillar Aβ have relatively little impact on the membrane. Changes to lipid membrane composition highlight a significant role for GM1-ganglioside in promoting Aβ-membrane interactions. The different effects of Aβ assembly forms observed align with the highlighted cytotoxicity reported for Aβ oligomers. The wide-scale incorporation of Aβ oligomers and curvilinear protofibrils into the lipid bilayer suggests a mechanism by which membrane integrity is lost.

Cryo-electron tomography 3D imaging of amyloid-β oligomers carpeting the surface of lipid bilayers in near native conditions.     

Impact of sphingosine and acetylsphingosines on the aggregation and toxicity of metal-free and metal-treated amyloid-β

Pathophysiological shifts in the cerebral levels of sphingolipids in Alzheimer''s disease (AD) patients suggest a link between sphingolipid metabolism and the disease pathology. Sphingosine (SP), a structural backbone of sphingolipids, is an amphiphilic molecule that is able to undergo aggregation into micelles and micellar aggregates. Considering its structural properties and cellular localization, we hypothesized that SP potentially interacts with amyloid-β (Aβ) and metal ions that are found as pathological components in AD-affected brains, with manifesting its reactivity towards metal-free Aβ and metal-bound Aβ (metal–Aβ). Herein, we report, for the first time, that SP is capable of interacting with both Aβ and metal ions and consequently affects the aggregation of metal-free Aβ and metal–Aβ. Moreover, incubation of SP with Aβ in the absence and presence of metal ions results in the aggravation of toxicity induced by metal-free Aβ and metal–Aβ in living cells. As the simplest acyl derivatives of SP, N-acetylsphingosine and 3-O-acetylsphingosine also influence metal-free Aβ and metal–Aβ aggregation to different degrees, compared to SP. Such slight structural modifications of SP neutralize its ability to exacerbate the cytotoxicity triggered by metal-free Aβ and metal–Aβ. Notably, the reactivity of SP and the acetylsphingosines towards metal-free Aβ and metal–Aβ is determined to be dependent on their formation of micelles and micellar aggregates. Our overall studies demonstrate that SP and its derivatives could directly interact with pathological factors in AD and modify their pathogenic properties at concentrations below and above critical aggregation concentrations.

The reactivity of sphingosine and acetylsphingosines towards both metal-free and metal-treated amyloid-β is demonstrated showing a correlation of their micellization properties.     

Target-driven supramolecular self-assembly for selective amyloid-β photooxygenation against Alzheimer's disease

Photo-oxygenation of β-amyloid (Aβ) has been considered an efficient way to inhibit Aβ aggregation in Alzheimer''s disease (AD). However, current photosensitizers cannot simultaneously achieve enhanced blood–brain barrier (BBB) permeability and selective photooxygenation of Aβ, leading to poor therapeutic efficacy, severe off-target toxicity, and substandard bioavailability. Herein, an Aβ target-driven supramolecular self-assembly (PKNPs) with enhanced BBB penetrability and switchable photoactivity is designed and demonstrated to be effective in preventing Aβ aggregation in vivo. PKNPs are prepared by the self-assembly of the Aβ-targeting peptide KLVFF and an FDA-approved porphyrin derivative (5-(4-carboxyphenyl)-10,15,20-triphenylporphyrin). Due to the photothermal effect of PKNPs, the BBB permeability of PKNPs under irradiation is 8.5-fold higher than that of porphyrin alone. Moreover, upon selective interaction with Aβ, PKNPs undergo morphological change from the spherical to the amorphous form, resulting in a smart transformation from photothermal activity to photodynamic activity. Consequently, the disassembled PKNPs can selectively oxygenate Aβ without affecting off-target proteins (insulin, bovine serum albumin, and human serum albumin). The well-designed PKNPs exhibit not only improved BBB permeability but also highly selective Aβ photooxygenation. Furthermore, in vivo experiments demonstrate that PKNPs can alleviate Aβ-induced neurotoxicity and prolong the life span of the commonly used AD transgenic Caenorhabditis elegans CL2006. Our work may open a new path for using supramolecular self-assemblies as switchable phototheranostics for the selective and effective prevention of Aβ aggregation and related neurotoxicity in AD.

Photo-oxygenation of β-amyloid (Aβ) has been considered an efficient way to inhibit Aβ aggregation in Alzheimer''s disease (AD). We present the first example of Aβ-responsive photodynamic therapy to treatment of AD by using PKNPs self-assemblies.     

Metal-chelating benzothiazole multifunctional compounds for the modulation and 64Cu PET imaging of Aβ aggregation

While Alzheimer''s Disease (AD) is the most common neurodegenerative disease, there is still a dearth of efficient therapeutic and diagnostic agents for this disorder. Reported herein are a series of new multifunctional compounds (MFCs) with appreciable affinity for amyloid aggregates that can be potentially used for both the modulation of Aβ aggregation and its toxicity, as well as positron emission tomography (PET) imaging of Aβ aggregates. Firstly, among the six compounds tested HYR-16 is shown to be capable to reroute the toxic Cu-mediated Aβ oligomerization into the formation of less toxic amyloid fibrils. In addition, HYR-16 can also alleviate the formation of reactive oxygen species (ROS) caused by Cu²⁺ ions through Fenton-like reactions. Secondly, these MFCs can be easily converted to PET imaging agents by pre-chelation with the ⁶⁴Cu radioisotope, and the Cu complexes of HYR-4 and HYR-17 exhibit good fluorescent staining and radiolabeling of amyloid plaques both in vitro and ex vivo. Importantly, the ⁶⁴Cu-labeled HYR-17 is shown to have a significant brain uptake of up to 0.99 ± 0.04 %ID per g. Overall, by evaluating the various properties of these MFCs valuable structure–activity relationships were obtained that should aid the design of improved therapeutic and diagnostic agents for AD.

A series of multifunctional compounds and their ⁶⁴Cu complexes exhibit good affinity for Aβ aggregates and can also control Aβ toxicity.     

Amyloid binding and beyond: a new approach for Alzheimer's disease drug discovery targeting Aβo–PrPC binding and downstream pathways

Amyloid β oligomers (Aβo) are the main toxic species in Alzheimer''s disease, which have been targeted for single drug treatment with very little success. In this work we report a new approach for identifying functional Aβo binding compounds. A tailored library of 971 fluorine containing compounds was selected by a computational method, developed to generate molecular diversity. These compounds were screened for Aβo binding by a combined ¹⁹F and STD NMR technique. Six hits were evaluated in three parallel biochemical and functional assays. Two compounds disrupted Aβo binding to its receptor PrP^C in HEK293 cells. They reduced the pFyn levels triggered by Aβo treatment in neuroprogenitor cells derived from human induced pluripotent stem cells (hiPSC). Inhibitory effects on pTau production in cortical neurons derived from hiPSC were also observed. These drug-like compounds connect three of the pillars in Alzheimer''s disease pathology, i.e. prion, Aβ and Tau, affecting three different pathways through specific binding to Aβo and are, indeed, promising candidates for further development.

A new approach combining virtual screening, ¹⁹F and STD NMR, and biochemical assays using hiPSC and targetting multiple pathways involving Aβ, PrP^C and Tau provides a more effective strategy for Alzheimer''s disease drug discovery than Aβ only approach.     

Modification of amyloid-beta peptide aggregation via photoactivation of strained Ru(ii) polypyridyl complexes

Alzheimer''s disease (AD) is a chronic neurodegenerative disorder characterized by progressive and irreversible damage to the brain. One of the hallmarks of the disease is the presence of both soluble and insoluble aggregates of the amyloid beta (Aβ) peptide in the brain, and these aggregates are considered central to disease progression. Thus, the development of small molecules capable of modulating Aβ peptide aggregation may provide critical insight into the pathophysiology of AD. In this work we investigate how photoactivation of three distorted Ru(ii) polypyridyl complexes (Ru1–3) alters the aggregation profile of the Aβ peptide. Photoactivation of Ru1–3 results in the loss of a 6,6′-dimethyl-2,2′-bipyridyl (6,6′-dmb) ligand, affording cis-exchangeable coordination sites for binding to the Aβ peptide. Both Ru1 and Ru2 contain an extended planar imidazo[4,5-f][1,10]phenanthroline ligand, as compared to a 2,2′-bipyridine ligand for Ru3, and we show that the presence of the phenanthroline ligand promotes covalent binding to Aβ peptide His residues, and in addition, leads to a pronounced effect on peptide aggregation immediately after photoactivation. Interestingly, all three complexes resulted in a similar aggregate size distribution at 24 h, forming insoluble amorphous aggregates as compared to significant fibril formation for peptide alone. Photoactivation of Ru1–3 in the presence of pre-formed Aβ_1–42 fibrils results in a change to amorphous aggregate morphology, with Ru1 and Ru2 forming large amorphous aggregates immediately after activation. Our results show that photoactivation of Ru1–3 in the presence of either monomeric or fibrillar Aβ_1–42 results in the formation of large amorphous aggregates as a common endpoint, with Ru complexes incorporating the extended phenanthroline ligand accelerating this process and thereby limiting the formation of oligomeric species in the initial stages of the aggregation process that are reported to show considerable toxicity.

Photoactivation of a series of Ru(ii) polypyridyl complexes leads to ligand exchange and modulation of amyloid-beta peptide aggregation of relevance to Alzheimer''s disease.     

Small molecule-mediated co-assembly of amyloid-β oligomers reduces neurotoxicity through promoting non-fibrillar aggregation

Amyloid-β (Aβ) oligomers, particularly low molecular weight (LMW) oligomers, rather than fibrils, contribute very significantly to the onset and progression of Alzheimer''s Disease (AD). However, due to the inherent heterogeneity and metastability of oligomers, most of the conventional anti-oligomer therapies have indirectly modulated oligomers'' toxicity through manipulating Aβ self-assembly to reduce oligomer levels, which are prone to suffering from the risk of regenerating toxic oligomers from the products of modulation. To circumvent this disadvantage, we demonstrate, for the first time, rational design of rigid pincer-like scaffold-based small molecules with blood–brain barrier permeability that specifically co-assemble with LMW Aβ oligomers through directly binding to the exposed hydrophobic regions of oligomers to form non-fibrillar, degradable, non-toxic co-aggregates. As a proof of concept, treatment with a europium complex (EC) in such a structural mode can rescue Aβ-mediated dysfunction in C. elegans models of AD in vivo. This small molecule-mediated oligomer co-assembly strategy offers an efficient approach for AD treatment.

A rational design of pincer-like scaffold-based small molecule with blood-brain barrier permeability that can specifically co-assemble with low molecular weight Aβ oligomers to form non-fibrillar, degradable, non-toxic co-aggregates.     

Thermodynamics and kinetics of the amyloid-β peptide revealed by Markov state models based on MD data in agreement with experiment

The amlyoid-β peptide (Aβ) is closely linked to the development of Alzheimer''s disease. Molecular dynamics (MD) simulations have become an indispensable tool for studying the behavior of this peptide at the atomistic level. General key aspects of MD simulations are the force field used for modeling the peptide and its environment, which is important for accurate modeling of the system of interest, and the length of the simulations, which determines whether or not equilibrium is reached. In this study we address these points by analyzing 30-μs MD simulations acquired for Aβ40 using seven different force fields. We assess the convergence of these simulations based on the convergence of various structural properties and of NMR and fluorescence spectroscopic observables. Moreover, we calculate Markov state models for the different MD simulations, which provide an unprecedented view of the thermodynamics and kinetics of the amyloid-β peptide. This further allows us to provide answers for pertinent questions, like: which force fields are suitable for modeling Aβ? (a99SB-UCB and a99SB-ILDN/TIP4P-D); what does Aβ peptide really look like? (mostly extended and disordered) and; how long does it take MD simulations of Aβ to attain equilibrium? (at least 20–30 μs). We believe the analyses presented in this study will provide a useful reference guide for important questions relating to the structure and dynamics of Aβ in particular, and by extension other similar disordered proteins.

The convergence of MD simulations is tested using varying measures for the intrinsically disordered amyloid-β peptide (Aβ). Markov state models show that 20–30 μs of MD is needed to reliably reproduce the thermodynamics and kinetics of Aβ.     

High-efficiency dynamic sensing of biothiols in cancer cells with a fluorescent β-cyclodextrin supramolecular assembly

A unique fluorescent supramolecular assembly was constructed using coumarin-modified β-cyclodextrin as a reversible ratiometric probe and an adamantane-modified cyclic arginine–glycine–aspartate peptide as a cancer-targeting agent via host–guest inclusion complexation. Importantly, the coumarin-modified β-cyclodextrin not only showed higher sensitivity than the parent coumarin derivatives owing to the presence of numerous hydroxyl groups on the cyclodextrin but also provided a hydrophobic cavity for encapsulation of a cancer-targeting agent. The assembly showed a reversible and fast response to biothiols with a micromolar dissociation constant, as well as outstanding cancer cell permeability, which can be used for high-efficiency real-time monitoring of biothiols in cancer cells. This supramolecular assembly strategy endows the fluorescent probe with superior performance for dynamic sensing of biothiols.

A unique fluorescent supramolecular assembly was constructed from coumarin-modified β-cyclodextrin and an adamantane-modified cyclic arginine–glycine–aspartate peptide for high-efficiency real-time monitoring of biothiols in cancer cells.     

Monitoring phagocytic uptake of amyloid β into glial cell lysosomes in real time

Phagocytosis by glial cells is essential to regulate brain function during health and disease. Therapies for Alzheimer''s disease (AD) have primarily focused on targeting antibodies to amyloid β (Aβ) or inhibitng enzymes that make it, and while removal of Aβ by phagocytosis is protective early in AD it remains poorly understood. Impaired phagocytic function of glial cells during later stages of AD likely contributes to worsened disease outcome, but the underlying mechanisms of how this occurs remain unknown. We have developed a human Aβ_1–42 analogue (Aβ^pH) that exhibits green fluorescence upon internalization into the acidic organelles of cells but is non-fluorescent at physiological pH. This allowed us to image, for the first time, glial uptake of Aβ^pH in real time in live animals. We find that microglia phagocytose more Aβ^pH than astrocytes in culture, in brain slices and in vivo. Aβ^pH can be used to investigate the phagocytic mechanisms responsible for removing Aβ from the extracellular space, and thus could become a useful tool to study Aβ clearance at different stages of AD.

Glial cell phagocytosis of pH-dependent amyloid-β, Aβ^pH, in live and fixed cultures, brain tissue sections, retina, cortex and in live animals useful for studying function in health and disease.     

Templating S100A9 amyloids on Aβ fibrillar surfaces revealed by charge detection mass spectrometry,microscopy, kinetic and microfluidic analyses

The mechanism of amyloid co-aggregation and its nucleation process are not fully understood in spite of extensive studies. Deciphering the interactions between proinflammatory S100A9 protein and Aβ₄₂ peptide in Alzheimer''s disease is fundamental since inflammation plays a central role in the disease onset. Here we use innovative charge detection mass spectrometry (CDMS) together with biophysical techniques to provide mechanistic insight into the co-aggregation process and differentiate amyloid complexes at a single particle level. Combination of mass and charge distributions of amyloids together with reconstruction of the differences between them and detailed microscopy reveals that co-aggregation involves templating of S100A9 fibrils on the surface of Aβ₄₂ amyloids. Kinetic analysis further corroborates that the surfaces available for the Aβ₄₂ secondary nucleation are diminished due to the coating by S100A9 amyloids, while the binding of S100A9 to Aβ₄₂ fibrils is validated by a microfluidic assay. We demonstrate that synergy between CDMS, microscopy, kinetic and microfluidic analyses opens new directions in interdisciplinary research.

Templating mechanism of S100A9 amyloids on Aβ fibrillar surfaces during amyloid co-aggregation process was revealed by synergy of biophysical methods including charge detection mass spectrometry, microscopy, kinetic and microfluidic analyses.     

Construction of chiral α-tert-amine scaffolds via amine-catalyzed asymmetric Mannich reactions of alkyl-substituted ketimines

Stereoselective Mannich reactions of aldehydes with ketimines provide chiral β-amino aldehydes that bear an α-tert-amine moiety. However, the structural variation of the ketimines is limited due to the formation of inseparable E/Z isomers, low reactivity, and other synthetic difficulties. In this study, a highly diastereodivergent synthesis of hitherto difficult-to-access β-amino aldehydes that bear a chiral α-tert-amine moiety was achieved using the amine-catalyzed Mannich reactions of aldehydes with less-activated Z-ketimines that bear both alkyl and alkynyl groups.

Stereoselective Mannich reactions of aldehydes with ketimines provide chiral β-amino aldehydes that bear an α-tert-amine moiety.     

Enterobactin- and salmochelin-β-lactam conjugates induce cell morphologies consistent with inhibition of penicillin-binding proteins in uropathogenic Escherichia coli CFT073

The design and synthesis of narrow-spectrum antibiotics that target a specific bacterial strain, species, or group of species is a promising strategy for treating bacterial infections when the causative agent is known. In this work, we report the synthesis and evaluation of four new siderophore-β-lactam conjugates where the broad-spectrum β-lactam antibiotics cephalexin (Lex) and meropenem (Mem) are covalently attached to either enterobactin (Ent) or diglucosylated Ent (DGE) via a stable polyethylene glycol (PEG₃) linker. These siderophore-β-lactam conjugates showed enhanced minimum inhibitory concentrations against Escherichia coli compared to the parent antibiotics. Uptake studies with uropathogenic E. coli CFT073 demonstrated that the DGE-β-lactams target the pathogen-associated catecholate siderophore receptor IroN. A comparative analysis of siderophore-β-lactams harboring ampicillin (Amp), Lex and Mem indicated that the DGE-Mem conjugate is advantageous because it targets IroN and exhibits low minimum inhibitory concentrations, fast time-kill kinetics, and enhanced stability to serine β-lactamases. Phase-contrast and fluorescence imaging of E. coli treated with the siderophore-β-lactam conjugates revealed cellular morphologies consistent with the inhibition of penicillin-binding proteins PBP3 (Ent/DGE-Amp/Lex) and PBP2 (Ent/DGE-Mem). Overall, this work illuminates the uptake and cell-killing activity of Ent- and DGE-β-lactam conjugates against E. coli and supports that native siderophore scaffolds provide the opportunity for narrowing the activity spectrum of antibiotics in clinical use and targeting pathogenicity.

Siderophore-β-lactam conjugates based on enterobactin and diglucosylated enterobactin enter the periplasm of uropathogenic E. coli CFT073 via the FepA and IroN transporters, and target penicillin-binding proteins.     

A hierarchical assembly strategy for near-infrared photothermal conversion: unconventional heterogeneous metalla[2]catenanes

Herein, we report a hierarchical assembly strategy for constructing heterogeneous half-sandwich organometallic D–A (D = π-donor, A = π-acceptor) interlocked structures, and their application in near-infrared (NIR) photothermal conversion. Thienothiophene and diketopyrrolopyrrole groups were selected as the D and A units, leading to two homogeneous metalla[2]catenanes with D–D–D–D and A–A–A–A stacks, respectively. By the ordered secondary assembly of homogeneous metalla[2]catenanes, two unprecedented heterogeneous D–A metalla[2]catenanes comprising an unusual mixed D–A–D–D and unconventional D–A–A–A stacks were realized by the combination of multiple noncovalent interactions, as all demonstrated by a detailed X-ray crystallographic study. Benefiting from the mixed D–A stacking modes, NIR absorption of heterogeneous D–A metalla[2]catenanes is significantly enhanced in contrast to homogeneous metalla[2]catenanes. Thanks to the enhanced NIR absorption and the fluorescence quenching effect from half-sandwich organometallic fragments, heterogeneous D–A metalla[2]catenanes displayed high-performance NIR photothermal conversion properties (η = 27.3%).

Herein, we report a hierarchical assembly strategy for constructing heterogeneous half-sandwich organometallic D–A (D = π-donor, A = π-acceptor) interlocked structures, and their application in near-infrared (NIR) photothermal conversion.     

Increasing protein stability by engineering the n → π* interaction at the β-turn

Abundant n → π* interactions between adjacent backbone carbonyl groups, identified by statistical analysis of protein structures, are predicted to play an important role in dictating the structure of proteins. However, experimentally testing the prediction in proteins has been challenging due to the weak nature of this interaction. By amplifying the strength of the n → π* interaction via amino acid substitution and thioamide incorporation at a solvent exposed β-turn within the GB1 proteins and Pin 1 WW domain, we demonstrate that an n → π* interaction increases the structural stability of proteins by restricting the ϕ torsion angle. Our results also suggest that amino acid side-chain identity and its rotameric conformation play an important and decisive role in dictating the strength of an n → π* interaction.

Amino acid residues adopt a right-handed α-helical conformation with increasing strength of the n → π* interaction. We also demonstrate a direct consequence of n → π* interactions on enhancing the structural stability of proteins.     

Extract from the Marine Seaweed Padina pavonica Protects Mitochondrial Biomembranes from Damage by Amyloidogenic Peptides

The identification of compounds which protect the double-membrane of mitochondrial organelles from disruption by toxic confomers of amyloid proteins may offer a therapeutic strategy to combat human neurodegenerative diseases. Here, we exploited an extract from the marine brown seaweed Padina pavonica (PPE) as a vital source of natural bioactive compounds to protect mitochondrial membranes against insult by oligomeric aggregates of the amyloidogenic proteins amyloid-β (Aβ), α-synuclein (α-syn) and tau, which are currently considered to be major targets for drug discovery in Alzheimer’s disease (AD) and Parkinson’s disease (PD). We show that PPE manifested a significant inhibitory effect against swelling of isolated mitochondria exposed to the amyloid oligomers, and attenuated the release of cytochrome c from the mitochondria. Using cardiolipin-enriched synthetic lipid membranes, we also show that dye leakage from fluorophore-loaded vesicles and formation of channel-like pores in planar bilayer membranes are largely prevented by incubating the oligomeric aggregates with PPE. Lastly, we demonstrate that PPE curtails the ability of Aβ42 and α-syn monomers to self-assemble into larger β-aggregate structures, as well as potently disrupts their respective amyloid fibrils. In conclusion, the mito-protective and anti-aggregator biological activities of Padina pavonica extract may be of therapeutic value in neurodegenerative proteinopathies, such as AD and PD.     

Asymmetric hydrogenation of exocyclic γ,δ-unsaturated β-ketoesters to functionalized chiral allylic alcohols via dynamic kinetic resolution

An iridium catalyzed asymmetric hydrogenation of racemic exocyclic γ,δ-unsaturated β-ketoesters via dynamic kinetic resolution to functionalized chiral allylic alcohols was developed. With the chiral spiro iridium catalysts Ir-SpiroPAP, a series of racemic exocyclic γ,δ-unsaturated β-ketoesters bearing a five-, six-, or seven-membered ring were hydrogenated to the corresponding functionalized chiral allylic alcohols in high yields with good to excellent enantioselectivities (87 to >99% ee) and cis-selectivities (93 : 7 to >99 : 1). The origin of the excellent stereoselectivity was also rationalized by density functional theory calculations. Furthermore, this protocol could be performed on gram scale and at a lower catalyst loading (0.002 mol%) without the loss of reactivity and enantioselectivity, and has been successfully applied in the enantioselective synthesis of chiral carbocyclic δ-amino esters and the β-galactosidase inhibitor isogalactofagomine.

An iridium catalyzed asymmetric hydrogenation of exocyclic γ,δ-unsaturated β-ketoesters via dynamic kinetic resolution was developed, providing efficient protocol for enantioselective synthesis of functionalized chiral allylic alcohols.     

CHIP-mediated hyperubiquitylation of tau promotes its self-assembly into the insoluble tau filaments

The tau protein is a highly soluble and natively unfolded protein. Under pathological conditions, tau undergoes multiple post-translational modifications (PTMs) and conformational changes to form insoluble filaments, which are the proteinaceous signatures of tauopathies. To dissect the crosstalk among tau PTMs during the aggregation process, we phosphorylated and ubiquitylated recombinant tau in vitro using GSK3β and CHIP, respectively. The resulting phospho–ub-tau contained conventional polyubiquitin chains with lysine 48 linkages, sufficient for proteasomal degradation, whereas unphosphorylated ub-tau species retained only one–three ubiquitin moieties. Mass-spectrometric analysis of in vitro reconstituted phospho–ub-tau revealed seven additional ubiquitylation sites, some of which are known to stabilize tau protofilament stacking in the human brain with tauopathy. When the ubiquitylation reaction was prolonged, phospho–ub-tau transformed into insoluble hyperubiquitylated tau species featuring fibrillar morphology and in vitro seeding activity. We developed a small-molecule inhibitor of CHIP through biophysical screening; this effectively suppressed tau ubiquitylation in vitro and delayed its aggregation in cultured cells including primary cultured neurons. Our biochemical findings point to a “multiple-hit model,” where sequential events of tau phosphorylation and hyperubiquitylation function as a key driver of the fibrillization process, thus indicating that targeting tau ubiquitylation may be an effective strategy to alleviate the course of tauopathies.

Multiple-hit model for tau aggregation, where sequential events of tau phosphorylation and hyperubiquitylation function as a key driver of the fibrillization process.