Using silver nanoparticle-decorated whey protein isolate amyloid fibrils to modify the electrode surface used for electrochemical detection of para-nitrophenol.

Due to their organized structures, remarkable stiffness, and nice biocompatibility and biodegradability, amyloid fibrils serve as building blocks for versatile sustainable materials. Silver nanoparticles (AgNPs) are commonly used as the nano-catalysts for various electrochemical reactions. Given their large specific surface area and high surface energy, AgNPs exhibit high aggregation propensity, which hampers their electrocatalytic performance. Food protein wastes have been identified to be associated with climate change and environmental impacts, and a surplus of whey proteins in dairy industries causes high biological and chemical demands, and greenhouse gas emissions. This study is aimed at constructing sustainable electrode surface modifiers using AgNP-deposited whey protein amyloid fibrils (AgNP/WPI-AFs). AgNP/WPI-AFs were synthesized and characterized via spectroscopic techniques, electron microscopy, and X-ray diffraction. Next, the electrocatalytic performance of AgNP/WPI-AF modified electrode was assessed via para-nitrophenol (p-NP) reduction combined with various electrochemical analyses. Moreover, the reaction mechanism of p-NP electrocatalysis on the surface of AgNP/WPI-AF modified electrode was investigated. The detection range, limit of detection, sensitivity, and selectivity of the AgNP/WPI-AF modified electrode were evaluated accordingly. This work not only demonstrates an alternative for whey valorization but also highlights the feasibility of using amyloid-based hybrid materials as the electrode surface modifier for electrochemical sensing purposes.

Recent advances in the biological depolymerization and upcycling of polyethylene terephthalate.

Polyethylene terephthalate (PET) is favored for its exceptional properties and widespread daily use. This review highlights recent advancements that enable the development of biological tools for PET decomposition, transforming PET into valuable platform chemicals and materials in upcycling processes. Enhancing PET hydrolases' catalytic activity and efficiency through protein engineering strategies is a priority, facilitating more effective PET waste management. Efforts to create novel PET hydrolases for large-scale PET depolymerization continue, but cost-effectiveness remains challenging. Hydrolyzed monomers must add additional value to make PET recycling economically attractive. Valorization of hydrolysis products through the upcycling process is expected to produce new compounds with different values and qualities from the initial polymer, making the decomposed monomers more appealing. Advances in synthetic biology and enzyme engineering hold promise for PET upcycling. While biological depolymerization offers environmental benefits, further research is needed to make PET upcycling sustainable and economically feasible.

Breakthrough Curve Modeling and Analysis for Lysozyme Adsorption by Tris(hydroxymethyl)aminomethane Affinity Nanofiber Membrane.

In this study, a polyacrylonitrile nanofiber membrane was first hydrolyzed and then functionalized with tris(hydroxymethyl)aminomethane (P-Tris), then used as an affinity nanofiber membrane for lysozyme adsorption in membrane chromatography. The dynamic adsorption behavior of lysozyme was investigated in a flow system under various operating parameters, including adsorption pHs, initial feed lysozyme concentration, loading flow rate, and the number of stacked membrane layers. Four different kinetic models, pseudo-first-order, pseudo-second-order, Elovich, and intraparticle diffusion kinetic models, were applied to experimental data from breakthrough curves of lysozyme. The results showed that the dynamic adsorption results were fitted well with the pseudo-second-order kinetic model. The breakthrough curve experimental results show significant differences in the breakthrough time, the dynamic binding capacity, the length of the mass transfer zone, and the utilization rate of the membrane bed under different operating parameters. Four dynamic adsorption models (i.e., Bohart-Adams, Thomas, Yoon-Nelson, and BDST models) were used to analyze the breakthrough curve characteristics of the dynamic adsorption experiments. Among them, the Yoon-Nelson model was the best model to fit the breakthrough curve. However, some of the theoretical results based on the Thomas and Bohart-Adams model analyses of the breakthrough curve fit well with the experimental data, with an error percentage of <5%. The Bohart-Adams model has the largest difference from the experimental results; hence it is not suitable for breakthrough curve analysis. These results significantly impact dynamic kinetics studies and breakthrough curve characteristic analysis in membrane bed chromatography.

Developing targeted drug delivery carriers for breast cancer using glutathione-sensitive doxorubicin-coupled glycated bovine serum albumin nanoparticles.

Incorporation of the nano-based carriers into drug delivery provides a promising alternative to overcome the limitations of the conventional chemotherapy. Doxorubicin (DOXO) is an effective chemotherapeutic drug widely used in chemotherapy for breast cancer treatment. A globular protein bovine serum albumin (BSA) holds great potential as carriers in pharmaceutical applications. This work is aimed at developing the DOXO-coupled glycated BSA nanoparticles via desolvation method for improving the capability of targeting the GLUT5 transporters over-expressed on breast cancer cells. Fructosamine assay and Fourier transform infrared spectroscopy were employed to determine the content of fructosamine structure and structural changes on the surfaces of nanoparticles, respectively. Additionally, the synthesized BSA nanoparticles were further characterized by electron microscopy and dynamic light scattering. Results revealed that the DOXO-coupled glycated BSA nanoparticles were spherically shaped with a hydrodynamic diameter of ~60.74 nm and a ζ-potential of ~ - 42.20 mV. Moreover, the DOXO release behavior of as-synthesized DOXO-coupled glycated BSA nanoparticles was examined under different conditions. Finally, the DOXO-coupled glycated BSA nanoparticles were found to exhibit cytotoxicity toward both MCF-7 and MDA-MB-231 cells. Our findings evidently suggested that the drug-coupled glycated BSA nanoparticles serve as the potential candidates for targeted drug delivery platform used in breast cancer therapy.

Modulation of Insulin Amyloid Fibrillization in Imidazolium-Based Ionic Liquids with Hofmeister Series Anions.

Amyloid fibrils have immense potential to become the basis of modern biomaterials. The formation of amyloid fibrils in vitro strongly depends on the solvent properties. Ionic liquids (ILs), alternative solvents with tunable properties, have been shown to modulate amyloid fibrillization. In this work, we studied the impact of five ILs with 1-ethyl-3-methylimidazolium cation [EMIM+] and anions of Hofmeisterseries hydrogen sulfate [HSO4-], acetate [AC-], chloride [Cl-], nitrate [NO3-], and tetrafluoroborate [BF4-] on the kinetics of insulin fibrillization and morphology, and the structure of insulin fibrils when applying fluorescence spectroscopy, AFM and ATR-FTIR spectroscopy. We found that the studied ILs were able to speed up the fibrillization process in an anion- and IL-concentration-dependent manner. At an IL concentration of 100 mM, the efficiency of the anions at promoting insulin amyloid fibrillization followed the reverse Hofmeister series, indicating the direct binding of ions with the protein surface. At a concentration of 25 mM, fibrils with different morphologies were formed, yet with similar secondary structure content. Moreover, no correlation with the Hofmeister ranking was detected for kinetics parameters. IL with the kosmotropic strongly hydrated [HSO4-] anion induced the formation of large amyloid fibril clusters, while the other kosmotropic anion [AC-] along with [Cl-] led to the formation of fibrils with similar needle-like morphologies to those formed in the IL-free solvent. The presence of the ILs with the chaotropic anions [NO3-] and [BF4-] resulted in longer laterally associated fibrils. The effect of the selected ILs was driven by a sensitive balance and interplay between specific protein-ion and ion-water interactions and non-specific long-range electrostatic shielding.

Application of Amyloid-Based Hybrid Membranes in Drug Delivery.

The properties of amyloid fibrils, e.g., unique structural characteristics and superior biocompatibility, make them a promising vehicle for drug delivery. Here, carboxymethyl cellulose (CMC) and whey protein isolate amyloid fibril (WPI-AF) were used to synthesize amyloid-based hybrid membranes as vehicles for the delivery of cationic and hydrophobic drugs (e.g., methylene blue (MB) and riboflavin (RF)). The CMC/WPI-AF membranes were synthesized via chemical crosslinking coupled with phase inversion. The zeta potential and scanning electron microscopy results revealed a negative charge and a pleated surface microstructure with a high content of WPI-AF. FTIR analysis showed that the CMC and WPI-AF were cross-linked via glutaraldehyde and the interacting forces between membrane and MB or RF was found to be electrostatic interaction and hydrogen bonding, respectively. Next, the in vitro drug release from membranes was monitored using UV-vis spectrophotometry. Additionally, two empirical models were used to analyze the drug release data and relevant rate constant and parameters were determined accordingly. Moreover, our results indicated that in vitro drug release rates depended on the drug-matrix interactions and transport mechanism, which could be controlled by altering the WPI-AF content in membrane. This research provides an excellent example of utilizing two-dimensional amyloid-based materials for drug delivery.

Reactive Green 19 dye-ligand immobilized on the aminated nanofiber membranes for efficient adsorption of lysozyme: Process development and optimization in batch and flow systems.

The performance of lysozyme adsorption by the aminated nanofiber membrane immobilized with Reactive Green 19 (RG19) dyes was evaluated in batch and flow systems. The physicochemical properties of the dye-immobilized nanofiber membrane were characterized. The parameters of batch-mode adsorption of lysozyme (e.g., pH, initial dye concentration, and lysozyme concentration) were optimized using the Taguchi method. In a flow process, the factors influencing the dynamic binding performance for lysozyme adsorption in the chicken egg white (CEW) solution include immobilized dye concentration, adsorption pH value, feed flow rate, and feed CEW concentration. The impact of these operating conditions on the lysozyme purification process was investigated. Under optimal conditions, the recovery yield and purification factor of lysozyme achieved from the one-step adsorption process were 98.52% and 143 folds, respectively. The dye-affinity nanofiber membrane also did not exhibit any significant loss in its binding capacity and purification performance after five consecutive uses.

Silver nanoparticle-deposited whey protein isolate amyloid fibrils as catalysts for the reduction of methylene blue.

The unique structural characteristics and superior biocompatibility make the protein nanofibers promising immobilization platforms/substrates for catalysts/enzymes. Metal nanoparticles have been employed as the catalysts in industries due to their excellent catalytic activity and stability, whereas their high surface energy leads to nanoparticle aggregation, thereby hampering their catalytic performance. Here, amyloid fibril (AF) derived from whey protein isolate (WPI) was chosen as the support of silver nanoparticles (AgNP) and utilized for the catalytic reduction of methylene blue (MB). The one-dimensional amyloid-based hybrid materials (AgNP/WPI-AF) were first synthesized via chemical or photochemical route. The characterization of AgNP/WPI-AF by UV-vis spectrophotometry and electron microscopy revealed that the sizes of AgNP on WPI-AF's surface ranged from 2 to 30 nm. Next, the catalytic performances of AgNP/WPI-AF prepared by various routes for MB degradation were investigated. Additionally, the kinetic data were analyzed using two different models and the apparent rate constants and thermodynamic parameters were further determined accordingly. Moreover, the reusability of AgNP/WPI-AF was assessed by monitoring the percentage removal of MB over consecutive filtering cycles. Our results indicated that Langmuir-Hinshelwood-type mechanism better described the catalytic MB reduction using AgNP/WPI-AF. This work provides a nice example of application of nanoparticle-amyloid fibril composite materials for catalysis.

Removal of Ionic Dyes by Nanofiber Membrane Functionalized with Chitosan and Egg White Proteins: Membrane Preparation and Adsorption Efficiency.

Electrospun polyacrylonitrile (PAN) nanofiber membrane was functionalized with chitosan and proteins for use in the treatment of dye-containing wastewater. The PAN nanofiber membrane was subjected to alkaline hydrolysis, before being grafted with chitosan and subsequently the proteins from chicken egg white. The resultant nanofiber membrane (P-COOH-CS-CEW) was comprehensively characterized using thermogravimetric analysis, Fourier-transform infrared spectroscopy, and scanning electron microscopy. The efficiency of P-COOH-CS-CEW in removing cationic dye toluidine blue O (TBO) and anionic dye acid orange 7 (AO7) in aqueous solution was evaluated. Based on the performance of model fitting, Langmuir and pseudo-second-order kinetic model could be used to describe the performance of P-COOH-CS-CEW in the removal of TBO (pH 10) and AO7 (pH 2) from the dye solutions. The adsorbed TBO and AO7 dyes can be completely desorbed by an elution solution made of 50% (v/v) ethanol and 1 M sodium chloride. After five consecutive adsorption-desorption cycles, the efficiency of dye removal by P-COOH-CS-CEW was maintained above 97%.

Kinetic and Thermodynamic Studies of Lysozyme Adsorption on Cibacron Blue F3GA Dye-Ligand Immobilized on Aminated Nanofiber Membrane.

The polyacrylonitrile (PAN) nanofiber membrane was prepared by the electrospinning technique. The nitrile group on the PAN nanofiber surface was oxidized to carboxyl group by alkaline hydrolysis. The carboxylic group on the membrane surface was then converted to dye affinity membrane through reaction with ethylenediamine (EDA) and Cibacron Blue F3GA, sequentially. The adsorption characteristics of lysozyme onto the dye ligand affinity nanofiber membrane (namely P-EDA-Dye) were investigated under various conditions (e.g., adsorption pH, EDA coupling concentration, lysozyme concentration, ionic strength, and temperature). Optimum experimental parameters were determined to be pH 7.5, a coupling concentration of EDA 40 µmol/mL, and an immobilization density of dye 267.19 mg/g membrane. To understand the mechanism of adsorption and possible rate controlling steps, a pseudo first-order, a pseudo second-order, and the Elovich models were first used to describe the experimental kinetic data. Equilibrium isotherms for the adsorption of lysozyme onto P-EDA-Dye nanofiber membrane were determined experimentally in this work. Our kinetic analysis on the adsorption of lysozyme onto P-EDA-Dye nanofiber membranes revealed that the pseudo second-order rate equation was favorable. The experimental data were satisfactorily fitted by the Langmuir isotherm model, and the thermodynamic parameters including the free energy change, enthalpy change, and entropy change of adsorption were also determined accordingly. Our results indicated that the free energy change had a negative value, suggesting that the adsorption process occurred spontaneously. Moreover, after five cycles of reuse, P-EDA-Dye nanofiber membranes still showed promising efficiency of lysozyme adsorption.

Examining the effect of bovine serum albumin on the properties and drug release behavior of ß-lactoglobulin-derived amyloid fibril-based hydrogels.

Herein, we report the use of ß-lactoglobulin (ß-LG) combined with bovine serum albumin (BSA) for the preparation of amyloid-based hydrogels with aim of delivering riboflavin. The incorporation of BSA enhanced ß-LG fibrillogenesis and protected ß-LG fibrils from losing fibrillar structure due to the pH shift. The mechanical properties of hydrogels were observed to be positively correlated with the number of amyloid fibrils. While the addition of BSA induced amyloid fibril formation, its presence between the fibril chains interfered with the entanglement of fibril chains, thus adversely affecting the hydrogels' mechanical properties. Hydrogels' surface microstructure became more compact as the number of amyloid fibrils rose and the presence of BSA could improve hydrogels' surface homogeneity. In vitro riboflavin (RF) release rate was found to be correlated with the number of fibrils and BSA-RF binding affinity. However, when the digestive enzymes were present, the influence of BSA-RF affinity was alleviated due to enzymes' destructive and/or degradative effects on BSA and/or hydrogels, thus the release rate relied on the number of fibrils, which could be adjusted by the amount of BSA. Results indicate that the additional component, BSA, plays an important role in modulating the properties and functions of ß-LG fibril-based hydrogels.

Protection of human γD-crystallin protein from ultraviolet C-induced aggregation by ortho-vanillin.

Cataract is known as one of the leading causes of vision impairment worldwide. While the detailed mechanism of cataratogenesis remains unclear, cataract is believed to be correlated with the aggregation and/or misfolding of human ocular lens proteins called crystallins. A 173-residue structural protein human γD-crystallin is a major γ-crystallin protein in the human eye lens and associated with the development of juvenile and mature-onset cataracts. This work is aimed at investigating the effect of a small molecule, e.g., ortho-vanillin, on human γD-crystallin aggregation upon exposure to ultraviolet-C irradiation. According to the findings of right-angle light scattering, transmission electron microscopy, and gel electrophoresis, ortho-vanillin was demonstrated to dose-dependently suppress ultraviolet-C-triggered aggregation of human γD-crystallin. Results from the synchronous fluorescence spectroscopy, tryptophan fluorescence quenching, and molecular docking studies revealed the structural change of γD-crystallin induced by the interaction/binding between ortho-vanillin and protein. We believe the outcome from this work may contribute to the development of potential therapeutics for cataract.

Investigating the effect of sugar-terminated nanoparticles on amyloid fibrillogenesis of ß-lactoglobulin.

In vivo tissue deposition of fibrillar protein aggregates is the cause of several degenerative diseases. Evidence suggests that interfering with the pathology-associated amyloid fibrillogenesis by inhibitory molecules is envisaged as the primary therapeutic strategy. Amyloid fibril formation of proteins has been demonstrated to be influenced by nanoparticles/nanomaterials. As compared with their molecular form counterpart, this work examined the effect of sucrose-terminated nanoparticles on the in vitro amyloid fibrillogenesis and structural properties of ß-lactoglobulin at pH 2.0 and 80 °C. ThT binding and electron microscopy results demonstrated that sucrose-terminated nanoparticles were able to suppress ß-lactoglobulin fibrillogenesis in a concentration-dependent fashion. Importantly, sucrose-terminated nanoparticles showed better ß-lactoglobulin fibril-inhibiting ability than sucrose molecules. ANS fluorescence and right-angle light scattering results showed reduced solvent exposure and decreased aggregation, respectively, in the ß-lactoglobulin samples upon treatment with sucrose-terminated nanoparticles. Moreover, fluorescence quenching analyses revealed that the static quenching mechanism and formation of a non-fluorescent fluorophore-nanoparticle complex are involved in the nanoparticle-ß-lactoglobulin interaction. We believe that the results from this study may suggest that the nanoparticle form of biocompatible sugar-related osmolytes may serve as effective inhibiting/suppressing agents toward protein fibrillogenesis.

Catalase immobilized in polypeptide/silica nanocomposites via emulsion and biomineralization with improved activities.

Polypeptide-mediated silica mineralization is an attractive approach to prepare polypeptide/silica nanocomposites for enzyme immobilization. Herein, a facile approach for in situ immobilization of catalase (CAT) in polypeptide/silica nanocomposites is developed via the preparation of cross-linked polypeptide/enzyme microgels using an emulsion process followed by silica mineralization. The efficient protein immobilization under benign condition (25-28 °C, pH 7.0, 0.05 N) was evidenced by high immobilization yield (> 99%) and no protein leakage. Our data showed that the immobilized CAT exhibited prolonged reusability and storage stability compared to free one, suggesting that the composite networks not only provide suitable microenvironments to facilitate enzymatic reactions but also confine the enzyme macromolecules to prevent subunit dissociation. Star-shaped topology exhibited better coverage onto the enzyme than linear counterpart, leading to the superior reusability (relative activity >95% for 30 cycling number) and storage stability (relative activity >95% for 60 days) of the immobilized CAT (~ 14 mg/g of support). The substrate affinity and enzymatic reaction rate for the immobilized CAT were also influenced by silica content and polypeptide topology. This strategy may provide a feasible and inexpensive approach to fabricate polypeptide/silica nanocomposites, which would be promising materials in biotechnological fields such as enzyme immobilization.

Exploring the influence of brilliant blue G on amyloid fibril formation of lysozyme.

Evidence suggests that amyloid fibril mitigation/inhibition is considered a promising approach toward treating amyloid diseases. In this work, we first examined how amyloid fibrillogenesis of lysozyme was affected by BBG, a safe triphenylmethane compound with nice blood-brain-barrier-permeability, and found that shorter fibrillar species were formed in the lysozyme samples treated with BBG. Next, alterations in the features including the secondary as well as tertiary structure, extent of aggregation, and molecular distribution of lysozyme triggered by the addition of BBG were examined by various spectroscopic techniques, right-angle light scattering, dynamic light scattering, and SDS-PAGE. In addition, we have investigated how BBG affected the lysozyme fibril-induced cytotoxicity in SH-SY5Y cells. We found that a large quantity of shorter fibrillar species and more lysozyme monomers were present in the samples treated with BBG. Also, the addition of BBG rescued SH-SY5Y cells from cell death induced by amyloid fibrils of lysozyme. Finally, information about the binding sites and interacting forces involved in the BBG-lysozyme interaction was further explored using synchronous fluorescence and molecular docking approaches. Molecular docking results revealed that, apart from the hydrophobic interaction(s), hydrogen bonding, electrostatic interactions, and van der Waal forces may also be involved in the binding interaction.

Examining the effects of dextran-based polymer-coated nanoparticles on amyloid fibrillogenesis of human insulin.

More than thirty human proteins and/or peptides can aggregate to form amyloid deposits that are linked to several amyloid diseases including clinical syndrome injection-localized amyloidosis, which is correlated with the aggregation of the 51-residue polypeptide insulin. While no cure is currently available toward tackling amyloid diseases, prevention or suppression of amyloid fibrillization is considered as the primary therapeutic strategy. Nanomaterials have been demonstrated to possess great potential in the fields of biomedical diagnosis and drug delivery, they are also able to affect the amyloid aggregation of proteins. This work explores the effects of three different magnetic nanoparticles coated with dextran-based polymers on the in vitro amyloid fibrillogenesis of human insulin. Surface modification of nanoparticles with dextran-based polymers was used to improve the biocompatibility of maghemite nanoparticles. We demonstrated that insulin fibrillization may be mitigated by the studied nanoparticles in a concentration-dependent fashion as verified by ThT binding assay and transmission electron microscopy. The extent of inhibitory activity against human insulin fibril formation was found to be associated with the physico-chemical properties of nanoparticles, with the highest inhibitory activity observed for diethylaminoethyl-dextran-coated nanoparticles. Using circular dichroism spectroscopy, ANS fluorescence spectroscopy, and right-angle light scattering, we probed the structural/conformational changes and investigated the aggregating behavior of insulin upon treatment with nanoparticles. This work demonstrates that nanoparticles with an appropriate surface modification can be utilized to suppress or even inhibit amyloid fibril formation of proteins.

Exploring the effects of methylene blue on amyloid fibrillogenesis of lysozyme.

The 129-residue lysozyme has been shown to form amyloid fibrils in vitro. While methylene blue (MB), a compound in the phenothiazinium family, has been shown to dissemble tau fibril formation, its anti-fibrillogenic effect has not been thoroughly characterized in other proteins/peptides. This study examines the effects of MB on the in vitro fibrillogenesis of lysozyme at pH 2.0 and 55 °C. Our results demonstrated that, upon 7-day incubation, the plateau ThT fluorescence of the sample was found to be ~8.69% or ~2.98% of the control when the molar ratio of lysozyme to MB was at 1:1.11 or 1:3.33, respectively, indicating that the inhibitory potency of MB against lysozyme fibrillogenesis is positively correlated with its concentration. We also found that MB is able to destabilize the preformed lysozyme fibrils. Moreover, molecular docking and molecular dynamics simulations results revealed that MB's mechanism of fibril formation inhibition may be triggered by binding with lysozyme's aggregation-prone region. Results reported here provide solid support for MB's effect on amyloid fibrillogenesis. We believe the additional insights gained herein may pave way to the discovery of other small molecules that may have similar action toward amyloid fibril formation and its associated diseases.

Effects of glycation on human γd-crystallin proteins by different glycation-inducing agents.

Human γd-crystallin (Hγd-crystallin), a major protein component of the human eye lens, is associated with the development of juvenile- and mature-onset cataracts. Evidence suggests that nonenzymatic protein glycation plays an important role in the aetiology of cataract and diabetic sequelae. This research compared the effects of various glycation modifiers on Hγd-crystallin aggregation, by treating samples of Hγd-crystallin with ribose, galactose, or methylglyoxal using several biophysical techniques. To measure advanced glycation end products, an Nε-(carboxyethyl)lysine enzyme-linked immunosorbent assay was performed on the glycating agent-treated Hγd-crystallin samples. Fructosamine production detection was performed for both ribose-treated and galactose-treated samples. Methylglyoxal-treated samples had the highest level of aggregation and the greatest extent of unfolding, and upon incubation for a minimum of 12 days, exhibited a marked enhancement in the amount of Nε-(carboxyethyl)lysine. The molecular profiles and morphological features of the glycated samples were highly correlated to the type of glycation agent used. These findings highlight a close connection between the type of glycation modifier and the various aggregation species that form. Thus, these results may facilitate deciphering of the molecular mechanism of diabetic cataractogenesis.

Lysozyme amyloid fibrillization in presence of tacrine/acridone-coumarin heterodimers.

Amyloid aggregates of proteins are one of the most abundant and important naturally occurring self-associated assemblies. Formation of poly/peptide amyloid aggregates is also associated with the widely spread diseases, so called amyloidosis, which include Alzheimer's disease, diabetes mellitus and lysozyme amyloidosis. These disorders are still incurable and novel therapeutical approaches are focused on using small molecules for inhibition of amyloid aggregation. We have observed effect of three structurally distinct groups of tacrine/acridone - coumarin heterodimers on hen egg white (HEW) lysozyme fibrillization in vitro. The ability of heterodimers to interfere with lysozyme amyloid aggregation was examined using Thioflavin T fluorescence assay, atomic force microscopy and docking method. The obtained data suggest that inhibitory effect of heterodimers on lysozyme fibrillization depends on their composition. We have shown that tacrine-coumarin heterodimers with alkylenediamine linker are the most effective inhibitors of lysozyme fibrillization. The inhibitory activities were quantified through IC50 values; the most potent heterodimers interfere with lysozyme aggregation in the scale of micromolar concentrations (19.2 µM-105.4 µM). The molecular docking showed that the modes of possible interactions involved in the binding are mainly hydrophobic interactions, hydrogen bonding and van der Waals interactions. Studied heterodimers had none or weak cytotoxic effect on human neuroblastoma cells. The obtained results can be helpful for the design and development of new therapeutics for amyloid-related diseases.

Brilliant blue R dye is capable of suppressing amyloid fibril formation of lysozyme.

Amyloid fibril formation is associated with an array of degenerative diseases. While no real cure is currently available, evidence suggests that suppression of amyloid fibrillogenesis is an effective strategy toward combating these diseases. Brilliant blue R (BBR), a disulfonated triphenylmethane compound, has been shown to interact with fibril-forming proteins but exert different effects on amyloid fibrillogenesis. These inconsistent findings prompted us to further evaluate BBR's effect on the inhibition/suppresion of protein fibrillogenesis. Using 129-residue hen lysozyme, which shares high sequence homology to human lysozyme associated with hereditary non-neuropathic systemic amyloidosis, as a model, this study is aimed at thoroughly examining the influence of BBR on the in vitro protein fibrillogenesis. We first showed that BBR dose-dependently attenuated lysozyme fibril formation probably by affecting the fibril growth rate, with the value of IC50 determined to be ~4.39 µM. Next, we employed tryptophan fluorescence quenching method to determine the binding constant and number of binding site(s) associated with BBR-lysozyme binding. In addition, we further conducted molecular docking studies to gain a better understanding of the possible binding site(s) and interaction(s) between lysozyme and BBR. We believe some of the information and/or knowledge concerning the structure-function relationship associated with BBR's suppressing activity obtained here can be applied for the future work in the subject matter related with the therapeutic strategies for amyloid diseases.