Molecular Design of Magnetic Resonance Imaging Agents Binding to Amyloid Deposits.

The ability to detect and monitor amyloid deposition in the brain using non-invasive imaging techniques provides valuable insights into the early diagnosis and progression of Alzheimer's disease and helps to evaluate the efficacy of potential treatments. Magnetic resonance imaging (MRI) is a widely available technique offering high-spatial-resolution imaging. It can be used to visualize amyloid deposits with the help of amyloid-binding diagnostic agents injected into the body. In recent years, a number of amyloid-targeted MRI probes have been developed, but none of them has entered clinical practice. We review the advances in the field and deduce the requirements for the molecular structure and properties of a diagnostic probe candidate. These requirements make up the base for the rational design of MRI-active small molecules targeting amyloid deposits. Particular attention is paid to the novel cryo-EM structures of the fibril aggregates and their complexes, with known binders offering the possibility to use computational structure-based design methods. With continued research and development, MRI probes may revolutionize the diagnosis and treatment of neurodegenerative diseases, ultimately improving the lives of millions of people worldwide.

Recent Advances in Protein-Protein Interactions.

Protein-protein interactions (PPIs) lead to formation of complexes and aggregates between a pair or multiple protein molecules [...].

Decasubstituted Pillar[5]arene Derivatives Containing L-Tryptophan and L-Phenylalanine Residues: Non-Covalent Binding and Release of Fluorescein from Nanoparticles.

Sensitive systems with controlled release of drugs or diagnostic markers are attractive for solving the problems of biomedicine and antitumor therapy. In this study, new decasubstituted pillar[5]arene derivatives containing L-Tryptophan and L-Phenylalanine residues have been synthesized as pH-responsive drug nanocarriers. Fluorescein dye (Fluo) was loaded into the pillar[5]arene associates and used as a spectroscopic probe to evaluate the release in buffered solutions with pH 4.5, 7.4, and 9.2. The nature of the substituents in the pillar[5]arene structure has a huge influence on the rate of delivering. When the dye was loaded into the associates based on pillar[5]arene derivatives containing L-Tryptophan, the Fluo release occurs in the neutral (pH = 7.4) and alkaline (pH = 9.2) buffered solutions. When the dye was loaded into the associates based on pillar[5]arene with L-Phenylalanine fragments, the absence of release was observed in every pH evaluated. This happens as the result of different packing of the dye in the structure of the associate. This fact was confirmed by different fluorescence mechanisms (aggregation-caused quenching and aggregation-induced emission) and association constants. It was shown that the macrocycle with L-Phenylalanine fragments binds the dye more efficiently (lgKa = 3.92). The experimental results indicate that the pillar[5]arene derivatives with amino acids fragments have a high potential to be used as a pH-responsive drug delivery devices, especially for promoting the intracellular delivering, due to its nanometric size.

Aggregation of Amyloidogenic Peptide Uperin-Molecular Dynamics Simulations.

Uperin 3.5 is a remarkable natural peptide obtained from the skin of toadlets comprised of 17 amino acids which exhibits both antimicrobial and amyloidogenic properties. Molecular dynamics simulations were performed to study the ß-aggregation process of uperin 3.5 as well as two of its mutants, in which the positively charged residues Arg7 and Lys8 have been replaced by alanine. All three peptides rapidly underwent spontaneous aggregation and conformational transition from random coils to beta-rich structures. The simulations reveal that the initial and essential step of the aggregation process involves peptide dimerization and the formation of small beta-sheets. A decrease in positive charge and an increase in the number of hydrophobic residues in the mutant peptides lead to an increase in the rate of their aggregation.

Highlighting the difference in nanostructure between domain-forming and domainless protic ionic liquids.

Nanoheterogeneity in some ionic liquids is a known phenomenon, but quantifying or sometimes even identifying it is not a straightforward task. We compared several known and suggested some novel approaches to identify and characterize domain segregation using the results of atomistic simulations. 10 ammonium-based protic ionic liquids with different propensity to form segregated polar and apolar domains as suggested by experimental studies were considered. They include butyl-, propyl-, 2-methoxyethylammonium nitrate, butyl- and propylammonium hydrogen sulfate, butylammonium thiocyanate (domain-forming liquids), ethylammonium and pyrrolidinium nitrate (weakly pronounced segregation), methylammonium and 2-hydroxyethylammonium nitrate (domainless liquids). Molecular dynamics simulations were performed using models based on the OPLS-AA force field with scaled ion charges. Results show that domains can be recognized and the characteristic domain length scale can be determined from peaks of Ripley's functions, peaks and large-period oscillations of finite-volume radial distribution function integral, or difference of such integrals for polar and apolar atoms, and peaks of local atom density variance. These peaks disappear with increasing temperature due to the disruption of segregated domains. In domain-forming liquids, apolar atoms are more homogeneously distributed in space than polar atoms. In addition, the probability of molecular-sized cavity formation is significantly higher in apolar domains, which determines better solubility of apolar species in domain-forming ILs. The suggested approaches can be applied to various nanostructured liquids including both ionic and molecular solvents and mixtures, as well as other systems with mesoscale ordering.

Molecular Mechanisms of Inhibition of Protein Amyloid Fibril Formation: Evidence and Perspectives Based on Kinetic Models.

Inhibition of fibril formation is considered a possible treatment strategy for amyloid-related diseases. Understanding the molecular nature of inhibitor action is crucial for the design of drug candidates. In the present review, we describe the common kinetic models of fibril formation and classify known inhibitors by the mechanism of their interactions with the aggregating protein and its oligomers. This mechanism determines the step or steps of the aggregation process that become inhibited and the observed changes in kinetics and equilibrium of fibril formation. The results of numerous studies indicate that possible approaches to antiamyloid inhibitor discovery include the search for the strong binders of protein monomers, cappers blocking the ends of the growing fibril, or the species absorbing on the surface of oligomers preventing nucleation. Strongly binding inhibitors stabilizing the native state can be promising for the structured proteins while designing the drug candidates targeting disordered proteins is challenging.

The Gibbs free energy of cavity formation in a diverse set of solvents.

The concept of the formation of a solute-sized cavity in a solvent is widely used in the theories of solvation processes; however, most of the studies of cavity formation using atomistic simulations were limited to water and hydrocarbon models. We calculated the Gibbs free energy of cavity formation ΔcavG for a structurally diverse set of 23 common organic solvents. For the calculation, molecular dynamics simulations of solvent boxes were conducted, and the Widom particle insertion method was applied. The results obtained with two different force fields for the same solvent were in good agreement with each other in most cases. The obtained cavity size dependences of ΔcavG allowed ranking the solvents by the free energy cost of creation of a cavity with a certain size. Surprisingly, this cost was somewhat higher in glycerol, formamide, and ethylene glycol than in water. In general, higher values of ΔcavG are observed for the solvents with a branched network of intermolecular hydrogen bonds and strongly polar aprotic solvents. The numerical results can be used to improve the accuracy of the calculation of the cavity term in non-aqueous continuum solvation models.

Fast scanning calorimetry of lysozyme unfolding at scanning rates from 5 K/min to 500,000 K/min.

BACKGROUND: Protein denaturation is often studied using differential scanning calorimetry (DSC). However, conventional instruments are limited in the temperature scanning rate available. Fast scanning calorimetry (FSC) provides an ability to study processes at much higher rates while using extremely small sample masses [ng]. This makes it a very interesting technique for protein investigation. METHODS: A combination of conventional DSC and fast scanning calorimeters was used to study denaturation of lysozyme dissolved in glycerol. Glycerol was chosen as a solvent to prevent evaporation from the micro-sized samples of the fast scanning calorimeter. RESULTS: The lysozyme denaturation temperatures in the range of scanning rates from 5 K/min to ca. 500,000 K/min follow the Arrhenius law. The experimental results for FSC and conventional DSC fall into two distinct clusters in a Kissinger plot, which are well approximated by two parallel straight lines. CONCLUSIONS: The transition temperatures for the unfolding process measured on fast scanning calorimetry sensor are significantly lower than what could be expected from the results of conventional DSC using extrapolation to high scanning rates. Evidence for the influence of the relative surface area on the unfolding temperature was found. GENERAL SIGNIFICANCE: For the first time, fast scanning calorimetry was employed to study protein denaturation with a range of temperature scanning rates of 5 orders of magnitude. Decreased thermal stability of the micro-sized samples on the fast scanning calorimeter raise caution over using bulk solution thermal stability data of proteins for applications where micro-sized dispersed protein solutions are used, e.g., spray drying.

New insights into the solubility of graphene oxide in water and alcohols.

One of the main advantages of graphene oxide (GO) over its non-oxidized counterpart is its ability to form stable solutions in water and some organic solvents. At the same time, the nature of GO solutions is not completely understood; the existing data are scarce and controversial. Here, we demonstrate that the solubility of GO, and the stability of the as-formed solutions depend not just on the solute and solvent cohesion parameters, as commonly believed, but mostly on the chemical interactions at the GO/solvent interface. By the DFT and QTAIM calculations, we demonstrate that the solubility of GO is afforded by strong hydrogen bonding established between GO functional groups and solvent molecules. The main functional groups taking part in hydrogen bonding are tertiary alcohols; epoxides play only a minor role. The magnitude of the bond energy values is significantly higher than that for typical hydrogen bonding. The hydrogen bond energy between GO functional groups and solvent molecules decreases in the sequence: water > methanol > ethanol. We support our theoretical results by several experimental observations including solution calorimetry. The enthalpy of GO dissolution in water, methanol and ethanol is -0.1815 ± 0.0010, -0.1550 ± 0.0012 and -0.1040 ± 0.0010 kJ g-1, respectively, in full accordance with the calculated trend. Our findings provide an explanation for the well-known, but poorly understood solvent exchange phenomenon.

Pillar[5]arene/albumin biosupramolecular systems for simultaneous native protein preservation and encapsulation of a water-soluble substrate.

The growing resistance of pathogens, bacteria, viruses, and fungi to a number of drugs has encouraged researchers to use natural and synthetic biomimetic systems to overcome this challenge. Multicomponent systems are an attractive approach for drug design and multitarget therapy. In this study, we report the assembly of a three-component (pillar[5]arene, bovine serum albumin, and methyl orange) biosupramolecular system as a potential drug delivery system. We estimated the cytotoxic activity and transfection ability of pillar[5]arene derivatives and investigated the effect of the nature of macrocycle functions (L-phenylalanine, glycine, L-alanine) on the native conformation of serum albumin in a three-component system. NMR, UV-vis, fluorescence, CD spectroscopy, DLS, and molecular docking studies were performed in order to confirm the structure and possible pillar[5]arene/bovine serum albumin/methyl orange interactions occurring during the association process. Results indicate that pillar[5]arene with L-phenylalanine fragments retains the native form of BSA to the maximum extent and forms more stable associates.

Rheological Properties of Fish and Mammalian Gelatin Hydrogels as Bases for Potential Practical Formulations.

Hydrogels have the ability to retain large amounts of water within their three-dimensional polymer matrices. These attractive materials are used in medicine and the food industry; they can serve as the basis for structured food products, additives, and various ingredients. Gelatin is one of widely used biopolymers to create hydrogels that exhibit biocompatibility and tunable rheological properties. In this study, we offer a comparative analysis of rheological properties of gelatin-based hydrogels (C = 6.67%), including mammalian gelatins from bovine and porcine skins and fish gelatins from commercial samples and samples extracted from Atlantic cod skin. Mammalian gelatins provide high strength and elasticity to hydrogels. Their melting point lies in the range from 22 to 34 °C. Fish gelatin from cod skin also provides a high strength to hydrogels. Commercial fish gelatin forms weak gels exhibiting low viscoelastic properties and strength, as well as low thermal stability with a melting point of 7 °C. Gelatins were characterized basing on the analysis of amino acid composition, molecular weight distribution, and biopolymer secondary structure in gels. Our research provides a unique rheological comparison of mammalian and fish gelatin hydrogels as a tool for the re-evaluation of fish skin gelatin produced through circular processes.

The Ability of Some Polysaccharides to Disaggregate Lysozyme Amyloid Fibrils and Renature the Protein.

The deposition of proteins in the form of insoluble amyloid fibril aggregates is linked to a range of diseases. The supramolecular architecture of such deposits is governed by the propagation of ß-strands in the direction of protofilament growth. In the present study, we analyze the structural changes of hen egg-white lysozyme fibrils upon their interactions with a range of polysaccharides, using AFM and FTIR spectroscopy. Linear anionic polysaccharides, such as κ-carrageenan and sodium alginate, are shown to be capable to disaggregate protofilaments with eventual protein renaturation. The results help to understand the mechanism of amyloid disaggregation and create a platform for both the development of new therapeutic agents for amyloidose treatment, and the design of novel functional protein-polysaccharide complex-based nanomaterials.

Enhancing Photostability of Complex Lead Halides through Modification with Antibacterial Drug Octenidine.

The high power-conversion efficiencies of hybrid perovskite solar cells encourage many researchers. However, their limited photostability represents a serious obstacle to the commercialization of this promising technology. Herein, we present an efficient method for improving the intrinsic photostability of a series of commonly used perovskite material formulations such as MAPbI3, FAPbI3, Cs0.12FA0.88PbI3, and Cs0.10MA0.15FA0.75PbI3 through modification with octenidine dihydroiodide (OctI2), which is a widely used antibacterial drug with two substituted pyridyl groups and two cationic centers in its molecular framework. The most impressive stabilizing effects were observed in the case of FAPbI3 and Cs0.12FA0.88PbI3 absorbers that were manifested in significant suppression or even blocking of the undesirable perovskite films' recrystallization and other decomposition pathways upon continuous 110 mW/cm2 light exposure. The achieved material photostability-within 9000 h for the Oct(FA)n-1PbnI3n+1 (n = 40-400) and 20,000 h for Oct(Cs0.12FA0.88)n-1PbnI3n+1 (where n = 40-400) formulations-matches the highest values ever reported for complex lead halides. It is important to note that the stabilizing effect is maintained when OctI2 is used only as a perovskite surface-modifying agent. Using a two-cation perovskite composition as an example, we showed that the performances of the solar cells based on the developed Oct(Cs0.12FA0.88)399Pb400I1201 absorber material are comparable to that of the reference devices based on the unmodified perovskite composition. These findings indicate a great potential of the proposed approach in the design of new highly photostable and efficient light absorbers. We believe that the results of this study will also help to establish important guidelines for the rational material design to improve the operational stability of perovskite solar cells.

Underused Marine Resources: Sudden Properties of Cod Skin Gelatin Gel.

The main object of this work was to characterize the structure and properties of laboratory-made fish gelatin from cod skin in comparison with known commercial gelatins of fish and mammalian origin. This is one way we can contribute to the World Food Program and characterize foodstuff resources from alternative natural sources. Our research was based on the combination of an expanded set of complementary physical-chemical methods to study the similarities and distinctions of hydrogels from traditional and novel gelatin sources from underused marine resources. In this work, we have compared the morphology, supramolecular structure and colloid properties of two commercial (mammalian and fish) gelatins with gelatin we extracted from cold-water cod skin in laboratory conditions. The obtained results are novel, showing that our laboratory-produced fish gelatin is much closer to the mammalian one in terms of such parameters as thermal stability and strength of structural network under temperature alterations. Especially interesting are our experimental observations comparing both fish gelatins: it was shown that the laboratory-extracted cod gelatin is essentially more thermally stable compared to its commercial analogue, being even closer in its rheological properties to the mammalian one.

Beta-rich intermediates in denaturation of lysozyme: accelerated molecular dynamics simulations.

Amyloid fibrillar aggregates play a critical role in many neurodegenerative disorders. Conversion of globular proteins into fibrils is associated with global conformational rearrangement and involves the transformation of α-helices to ß-sheets. In the present work, the accelerated molecular dynamics technique was applied to study the unfolding of hen egg-white lysozyme at elevated temperatures, and the transformation of the native structure to a disordered one was analyzed. The influence of the disulfide bonds on the conformational dynamics and the energy landscape of denaturation process was considered. Our results show that formation of the metastable ß-enriched conformers of individual protein molecules may precede the aggregation process. These ß-rich intermediates can play a role of bricks making up fibrils.Communicated by Ramaswamy H. Sarma.

Effect of ligands with different affinity on albumin fibril formation.

The effect of binding of several ligands to bovine serum albumin on the kinetics of fibril formation at denaturing conditions is studied. The considered ligands are clinical drugs with different binding constants to albumin: relatively strong binders (naproxen, ibuprofen, warfarin with 105 to 107 binding constant values) and weak binders (isoniazid, ranitidine with 103 to 104 binding constant values). The data of thioflavin fluorescence binding assay, Congo red binding assay, and circular dichroism spectroscopy indicate ligand concentration-dependent suppression of fibril formation in the presence of strong binders and no effects in the presence of weak binders. Analysis of kinetic curves shows no induction lag associated with fibril nucleation and the first-order kinetics of fibril formation with respect to albumin concentration for all the studied systems. Using DSC method, the fractions of unfolded albumin at incubation temperature were determined for each albumin-ligand system and ligand concentration. Their magnitudes ranging from 0 to 1 correlate with the initial rates of fibril formation and with equilibrium concentrations of fibrils formed in the system after incubation for at least 120 min. The results indicate that fibrils are formed from partially or completely denatured albumin form with the rate proportional to the fraction of this form. Strong albumin binders act as thermodynamic inhibitors of fibrillation shifting the unfolding equilibrium to the side of the native ligand-bound protein.

Fibril fragments from the amyloid core of lysozyme: An accelerated molecular dynamics study.

Protein aggregation and formation of amyloid fibrils are associated with many diseases and present a ubiquitous problem in protein science. Hen egg white lysozyme (HEWL) can form fibrils both from the full length protein and from its fragments. In the present study, we simulated unfolding of the amyloidogenic fragment of HEWL encompassing residues 49-101 to study the conformational aspects of amyloidogenesis. The accelerated molecular dynamics approach was used to speed up the sampling of the fragment conformers under enhanced temperature. Analysis of conformational transformation and intermediate structures was performed. During the unfolding, the novel short-living and long-living ß-structures are formed along with the unstructured random coils. Such ß-structure enriched monomers can interact with each other and propagate into fibril-like forms. The stability of oligomers assembled from these monomers was evaluated in the course of MD simulations with explicit water. The residues playing a key role in fibril stabilization were determined. The work provides new insights into the processes occurring at the early stages of amyloid fibril assembly.

Crystal Nucleation and Growth in Cross-Linked Poly(ε-caprolactone) (PCL).

The crystal nucleation and overall crystallization kinetics of cross-linked poly(ε-caprolactone) was studied experimentally by fast scanning calorimetry in a wide temperature range. With an increasing degree of cross-linking, both the nucleation and crystallization half-times increase. Concurrently, the glass transition range shifts to higher temperatures. In contrast, the temperatures of the maximum nucleation and the overall crystallization rates remain the same, independent of the degree of cross-linking. The cold crystallization peak temperature generally increases as a function of heating rate, reaching an asymptotic value near the temperature of the maximum growth rate. A theoretical interpretation of these results is given in terms of classical nucleation theory. In addition, it is shown that the average distance between the nearest cross-links is smaller than the estimated lamellae thickness, which indicates the inclusion of cross-links in the crystalline phase of the polymer.

Evaluation of the binding properties of drugs to albumin from DSC thermograms.

There is a demand in rapid and robust methods to determine the affinity of drugs to receptors, enzymes, and transport proteins. Differential scanning calorimetry (DSC) is a common method to prove the existence of ligand-protein binding from the shift of denaturation peak, but it is rarely used to obtain the binding constant values. The work is aimed to prove that the DSC experiments can be a source of reliable values of the binding constants and information on the stoichiometry of drug-albumin binding. DSC thermograms of bovine serum albumin denaturation in the presence of several drugs with different affinity and stoichiometry of binding to albumin: naproxen, warfarin, ibuprofen, and isoniazid were recorded. The dependences of the denaturation peak maximum temperature and area on the molar drug/protein ratio, which varied from 0 to 100, were considered. With the help of numerical modeling of the DSC curves, these dependences were predicted using the binding parameters determined in independent experiments and a simple two-state model of denaturation. The DSC data at relatively small concentrations of ligands are in good agreement with the calculation results. The deviations from the model predictions at high ligand concentrations in the cases of naproxen and ibuprofen indicate that albumin is able to bind several additional molecules of these drugs with its low-affinity sites. The fit was improved by using a sequential binding model with two binding constants K1 = 1.0 × 107 and K2 = 1.0 × 104 for naproxen and a cooperative binding model for ibuprofen. The stoichiometry of drug-albumin complexes fully saturated with drug ligand was calculated from the dependence of the denaturation temperature on the drug concentration. In the case of isoniazid, DSC thermograms indicated very weak binding to albumin.