ANS Binding Research Articles

Platinum stabilises a molten-globule conformation of a small globular cytosolic protein SUMO1.

Proteins are generally resistant to large conformational changes under physiological conditions. Here, we show that platinum (Pt(II)), which is widely-used metal centre in cancer therapeutic drugs, binds to a cytosolic protein, small ubiquitin-like modifier 1 (SUMO1), under physiological conditions and changes its conformation to a molten globule (MG). Mass spectrometry (ICP-MS) studies confirmed stoichiometric Pt(II) binding to SUMO1. Fluorescence spectroscopy showed Tyr fluorescence quenching and increased ANS binding. Fluorescence assays on Trp-mutants indicated conformational changes and circular dichroism (CD) suggested MG formation upon Pt(II) binding. In contrast, structural homologues of SUMO1 (ubiquitin (Ubq) and SUMO2) showed no conformational changes on Pt(II) titration. Further studies compared the impact of distinct His residues in SUMO1 on Pt(II) binding and protein structure to SUMO2 and Ubq. Experiments at low pH (5.0) implicated His residues interacting with Pt(II), corroborated by the absence of conformational change in the H75L mutant of SUMO1. Pt(II)-His binding in SUMO1 unravels key molecular determinants of Pt(II)-protein interactions and their conformational consequences. SUMO1 with other SUMOylation components are shown to have enhanced expression in several cancers, hence, our study suggests a possible fate of the non-targetability of Pt(II)-based drugs on SUMOylation in cancer cells, upon interaction with SUMO1.

Quercetin-Loaded Nanocarriers as Effective Inhibitors for Copper Metal Ion-Induced γD-Crystallin Aggregation.

Cataract is one of the leading causes of blindness worldwide. Till date, the only solution for cataracts is surgery, which is a resource-intensive solution. A much simpler solution is to find a potential drug that could inhibit aggregation. It is well established that nonamyloid aggregates of eye lens protein result in cataract. γD-Crystallin, a thermodynamically stable protein, is one of the most abundant proteins in the core of the eye lens and is found to aggregate under stress conditions, leading to the cataract. It has also been found that in cataractous lens, the concentration of metals like copper is elevated significantly as compared to healthy eye lens, suggesting their role in inducing aggregation. In our present study, aggregation of γD-Crystallin was carried out in the presence of Cu (II). Using techniques like turbidity assay, CD spectroscopy, ANS binding assay, and microscopic studies like TEM, it could be confirmed that protein aggregates in the presence of Cu (II) and the nature of aggregates is amorphous. Various polyphenols were tested to suppress aggregation of the protein. Quercetin was observed to be the most efficient. To overcome the problems associated with the delivery of polyphenols, such as solubility and bioavailability, quercetin was encapsulated in two types of nanocarriers. Their characterization was done using TEM, DLS, and other techniques. The potency of quercetin-loaded CS-TPP/CS-PLGA NPs as inhibitors of γD-Crystallin aggregation was confirmed by various experiments.

Biophysical insight into the binding mechanism of epigallocatechin-3-gallate and cholecalciferol to albumin and its preventive effect against AGEs formation: An in vitro and in silico approach

Advanced glycation end products (AGEs) are produced non-enzymatically through the process of glycation. Increased AGEs production has been linked to several diseases including polycystic ovary syndrome (PCOS). PCOS contributes to the development of secondary comorbidities, such as diabetes, cardiovascular complications, infertility, etc. Consequently, research is going on AGEs-inhibiting phytochemicals for their potential to remediate and impede the progression of hyperglycaemia associated disorders. In this study human serum albumin is used as a model protein, as albumin is predominantly present in follicular fluid. This article focusses on the interaction and antiglycating potential of (−)-Epigallocatechin-3-gallate (EGCG) and vitamin D in combination using various techniques. The formation of the HSA-EGCG and HSA-vitamin D complex was confirmed by UV and fluorescence spectroscopy. Thermodynamic analysis verified the spontaneity of reaction, and presence of hydrogen bonds and van der Waals interactions. FRET confirms high possibility of energy transfer. Cumulative antiglycation resulted in almost 60 % prevention in AGEs formation, decreased alterations at lysine and arginine, and reduced protein carbonylation. Secondary and tertiary structural changes were analysed by circular dichroism, Raman spectroscopy and ANS binding assay. Type and size of aggregates were confirmed by Rayleigh and dynamic light scattering, ThT fluorescence, SEM and SDS-PAGE. Effect on cellular redox status, DNA integrity and cytotoxicity was analysed in lymphocytes using dichlorofluorescein (DCFH-DA), DAPI and MTT assay which depicted an enhancement in antioxidant level by cumulative treatment. These findings indicate that EGCG and vitamin D binds strongly to HSA and have antiglycation ability which enhances upon synergism.

Inhibition of cytotoxic self-assembly of HEWL through promoting fibrillation by new synthesized α-hydroxycarbamoylphosphinic acids.

The main objective of the present study is to investigate the potency of new synthesized hydroxycarbamoyl phosphinic acid derivatives in modulating cytotoxic fibrillogenesis of hen egg white lysozyme (HEWL), as a common model in protein aggregation studies. Hydroxycarbamoyl phosphinic acid derivatives were prepared by the reaction of α-hydroxyalkylphosphinic acids with isocyanates (or isothiocyanates) in the presence of trimethylsilyl chloride (TMSCl). The designed process involves the condensation reaction leading to formation of new C sp2-P bond formation. The synthesis and purity of novel designed compounds were confirmed by NMR, LC-MS, and HPLC techniques. A range of experiments, including thioflavin T (ThT) and 8-anilino-1-naphthalenesulfonic acid (ANS) fluorescence assays, Congo red binding measurement, atomic force microscopy imaging, MTT-based cell viability and hemolysis assays were employed to investigate anti-amyloidogenic effects of tested compounds. The obtained results demonstrate that these compounds are able to significantly modulate the self-assembly process of HEWL via shortening of nucleation phase leading to the acceleration of fibrillation and appearance of very large and thick fibrils with decreased surface hydrophobicity and cytotoxicity. Based on ANS binding data, we suggest that increased exposure of hydrophobic patches of oligomeric species is the possible mechanism by which tested compounds promote self-assembly process of HEWL. Fluorescence anisotropy and molecular docking studies indicate the interaction of both synthesized compounds with HEWL, and more specifically with residues that are situated in the highly aggregation-prone β-domain region of protein. This study unveils the potential of hydroxyalkylphosphinic acids as modulators of amyloid fibrillation highlighting these compounds as a promising approach for targeting protein aggregates associated with neurodegenerative diseases.

Endotoxin accelerates insulin amyloid formation and inactivates insulin signal transduction

Aims and objectivesThe aim of this study is to discuss the influence of endotoxin on insulin amyloid formation, to provide guidance for therapeutic insulin preparation and storage. Materials and methodsThe ThT and ANS binding assays were applied to characterize the dynamics curve of insulin amyloid formation with the presence or absence of endotoxin. The morphological structures of intermediate and mature insulin fibrils were observed with SEM and TEM. Secondary structural changes of insulin during fibriliation were examined with CD, FTIR and Raman spectral analysis. The cytotoxic effects of oligomeric and amyloidogenic insulin aggregates were detected using a cck-8 cell viability assay kit. The influence of endotoxin on insulin efficacy was analyzed by monitoring the activation of insulin signal transduction. Key findingsThT analysis showed that endotoxin, regardless of species, accelerated insulin fibrils formation in a dose-dependent manner, as observed with a shorter lag phase. ANS binding assay demonstrated endotoxin provoked the exposure of insulin hydrophobic patches. The results of SEM and TEM data displayed that endotoxin drove insulin to cluster into dense and viscous form, with thicker and stronger filaments. Based on CD, FTIR and Raman spectra, endotoxin promoted the transition of α-helix to random coil and β-strand secondary structures during insulin aggregation. Insulins in both oligomeric and amyloidogenic forms were cytotoxic to HepG2 cells, with the former being more severe. Finally, the efficacy of endotoxin treated insulin obviously decreased. SignificanceOur studies revealed that endotoxin disrupts the structural integrity of insulin and promotes its amyloidosis. These findings offered theoretical guidance for insulin storage and safe utilization, as well as pointing up a new direction for insulin resistance research.

Targeting the binding pocket of the fluorophore 8-anilinonaphthalene-1-sulfonic acid in the bacterial enzyme MurA.

8-Anilinonaphthalene-1-sulfonic acid (ANS) has been extensively used as a fluorescent probe to detect conformational changes of proteins. It has been cocrystallized with several of the proteins it is used to monitor, including the bacterial cell wall synthesis enzyme MurA. MurA catalyzes the first committed step of peptidoglycan biosynthesis, converting UDP-N-acetylglucosamine (UDP-GlcNAc) into enolpyruvyl UDP-GlcNAc. It has been reported before that ANS binds to MurA from Enterobacter cloacae without inhibiting the enzyme's activity up to a concentration of 1 mM ANS. In this study, we present evidence that ANS inhibits the activity of several isoforms of MurA with IC50 values of 18, 22, and 31 µM against wild-type Escherichia coli, C115D E. coli, and E. cloacae MurA, respectively. This prompted us to test a larger series of structural analogs of ANS for the inhibition of these MurA enzymes, which led to the discovery of compound 26. This ANS analog showed enhanced inhibition of MurA (WT and C115D MurA from E. coli, and E. cloacae MurA) with IC50 values of 2.7, 10, and 14 µM, respectively. Based on our results, the ANS binding pocket was identified as a novel target site for the development of potential antibiotics.

Assessment of DPPC Liposome Disruption by Embedded Tocopheryl Malonate

In this study, the effect of α-tocopheryl malonate (TM) on physical and structural properties of DPPC liposomes was investigated using ANS fluorescence, DPH, and TMA–DPH anisotropy fluorescence and differential scanning calorimetry (DSC) methods. The presence of embedded TM in DPPC liposomes caused alteration in its phase transition temperatures, structural order, dynamics, and hydration of head groups increasingly with growing TM concentration. The ANS fluorescence results demonstrated that increasing TM presence in the DPPC gel phase due to interrupted membrane structure caused the formation of new binding sites. Temperature investigations in the range of 20 °C to 60 °C showed that increasing temperature rises ANS fluorescence which reaches local and global maxima at 36 °C and 42 °C, respectively. The rising TM concentration at the phase transition temperature of DPPC led to the lowering of ANS fluorescence, indicating a decreased binding of ANS. Simultaneously, during heating, a roughly 10-nm shift of ANS emission maximum was observed. The results indicated that in the fluid phase, the observed quenching appears as a result of increasing accessibility of water molecules into ANS in this region. The DPH results indicated that in the gel phase presence of TM introduced disorder in the hydrophobic acyl chain region led to its fluidization. The TMA–DPH results indicated an increasing disorder in the interface region and an increasing hydration of head group atoms at the surface of the membrane. The increasing concentration of TM results in the formation of multicomponent DSC traces, suggesting the formation of another structural phase. The applied methods proved that the incorporation of TM into DPPC membrane results in the interaction of malonate moiety with DPPC head group atoms in the interphase layer and induces the interruption in the membrane packing order, leading to its structural changes. The presented results show that TM presence could regulate the membrane properties, thus it may indicate one of the possible mechanisms responsible for the effective disruption of cell membranes by TM. The knowledge of molecular mechanism how TM interacts with the membrane will help to elucidate its possible pharmacological activity.

Thermodynamic modulation of folding and aggregation energy landscape by DNA binding of functional domains of TDP-43

TDP-43 is a vital nucleic acid binding protein which forms stress-induced aberrant aggregates in around 97% cases of ALS, a fatal neurodegenerative disease. The functional tandem RRM domain of the protein (TDP-43tRRM) has been shown to undergo amyloid-like aggregation under stress in a pH-dependent fashion. However, the underlying thermodynamic and molecular basis of aggregation and how the energy landscape of folding, stability, and aggregation are coupled and modulated by nucleic acid binding is poorly understood. Here, we show that the pH stress thermodynamically destabilizes the native protein and systematically populates the unfolded-like aggregation-prone molecules which leads to amyloid-like aggregation. We observed that specific DNA binding inhibits aggregation and populates native-like compact monomeric state even under low-pH stress as measured by circular dichroism, ANS binding, size exclusion chromatography, and transmission electron microscopy. We show that DNA-binding thermodynamically stabilizes and populates the native state even under stress and reduces the population of unfolded-like aggregation-prone molecules which leads to systematic aggregation inhibition. Our results suggest that thermodynamic modulation of the folding and aggregation energy landscape by nucleic-acid-like molecules could be a promising approach for effective therapeutic intervention in TDP-43-associated proteinopathies.

Biophysical Correlates of Enhanced Immunogenicity of a Stabilized Variant of the Receptor Binding Domain of SARS-CoV-2.

The receptor binding domain (RBD) of SARS-CoV-2 is the primary target of neutralizing antibodies. We have previously reported the design and characterization of a mammalian cell expressed RBD derivative, mRBD1-3.2, that has higher thermal stability and greatly enhanced immunogenicity relative to the wild type mRBD. The protein is highly thermotolerant and immunogenic and is being explored for use in room temperature stable Covid-19 vaccine formulations. In the current study, we have investigated the folding pathway of both WT and stabilized RBD. It was found that chemical denaturation of RBD proceeds through a stable equilibrium intermediate. Thermal and chemical denaturation is reversible, as assayed by binding to the receptor ACE2. Unusually, in its native state, RBD binds to the hydrophobic probe ANS, and enhanced ANS binding is observed for the equilibrium intermediate state. Further characterization of the folding of mRBD1-3.2, both in solution and after reconstitution of lyophilized protein stored for a month at 37 °C, revealed a higher stability represented by higher Cm, faster refolding, slower unfolding, and enhanced resistance to proteolytic cleavage relative to WT. In contrast to WT RBD, the mutant showed decreased interaction with the hydrophobic moiety linoleic acid. Collectively, these data suggest that the enhanced immunogenicity results from reduced conformational fluctuations that likely enhance in vivo half-life as well as reduce the exposure of irrelevant non-neutralizing epitopes to the immune system.

Cyclic-NDGA effectively inhibits human gamma-synuclein fibrillation, forms off-pathway species, and disintegrates pre-formed mature fibrils.

Parkinson's disease is one of the various diseases resulting from protein misfolding, aggregation and fibrillation and is characterized by Lewy body deposits which contain the protein α-synuclein (α-syn) as their major component. γ-synuclein (γ-syn), one of the three synucleins is also present in the Lewy bodies along with α-syn and is also implicated in various types of cancers especially breast cancer. It is known to seed the fibrillation of α-syn after its oxidation at methionine residue leading to synucleinopathy. Despite its involvement in synucleinopathy, the search for small molecule therapeutics against γ-syn fibrillation remains largely unexplored. We have investigated the modulatory properties of cyclic-Nordihydroguaiaretic acid (cNDGA), a plant polyphenol, on the structural and aggregational properties of human γ-syn employing various techniques namely ThT fluorescence, Rayleigh Light scattering, ANS binding, far-UV CD and FTIR spectroscopy, atomic force microscopy and MTT assay. cNDGA inhibits the fibrillation of γ-syn and unlike mature control fibrils, γ-syn forms large aggregates with no ThT binding property, negligible hydrophobicity, with lesser β-sheet content and reduced cytotoxicity when incubated with cNDGA. These species formed as a result of weak interaction of γ-syn with cNDGA (Kd ∼10−5M) as investigated by isothermal titration calorimetry are off pathway in nature suggested by seeding experiments. Increasing concentration of cNDGA resulted in an increased recovery of monomeric γ-syn as shown by SDS-PAGE and Native-PAGE. cNDGA was most effective in suppression of fibrillation when added at the beginning of the fibrillation kinetics and was also capable of disintegrating the pre-formed mature fibrils. It can be concluded that cNDGA effectively suppresses γ-syn fibrillation and forms species that are less toxic in nature. These findings could be further explored for the use of cNDGA and similar flavonoids as therapeutics against γ-synucleinopathies.

Inhibition of lysozyme amyloid fibrillation by curcumin-conjugated silver nanoparticles: A multispectroscopic molecular level study

Inhibition of amyloid fibrillation of hen egg white lysozyme (HEWL) was targeted by using curcumin-conjugated silver nanoparticles (c-AgNPs). Fluorescence assays (thioflavin T, ANS binding, tryptophan (Trp) fluorescence), circular dichroism and microscopic imaging techniques (HRTEM and fluorescence) were adopted to study fibrillation inhibition. Results suggested that c-AgNPs having hydrodynamic radius ∼ 22 nm can inhibit fibrillation of HEWL and restricted the protein fibrillation to smaller fibrils. Moreover, in the form of c-AgNPs, curcumin remained water-soluble and was more effective than curcumin. Further, interactions between HEWL and c-AgNPs were studied using fluorescence and circular dichroism spectroscopic techniques. The nanoparticles had caused static quenching of Trp fluorescence of native HEWL. This was also confirmed through determination of excited state lifetime of Trp of HEWL in absence and the presence of c-AgNPs. The association and quenching constant for the conjugation of HEWL with c-AgNPs were determined ∼ 107 M−1. Hydrophobic interactions were found to be involved in HEWL-c-AgNPs complex. Polarity of Trp microenvironment in HEWL was not perturbed by c-AgNPs as supported by synchronous and three-dimensional fluorescence spectroscopy. Far-UV CD spectra suggested that the secondary structure of native HEWL was not affected by c-AgNPs.

Synthesis of 4-hydroxy-L-proline derivatives as new non-classical inhibitors of human carbonic anhydrase II activity: an in vitro study

Carbonic anhydrase (CA) is a zinc metalloenzyme that facilitates the rapid conversion of water and carbon dioxide into proton and bicarbonate ion. CA isozymes have been broadly studied in many pathological/physiological processes. In the current research, a series of 4-hydroxy-L-proline derivatives were designed and chemically synthetized, and interaction of these carboxylic acid-based compounds with hCA II were evaluated. Results indicated that different derivatives had different potencies on hCAII inhibitory activity and among them, compounds 3 b and 3c had the lowest IC50 and K d values than 4-hydroxy-L-proline and other derivatives and therefore had the most affinity to the hCA II enzyme. As a result, compounds 3 b and 3c were chosen for additional testing in this research. The Kinetic data demonstrated that 3 b and 3c inhibit the hCA II esterase activity in a linear competitive way, with K i values in the low micromolar range. Fluorescence tests showed that the hCA II surface hydrophobicity is diminished in the presence of compounds 3 b and 3c, as confirmed by the decrease in ANS binding to hCA II in their presence. Docking results revealed that 3 b and 3c had more binding energy than 4-hydroxy-L-proline. Furthermore, these compounds could occupy the active site of hCA II, where they would interact with critical amino acid residues via non-covalent forces to inhibit hCA II. Overall, the strengthening of inhibitory activity and the binding power of these carboxylic acid derivatives (3 b and 3c) for the hCA II makes these compounds interesting for designing novel hCA II inhibitors. Communicated by Ramaswamy H. Sarma

Role of amino acid oxidation and protein unfolding in peroxyl radical and peroxynitrite-induced inactivation of glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides.

The mechanisms underlying the inactivation of Leuconostoc mesenteroides glucose 6-phosphate dehydrogenase (G6PDH) induced by peroxyl radicals (ROO●) and peroxynitrite (ONOO-), were explored. G6PDH was incubated with AAPH (2,2' -azobis(2-methylpropionamidine)dihydrochloride), used as ROO● source, and ONOO-. Enzymatic activity was assessed by NADPH generation, while oxidative modifications were analyzed by gel electrophoresis and liquid chromatography (LC) with fluorescence and mass detection. Changes in protein conformation were studied by circular dichroism (CD) and binding of the fluorescent dye ANS (1-anilinonaphthalene-8-sulfonic acid). Incubation of G6PDH (54.4μM) with 60mM AAPH showed an initial phase without significant changes in enzymatic activity, followed by a secondary time-dependent continuous decrease in activity to ∼59% of the initial level after 90min. ONOO- induced a significant and concentration-dependent loss of G6PDH activity with ∼46% of the initial activity lost on treatment with 1.5mM ONOO-. CD and ANS fluorescence indicated changes in G6PDH secondary structure with exposure of hydrophobic sites on exposure to ROO●, but not ONOO-. LC-MS analysis provided evidence for ONOO--mediated oxidation of Tyr, Met and Trp residues, with damage to critical Met and Tyr residues underlying enzyme inactivation, but without effects on the native (dimeric) state of the protein. In contrast, studies using chloramine T, a specific oxidant of Met, provided evidence that oxidation of specific Met and Trp residues and concomitant protein unfolding, loss of dimer structure and protein aggregation are involved in G6PDH inactivation by ROO●. These two oxidant systems therefore have markedly different effects on G6PDH structure and activity.

A combined spectroscopic and molecular modeling Study on structure-function-dynamics under chemical modification: Alpha-chymotrypsin with formalin preservative.

Enzyme function can be altered via modification of its amino acid residues, side chains and large-scale domain modifications. Herein, we have addressed the role of residue modification in catalytic activity and molecular recognition of an enzyme alpha-chymotrypsin (CHT) in presence of a covalent cross-linker formalin. Enzyme assay reveals reduced catalytic activity upon increased formalin concentration. Polarization gated anisotropy studies of a fluorophore 8-Anilino-1-naphthalenesulfonic acid (ANS) in CHT show a dip rise pattern in presence of formalin which is consistent with the generation of multiple ANS binding sites in the enzyme owing to modifications of its local amino acid residues. Molecular docking study on amino acid residue modifications in CHT also indicate towards the formation of multiple ANS binding site. The docking model also predicted no change in binding behavior for the substrate Ala-Ala-Phe-7-amido-4-methylcoumarin (AMC) at the active site upon formalin induced amino acid cross-linking.

Insights into the C-terminal domain of apolipoprotein E from chimera studies with apolipophorin III.

Apolipoprotein E3 (apoE) is a critical cholesterol transport protein in humans and is composed of two domains: a well characterized N-terminal (NT) domain that harbors the low-density lipoprotein LDL receptor, and a less understood C-terminal (CT) domain that is the site of protein oligomerization and initiation of lipid binding. To better understand the domain structure of apoE, the CT domain was fused to apolipophorin III (apoLp-III), a single-domain, monomeric apolipoprotein of insect origin, to yield a chimeric protein, apoLp-III/CT-apoE. Recombinant apoLp-III/CT-apoE maintained an overall helical content similar to that of the parent proteins, while chemical induced unfolding studies indicated that its structural integrity was not compromised. Analysis using 1-anilinonaphthalene-8-sulfonic acid (ANS), a sensitive fluorescent indicator of exposed hydrophobic sites and protein folding, demonstrated that whereas apoLp-III provided few ANS binding sites, apoLp-III/CT-apoE harbored an abundance of ANS binding sites. Thus, this indicated tertiary structure formation in CT-apoE when part of the chimera. Size-exclusion chromatography and chemical crosslinking analysis demonstrated that while apoLp-III is monomeric, the chimeric protein formed large oligomeric complexes, similar to native apoE3. Compared to apoLp-III, the chimera showed a two-fold enhancement in phospholipid vesicle solubilization rates and a significantly improved ability to bind to lipolyzed low-density lipoprotein, preventing the onset of lipoprotein aggregation at concentrations comparable to that of parent CT-apoE. These results confirm that high lipid binding and self-association sites are located in the CT domain of apoE, and that these properties can be transferred to an unrelated apolipoprotein, demonstrating that these properties operate independently from the NT domain.

Mechanistic insight into differential interactions of iron oxide nanoparticles with native, glycated albumin and their effect on erythrocytes parameters

Nanoparticles and protein bioconjugates have been studied for multiple biomedical applications. We sought to investigate the interaction and structural modifications of bovine serum albumin (BSA) with iron oxide nanoparticles (IONPs). The IONPs were green synthesized using E. crassipes aqueous leaf extract following characterization using transmission electron microscopy, energy dispersive X-ray analysis and X-ray diffraction. Two different concentrations of native/glycated albumin (0.5 and 1.5 mg/ml) with IONPs were allowed to interact for 1 h at 37 °C. Glycation markers, protein modification markers, cellular antioxidant, and hemolysis studies showed structural modifications and conformational changes in albumin due to the presence of IONPs. UV–visible absorbance resulted in hyperchromic and bathochromic effects of IONPs-BSA conjugates. Fluorescence measurements of tyrosine, tryptophan, advanced glycated end products, and ANS binding assay were promising and quenching effects proved IONPs-BSA conjugate formation. In FTIR of BSA-IONPs, transmittance was increased in amide A and B bands while decreased in amide I and II bands. In summary, native PAGE, HPLC, and FTIR analysis displayed a differential behaviour of IONPs with native and glycated BSA. These results provided an understanding of the interaction and structural modifications of glycated and native BSA which may provide fundamental repercussions in future studies.

Elucidating the binding and inhibitory potential of p-coumaric acid against amyloid fibrillation and their cytotoxicity: Biophysical and docking analysis

P-Coumaric acid (p-CA) is a plant metabolite with anti-inflammatory and antioxidant effects. Due to its therapeutic potential, p-CA has attracted much attention from the scientific community lately. Oxidative stress, amyloid formation, and impaired proteasomal degradation are hallmarks of neurodegenerative diseases like Alzheimer's (AD) and are targets for developing therapeutics against such conditions. Here, we have investigated the anti-amyloidogenic properties of p-coumaric acid on hen egg white lysozyme (HEWL). Heat, pH, and agitation (55 °C, pH 2.0, 600 rpm) stress were used to induce amyloid formation in lysozyme. The aggregates characterization was done by turbidity, Rayleigh light scattering (RLS), and thioflavin-T (ThT) assays. Moreover, ANS (1-anilino naphthalene sulphate) binding assay and circular dichroism (CD) were employed to unveil protein hydrophobicity and secondary structure perturbation, respectively. Lysozyme demonstrated increased hydrophobicity and transition of α-helix to β-sheet under aggregating conditions. Moreover, co-incubation of lysozyme with p-coumaric acid attenuates the process of amyloid in a concentration dependent manner. At 50 and 200 μM concentrations of p-coumaric acid, lysozyme retained its native-like folded structure. Cytotoxicity protection on human SK-N-SH neuroblastoma cell line was also observed using MTT assay and phase contrast microscopy. In addition, transmission electron microscopy (TEM) reaffirms the fibrillar nature of lysozyme aggregates and their attenuation by p-coumaric acid. The steady state fluorescence revealed that the mode of fluorescence quenching for the HEWL-p-coumaric acid interaction is static rather than dynamic. Moderate strength of binding in order of 104 M−1 exists between HEWL and p-coumaric acid. Thermodynamic parameters (∆H and ∆S) obtained from van't Hoff plot suggested spontaneous reaction with hydrophobic interaction. A slight micro-environmental change in HEWL around Tyr residue was observed during the binding process with the help of synchronous fluorescence. Molecular docking analysis reported the involvement of amino acid residues (TRP63, LEU75, ASP101, LYS97) to form a complex between HEWL-p-coumaric acid. The observed anti-amyloidogenic and inherent antioxidative properties of p-coumaric acid could be helpful to design a neuroprotective agent.

Structural and mechanistic insights into modulation of α-Synuclein fibril formation by aloin and emodin

α-Synuclein (α-Syn) aggregation/fibrillation is a leading cause of neuronal death and is one of the major pathogenic factors involved in the progression of Parkinson's' disease (PD). Against this backdrop, discovering new molecules as inhibitors or modulators of α-Syn aggregation/fibrillation is a subject of enormous research. In this study, we have shown modulation, disaggregation, and neuroprotective potential of aloin and emodin against α-Syn aggregation/fibrillation. Thioflavin T (ThT) fluorescence assay showed an increase in lag phase from (51.14 ± 2) h to (68.58 ± 2) h and (74.14 ± 3) h in the presence of aloin and emodin respectively. ANS binding assay represents a modulatory effect of these molecules on hydrophobicity which is crucial for aggregates/fibril formation. NMR spectroscopy and tyrosine quenching studies reveal the binding of aloin/emodin with monomeric α-Syn. TEM and DLS micrographs illustrate the attenuating effect of aloin/emodin against the development of large aggregates/fibrils. Our seeding experiments suggest aloin/emodin generate seeding incompetent oligomers that direct the off-pathway aggregation/fibrillation. Also, aloin/emodin capably reduces the fibrils-induced cytotoxicity and disassembles the preexisting amyloid fibrils. These findings provide deep insight into the modulatory mechanism of α-Syn aggregation/fibrillation in the presence of aloin and emodin, thereby suggesting their potential roles as promising therapeutic molecules against aggregation/fibrillation related disorders.

Design and synthesis of novel stilbene-hydroxypyridinone hybrids as tyrosinase inhibitors and their application in the anti-browning of freshly-cut apples

In order to develop the tyrosinase inhibitors with potential application in food industry, a series of stilbene-hydroxypyridinone hybrids were prepared. Among these compounds, 1h was found to possess the most potent tyrosinase inhibitory effect on both monophenolase and diphenolase activities, with IC50 values of 2.72 μM and 15.86 μM, respectively. The inhibitory effect of 1h on monophenolase activity was 4.6 times that of kojic acid. An inhibition kinetic assay indicated that 1h was a mixed-type and reversible inhibitor. The copper-binding and reducing ability assays, molecular docking study, intrinsic and ANS–binding fluorescence assays indicated that copper coordination and reduction is likely to be the causative mechanism for 1h-induced inhibition on tyrosinase. The results of color measurement and browning index determination indicated that treatment with 1h retarded effectively the browning of freshly-cut apples during their storage. Meanwhile, PPO and POD activities in apple slices were found to be effectively inhibited.

Rate and Equilibrium Constants for Bupivacaine’s Binding to Isolated Alpha-1-Acid Glycoprotein: An In vitro study.

Binding of local anesthetics to plasma proteins has been presented as an important determinant of their bioavailability. Local anesthetics with a high potential for systemic toxicity, e.g. bupivacaine (BUP), are bound strongly by alpha1-acid glycoprotein (AAG), more weakly by serum albumin, but drug dissociation may be rapid, thus limiting the importance of protein binding. The purpose of this study was to determine the binding kinetics of BUP to AAG. Bupivacaine binding to AAG was monitored by its displacement of the fluorescent probe 1-anilinonaphthalene-8-sulfonic acid (ANS). The increased fluorescence of ANS (λ excit/em = 380/480 nm) upon binding to AAG was used to determine the equilibrium and kinetic characteristics of this reaction. By studying how BUP altered the binding kinetics of ANS to AAG it was possible to calculate the BUPs equilibrium and kinetic rate constants for AAG binding. ANS fluorescence increased ca. 50-fold when bound to AAG. Increasing [BUP] with a constant [AAG] + [ANS] returned ANS fluorescence to its unbound status, due to complete displacement of ANS from AAG; bupivacaine’s competitive equilibrium constant, Ki , equals 1-2 μM (pH 7.4, 23oC). Pre-equilibrating AAG with BUP before the rapid (0.008s) addition of excess ANS slowed the binding of ANS to a rate limited by BUP’s dissociation: koff = 12.0 ± 0.5 s-1, corresponding to a half-time ~0.06 seconds. Therefore, although much of the total serum BUP at toxic levels (2-4 µg/mL) will be bound by plasma proteins, dissociation from the tightest binding protein shows that drug is rapidly freed during organ perfusion, allowing newly unbound drug to permeate into the perfused tissues. The very rapid dissociation of BUP from AAG means that equilibrium binding is a very poor index of bio-availability and systemic toxicity of that local anesthetic.