Investigating Chaperone like Activity of Green Silver Nanoparticles: Possible Implications in Drug Development.

Protein aggregation and amyloidogenesis have been associated with several neurodegenerative disorders like Alzheimer's, Parkinson's etc. Unfortunately, there are still no proper drugs and no effective treatment available. Due to the unique properties of noble metallic nanoparticles, they have been used in diverse fields of biomedicine like drug designing, drug delivery, tumour targeting, bio-sensing, tissue engineering etc. Small-sized silver nanoparticles have been reported to have anti-biotic, anti-cancer and anti-viral activities apart from their cytotoxic effects. The current study was carried out in a carefully designed in-vitro to observe the anti-amyloidogenic and inhibitory effects of biologically synthesized green silver nanoparticles (B-AgNPs) on human serum albumin (HSA) aggregation taken as a model protein. We have used different biophysical assays like thioflavin T (ThT), 8-Anilino-1-naphthalene-sulphonic acid (ANS), Far-UV CD etc. to analyze protein aggregation and aggregation inhibition in vitro. It has been observed that the synthesized fluorescent B-AgNPs showed inhibitory effects on protein aggregation in a concentration-dependent manner reaching a plateau, after which the effect of aggregation inhibition was significantly declined. We also observed meaningful chaperone-like aggregation-inhibition activities of as-synthesized florescent B-AgNPs in astrocytes.

Aggregation as a consequence of glycation: insight into the pathogenesis of arthritis.

Advanced glycation end products (AGEs) as a result of Maillard reaction are currently at the heart of the pathogenesis of several diseases and hence are the objective of numerous investigations. Glycation of proteins has been an implication in long-term complications. Collagen is the most abundant protein in the human body. The purpose of this study was to monitor and characterize the oligomeric aggregates and AGEs of human collagen on addition of glyoxal using ultraviolet, fluorescence, circular dichroism (CD) spectroscopy, docking studies, ITC, and microscopy. Collagen was incubated for varying time periods up to 21 days with three different concentrations (5, 20, and 40 mM) of glyoxal. Collagen exists as molten globule at day 6, evident from native-like secondary structure, altered tryptophan, and high ANS fluorescence due to surface-exposed hydrophobic residues. Glycated collagen as AGEs and aggregates was observed at day 18 and 21, respectively. Formation of AGE and aggregates were confirmed by UV and fluorescence spectroscopy. The obtained AGEs were characterized with respect to the extent of side chain modifications (lysine and arginine) forming the Schiff base, the carboxymethyl lysine, and carbonyl content. Non-tryptophan fluorescence for AGEs was also monitored as the emission peak at 400 and 440 nm, respectively. SEM and TEM confirmed the oligomeric nature of aggregates. Glyoxal at 40 mM shows maximum alterations in protein structure followed by 20 and 5 mM concentration. In the present paper, we propose that a high concentration of glyoxal for a prolonged time results in the formation of harmful aggregates and AGEs.

Synthesis and Pharmacological Properties of Modified A- and D-Ring in Dehydroepiandrosterone (DHEA): A Review.

Dehydroepiandrosterone (3ß-hydroxyandrost-5-en-17-one) (DHEA) is a naturally occurring steroid hormone primarily produced in the zona reticularis of the human adrenal glands. It serves as a crucial precursor for sex hormones, such as testosterone, estradiol, and androstenedione. Recent findings indicate that DHEA serves as the primary source of sex steroids in women during both pre- and postmenopausal stages. Additionally, a decline in DHEA levels with age is linked to various hormone-deficiency symptoms. Despite the wide array of biological activities that make DHEA a valuable polycyclic natural steroid, particularly for pharmaceutical and cosmetic applications, reports suggest that oral DHEA has limited clinical effect. Thus, A- and D-ring modified DHEA are synthesized and their biological activities are carried out by different research groups and enhanced biological activity reported in the literature. Here, in this review, we have tried to cover all of the synthetic routes and biological studies of modified A- and D-ring DHEA from 2015 to mid-2022.

Inhibition of protein misfolding and aggregation by steroidal quinoxalin-2(1H)-one and their molecular docking studies.

A series of D-ring fused 16-substituted steroidal quinoxalin-2(1H)-one attached to an electron-releasing (ER) or electron-withdrawing (EW) groups via steroidal oxoacetate intermediate were synthesized to investigate their protein aggregation inhibition potential using human lysozyme (HLZ). The influence of the type of substituent at the C-6 positions of the quinoxalin-2(1H)-one ring on the protein aggregation inhibition potential was observed, showing that the EW moiety improved the protein aggregation inhibition potency. Of all the evaluated compounds, NO2-substituted quinoxalin-2(1H)-one derivative 13 was the most active compound and had a maximum protein aggregation inhibition effect. Significant stabilization effects strongly support the binding of the most biologically active steroidal quinoxalin-2(1H)-one with docking studies. The predicted physicochemical and ADME properties lie within a drug-like space which shows no violation of Lipinski's rule of five except compounds 12 and 13. Combined, our results suggest that D-ring fused 16-substituted steroidal quinoxalin-2(1H)-one has the potential to modulate the protein aggregation inhibition effect.

Microwave-assisted one-pot multicomponent synthesis of steroidal pyrido[2,3-d]pyrimidines and their possible implications in drug development.

Protein misfolding can lead to fibrillar and non-fibrillar deposits which are the signs of countless human diseases. A promising strategy for the prevention of such diseases is the inhibition of protein aggregation, and the most crucial step toward effective prevention is the development of small molecules having the potential for protein-aggregation inhibition. In this search, a series of novel steroidal pyrido[2,3-d]pyrimidines have been synthesized employing steroidal ketone, substituted aldehydes, and 2,6-diaminopyrimidin-4(3H)-one through the microwave-assisted one-pot multicomponent methodology. The aggregation inhibition potential of newly synthesized compounds was evaluated on human lysozyme (HLZ). All the synthesized compounds were found to be efficient in the inhibition of protein aggregation in carefully designed in vitro experiments. Moreover, molecular docking studies also determine the binding interactions between all the synthesized compounds and native HLZ through hydrogen bonding. The structures of synthesized compounds were also elucidated using various spectroscopic techniques.

Peroxidase improves the activity of catalase by preventing aggregation during TFE-induced denaturation.

Catalase, a ubiquitous enzyme of the free radical scavenging machinery unfolds and aggregates in the presence of 2,2,2-triflouroethanol (TFE). Catalase molecule aggregates at 50% TFE as evident by high thioflavin T fluorescence, shifted congo red absorbance, change in circular dichroism and soret spectra. TEM images confirmed the nature of catalase aggregates to be oligomers. Organic solvent-induced aggregation of catalase is prevented by the presence of peroxidase (another enzyme of the free radical scavenging machinery). To alter the progress of aggregation in presence of increasing concentration of TFE, we determined the effect of peroxidase on catalase oligomerization by several different techniques, including turbidity measurement, activity assay, thioflavin T fluorescence, circular dichroism, shift in congo red absorbance, transmission electron microscopy (TEM), Rayleigh scattering, soret absorption spectra, and ANS fluorescence. The presence of peroxidase in the vicinity of folded catalase helps it to remain functionally active and inhibited aggregation in the presence of TFE, suggesting that proteins are stable in crowded environments. Moreover, this catalase-peroxidase interaction is biologically significant as it provides insights into how the aggregation process may be altered.

Green synthesis of silver nanoparticles, its characterization, and chaperone-like activity in the aggregation inhibition of α-chymotrypsinogen A.

Consumption of silver nanoparticles (AgNPs) has been increased many folds due to its antimicrobial actions resulting in its widespread incorporation into a wide range of biomedical and consumer products. Still, enough research is needed to clearly understand the effect of these nanoparticles on the conformations of important macromolecules like proteins under different pathophysiological conditions. Pointing towards the situation, we carefully designed an in vitro study to elucidate the effect of green AgNPs on the aggregation pattern of α-chymotrypsinogen A at a human pathological body temperature. We observed that the B-AgNPs inhibited the aggregation in αCgn-A in a concentration-dependent manner showing maximum inhibition at 30 µg/ml above which the effect of aggregation inhibition was reduced as evident at 40 and 50 µg/ml concentrations of B-AgNPs. Further, in our in vitro analysis, we found that the B-AgNPs of lower sizes has potential chaperone-like activity at pathological body temperature, which can be used as a component of the drug to prevent protein aggregation after further verification in animal models.

A Biophysical and Computational Study of Concanavalin A Immobilized Zinc Oxide Nanoparticles.

BACKGROUND: Con A, a lectin extract from jackbean Canavalia ensiformis is known for its agglutination activity. ZnO nanoparticles promote the faster electron transfer between the lectin immobilized and the target cells. Hence, Con A immobilized on ZnO nanoparticles will agglutinate cells more effectively than the native protein. OBJECTIVES: Concanavalin A (Con A), a lectin was immobilized on the hexagonal zinc oxide (ZnO) nanoparticles to monitor its activity on RBCs and lymphocytes. METHODS: The immobilization of Con A and zinc oxide nanoparticles has been studied by molecular docking, microscopic and genotoxicity assessment techniques. RESULTS: Qualitative assessment using various techniques like atomic force microscopy, scanning electron microscopy and X-ray diffraction showed minor changes in morphology of Con A and ZnO nanoparticles. FT-IR spectroscopy confirmed the linking of Con A amino groups with ZnO nanoparticles. Con A immobilized nanoparticles in contrast to native lectin showed minor changes in hemagglutination activity as confirmed by pH dependence studies using fluorimetry. Con Aimmobilized nanoparticles retained the agglutination activity, this can be indicative of their potential application in detection of virus transformed and neoplastic cells. The Con A immobilized ZnO nanoparticles did not induce any significant but minor damage to whole cell DNA as revealed from comet assay or plasmid DNA. CONCLUSION: Con A immobilized on ZnO nanoparticles showed minor changes in the structure of ZnO nanoparticles and in the conformational of native Con A. However, Con A immobilized ZnO nanoparticles interestingly, showed pH resistance and better hemagglutination activity as well as minor DNA damage to whole cell lymphocytes. Thus, this novel bioaffinity support has prospective clinical implications.

Thermal unfolding of human lysozyme induces aggregation: Recognition of the aggregates by antisera against the native protein.

Protein aggregates are formed due to the inappropriate folding of polypeptides. Human lysozyme (HLZ) plays an important role in the innate immune response of the body and has been used extensively as a model protein to study aggregation. In this study, we showed that HLZ undergoes unfolding induced aggregation when heated by using spectroscopic and microscopic techniques. We further showed that the aggregates were recognized by polyclonal antibodies against the native HLZ. The consequences of these observations are further co-related with mammalian physiology.

Aloe emodin, an anthroquinone from Aloe vera acts as an anti aggregatory agent to the thermally aggregated hemoglobin.

Aggregation of proteins is a physiological process which contributes to the pathophysiology of several maladies including diabetes mellitus, Huntington's and Alzheimer's disease. In this study we have reported that aloe emodin (AE), an anthroquinone, which is one of the active components of the Aloe vera plant, acts as an inhibitor of hemoglobin (Hb) aggregation. Hb was thermally aggregated at 60°C for four days as evident by increased thioflavin T and ANS fluorescence, shifted congo red absorbance, appearance of ß sheet structure, increase in turbidity and presence of oligomeric aggregates. Increasing concentration of AE partially reverses the aggregation of the model heme protein (hemoglobin). The maximum effect of AE was observed at 100µM followed by saturation at 125µM. The results were confirmed by UV-visible spectrometry, intrinsic fluorescence, ThT, ANS, congo red assay as well as transmission electron microscopy (TEM). These results were also supported by fourier transform infrared spectroscopy (FTIR) and circular dichroism (CD) which shows the disappearance of ß sheet structure and appearance of α helices. This study will serve as baseline for translatory research and the development of AE based therapeutics for diseases attributed to protein aggregation.

Serotonin abrogates dopamine induced aggregation of cytochrome c.

Importance of cytochrome c arises from its involvement in apoptosis, sequence homology and conserveness during molecular evolution. Dopamine at 25µM concentration for 30h resulted in cytochrome c aggregation as evident by increase in ANS and ThT binding, structural transition from α helix to ß sheet and shift in congo red assay. Interestingly, serotonin at 25µM concentration incubated for 60h abrogates the aggregatory effect on cytochrome c. Fibrillation of human cytochrome c in the presence of dopamine gives the clue about conformational changes taking place in protein and example of natural aggregation and inhibitory effect of serotonin gives the clue about natural defence mechanism occurring in human body.