Applications of Solution NMR in Drug Discovery.

During the past decades, solution nuclear magnetic resonance (NMR) spectroscopy has demonstrated itself as a promising tool in drug discovery. Especially, fragment-based drug discovery (FBDD) has benefited a lot from the NMR development. Multiple candidate compounds and FDA-approved drugs derived from FBDD have been developed with the assistance of NMR techniques. NMR has broad applications in different stages of the FBDD process, which includes fragment library construction, hit generation and validation, hit-to-lead optimization and working mechanism elucidation, etc. In this manuscript, we reviewed the current progresses of NMR applications in fragment-based drug discovery, which were illustrated by multiple reported cases. Moreover, the NMR applications in protein-protein interaction (PPI) modulators development and the progress of in-cell NMR for drug discovery were also briefly summarized.

Potential TSPO Ligand and Photooxidation Quencher Isorenieratene from Arctic Ocean Rhodococcus sp. B7740.

Due to its special aromatic structure, isorenieratene is thought to be an active natural antioxidant and photo/UV damage inhibitor. In this work, isorenieratene that was extracted from Rhodococcus sp. B7740 isolated from the Arctic Ocean, showed excellent scavenging ability of both singlet oxygen and hydroxyl radical in the UVB-induced auto-oxidation process using the EPR method. Within an ARPE-19 cell model damaged by UVB radiation, isorenieratene showed fine protective effects (1.13 ± 0.03 fold) compared with macular xanthophylls (MXs) through upregulating of tspo. The molecular docking was firstly performed to investigate the interaction of isorenieratene with TSPO as a special ligand. Results showed isorenieratene might form a better binding conformation (S-score -8.5438) than MXs and indicate that isorenieratene not only can function as a direct antioxidant but also activate tspo in ARPE-19 cells. Thus, isorenieratene might ease the UV-related damages including age-related macular degeneration (AMD).

Comprehensive chemical profiling of Yindan Xinnaotong soft capsule and its neuroprotective activity evaluation in vitro.

Discovering effective combinational components (ECCs) and quality control markers of TCMs is still facing challenges because the holistic healing system comprises hundreds of compounds. Here, taking Yindan Xinnaotong soft capsule (YDXNT), a TCMs preparation composed by 8 herbs, as a case, a strategy that integrated multiple chromatographic analysis and bioactivity assay was proposed for potential ECCs of neuroprotection discovery. Firstly, ultra-high performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry (UHPLC-QTOF MS) and gas chromatography-mass spectrometry (GC-MS) were applied for comprehensive profiling of the chemical constituents in YDXNT. Given the fact that the complex matrix interference makes it more difficult to identify potentially active compounds, we proposed a structure-diagnostic ions-oriented strategy to remove interference ions from the raw UHPLC-MS data. The proposed strategy consisted of different filtering methods, including diagnostic fragment ions filtering (DFIF), mass defect filtering (MDF) and neutral loss (NL). Using this strategy, a total of 124 compounds were rapidly identified. Among them, 62 non-volatile and 5 volatile constituents in 30 batches of YDXNT were quantified by UHPLC tandem triple quadrupole mass spectrometry (QQQ-MS) and GC-MS methods, respectively. In order to facilitate the quality control of YDXNT, candidate ECCs were selected based on the threshold setting of absolute -contents, and their neuroprotective effects were examined. Finally, a combination of 16 compounds, accounts for 2.80% (w/w) of original YDXNT, was identified as its potential ECCs, which could be considered for the improvement of quality standardization of YDXNT.

Nanostructured biomedical selenium at the biological interface (Review).

This paper critically reviews the current evidence of research in biomedical applications of selenium nanoparticles (SeNPs) and their effects at cellular and tissue levels. In recent years, interest in SeNPs as a natural trace element nanomaterial for nanomedicine has resulted in a number of studies evaluating their bioactivities, such as anticancer, antimicrobial, and antioxidant properties. Significant data have been generated to demonstrate the effectiveness of SeNPs alone or in combination with other reagents. Their activities are demonstrated through in vitro and in vivo experimentation; yet, the levels of efficacy need to be improved, particularly when compared with those of pharmaceutical drugs (such as antibiotics and cytotoxic chemotherapeutic drugs). However, promising evidence suggests decreased toxicity when using SeNPs, and more importantly their ability to perform as an interfacing biomaterial with cells and tissues. SeNPs have demonstrated unique antibacterial properties: they inhibit bacterial adhesion, growth, and/or quorum sensing and as a result prevent biofilm formation on medical devices, to name a few. Therefore, as with other nanomaterials, SeNPs warrant further study as part of the biomaterial-based therapeutic toolkit as an alternative to traditional pharmaceutical agents. This paper will provide a succinct review of recent studies on SeNPs to critically assess the findings in the light of effectiveness, particularly highlighting the roles of the cellular interface. Finally, an outlook of the potential of SeNPs will be presented to highlight the need for more intensive studies of material stability, mechanistic understanding at subcellular levels, and investigations into their combinational and/or synergistic effects with other bioactive reagents including pharmaceutical drugs.

A efficient method to identify cardioprotective components of Astragali Radix using a combination of molecularly imprinted polymers-based knockout extract and activity evaluation.

Although herb medicines have become the major source for new drug discovery, many of them are largely under-explored due to the purity-activity relationship. Efficient identification of bioactive compounds in conventional stepwise separation and isolation has not yet been elucidated. Therefore, we proposed a new separation strategy for holism understanding of herb pharmacology using molecularly imprinted polymers (MIPs). Astragali Radix (AR), important in traditional Chinese medicine, was chosen in this study for multicomponent knockout followed by bioactivity evaluation. We prepared calycosin molecularly imprinted polymers (calycosin-MIPs) which could selectively recognize flavonoid aglycons in AR. The molecular selectivity of calycosin-MIPs as a critical parameter was evaluated using the template and other high content compounds in AR. Based on it, using the calycosin-MIPs material via solid-phase extraction procedures was applied for the knockout of flavonoid aglycons in AR. Finally, hypoxia/reoxygenation model in H9c2 cells was used to evaluate the activity of the AR extract before and after knockout. The results showed that calycosin-MIPs as recognition materials for flavonoid aglycons in AR are applied in one-step separation with high selectivity and tunability. The subextract in the absence of flavonoid aglycons has been demonstrated to clarify the cardio-protective components of AR. In conclusion, this proof-of-principle study is the first one showing that molecular imprinting technology coupled with a bioassay can be used to explore the bioactive variability from the perspective of multicomponent separation of herbal medicine.

Water extract of Semecarpus parvifolia Thw. leaves inhibits cell proliferation and induces apoptosis on HEp-2 cells.

BACKGROUND: Semecarpus parvifolia Thw is used as an ingredient of poly herbal decoctions to treat cancer in traditional medicine. The present study aims to investigate the antiproliferative activity on HEp 2 cells by the water extract of S. parvifolia leaves and to evaluate potential mechanisms. METHODS: The plant extract was exposed to S. parvifolia for 24 hours and antiproliferative activity was quantified by Sulforhodamine B (SRB), 3-(4, 5-dimethythiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) and Lactate dehydrogenase (LDH) assays. Morphological changes were observed after staining cells with ethidium bromide/acridine orange (EB/AO) and Giemsa dye. Comet assay was performed to evaluate the DNA damage. The toxicity of the plant extract was determined by brine shrimp lethality assay. RESULTS: S. parvifolia leaves reduced the cell proliferation in a dose and time dependent manner. A two fold increase in NO level was observed at higher concentrations. Morphological changes characteristic to apoptosis were observed in light microscopy, Giemsa and EB/AO stained cells. Fragmented DNA further confirmed its capacity to induce apoptosis. No lethality was observed with brine shrimps. CONCLUSION: The results suggest that Semecarpus parvifolia Thw induces apoptosis in HEp-2 cells through a NO dependent pathway.

Inhibitory effects of selenium on cadmium-induced cytotoxicity in PC12 cells via regulating oxidative stress and apoptosis.

Purpose of this study is to investigate mechanism/s of cyto-protection by selenium (Na2SeO3; Se4+) against cadmium (CdCl2; Cd2+)-induced cytotoxicity using PC12 cells. In addition, Se (5, 10, 20 and 40 µM) and Cd (2.5, 5 and 10 µM)-induced cytotoxicity is determined. Cytotoxicity assays and western blot analyses confirmed that Se (≥10 µM) promotes autophagic cell death via inhibition of mTOR activation and p62 accumulation due to increase of cellular oxidative stress. On the other hand, co-presence of non-toxic Se (5 µM) and toxic Cd (5 µM) showed to increase cell viability, glutathione and glutathione peroxidase 1 (GPx1) levels, and to decrease DNA fragmentation and lactate dehydrogenase (LDH) activity compared to Cd-treated (5 µM) cells alone. Furthermore, western blot analyses of cytochrome c and ERK1 indicated that Cd-induced apoptotic cell death in PC12 cells. However, the co-exposure of Se with Cd significantly decreases the release of cytochrome c into cytosol from mitochondria, and up-regulates ERK1 protein to inhibit Cd-induced apoptosis. In conclusion, Se (≥10 µM) possess cytotoxicity in PC12 cells; however, co-presence of Se (5 µM) with Cd (5 µM) protects against Cd-induced apoptosis in PC12 cells due to inhibition of Cd-induced oxidative stress and subsequently suppression of mitochondrial apoptosis pathway.

Proanthocyanidins purified from fruit pericarp of Clausena lansium (Lour.) Skeels as efficient tyrosinase inhibitors: structure evaluation, inhibitory activity and molecular mechanism.

Fruit pericarp of Clausena lansium (Lour.) Skeels, a food waste, was selected as a raw material for proanthocyanidins. The proanthocyanidins' structures were integrally analyzed using three methods: matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), high performance liquid chromatography electrospray ionization mass spectrometry (HPLC-ESI-MS) and 13C nuclear magnetic resonance (NMR). The results elucidated that these compounds were composed of prodelphinidin (75%) and procyanidin (25%) with a degree of polymerization (DP) up to the 20-mers. They were proved to be remarkable, reversible and mixed competitive inhibitors of tyrosinase according to results from enzyme experiments. The IC50 values were calculated to be 23.6 ± 1.2 and 7.0 ± 0.2 µg mL-1 for the monophenolase and diphenolase activities, respectively. In addition, the proanthocyanidins had a good inhibitory effect on cell proliferation, cellular tyrosinase activity and melanin production of B16 mouse melanoma cells. Chelation between the hydroxyl group on the B ring of the proanthocyanidins and dicopper irons of the enzyme provided one of the feasible mechanisms for the inhibition on the basis of fluorescence quenching and molecular docking analyses. This research would supply the scientific basis to these compounds application in the pharmaceutical, insecticides, and preservative fields.

Biophysical characteristics of proteins and living cells exposed to the green tea polyphenol epigallocatechin-3-gallate (EGCg): review of recent advances from molecular mechanisms to nanomedicine and clinical trials.

Herbs and traditional medicines have been applied for thousands of years, but researchers started to study their mode of action at the molecular, cellular and tissue levels only recently. Nowadays, just like in ancient times, natural compounds are still determining factors in remedies. To support this statement, the recently won Nobel Prize for an anti-malaria agent from the plant sweet wormwood, which had been used to effectively treat the disease, could be mentioned. Among natural compounds and traditional Chinese medicines, the green tea polyphenol epigallocatechin gallate (EGCg) is one of the most studied active substances. In the present review, we summarize the molecular scale interactions of proteins and EGCg with special focus on its limited stability and antioxidant properties. We outline the observed biophysical effects of EGCg on various cell lines and cultures. The alteration of cell adhesion, motility, migration, stiffness, apoptosis, proliferation as well as the different impacts on normal and cancer cells are all reviewed. We also handle the works performed using animal models, microbes and clinical trials. Novel ways to develop its utilization for therapeutic purposes in the future are discussed too, for instance, using nanoparticles and green tea polyphenols together to cure illnesses and the combination of EGCg and anticancer compounds to intensify their effects. The limitations of the employed experimental models and criticisms of the interpretation of the obtained experimental data are summarized as well.

Protective effects of five allium derived organosulfur compounds against mutation and oxidation.

In this study, we examined the ability of five allium-derived organosulfur compounds to protect cells against mutation and oxidation. The compounds tested were 1-propylmercaptan (PM), dimethyl disulfide (DMDS), diallyl disulfide (DADS), propyl disulfide (PDS), and 2,5-dimethylthiophene (DMT). Our results showed that when used at concentrations of 100-400 µmol/l, the five compounds inhibited the mutagenicity of 4-nitroquinoline-N-oxide, a direct mutagen, and benzo[a]pyrene, an indirect mutagen, toward Salmonella typhimurium TA 98 and TA 100. Furthermore, at these concentrations, all five of the compounds protected HepG2 cells against tert-butyl hydroperoxide-induced oxidative cytotoxicity. The compounds likely enhanced cell viability by suppressing the formation of reactive oxygen species and the depletion of glutathione depletion in cells. DMT and PM inhibited mutation and oxidation to a greater extent than DMDS, DADS, and PDS. These results demonstrate for the first time that DMT and PM can contribute to the antimutagenic and the antioxidative property of Allium vegetables.

The effect of five Taraxacum species on in vitro and in vivo antioxidant and antiproliferative activity.

Plants belonging to the genus Taraxacum are considered a nutritious food, being consumed raw or cooked. Additionally, these plants have long been used in folk medicine due to their choleretic, diuretic, antitumor, antioxidant, antiinflammatory, and hepatoprotective properties. This genus, with its complex taxonomy, includes several species that are difficult to distinguish. Its traditional use must be related not only to T. officinale F.H. Wigg., the most studied species, but also to others. The aim of this work is to compare five different common South European species of Taraxacum (T. obovatum (Willd.) DC., T. marginellum H. Lindb., T. hispanicum H. Lindb., T. lambinonii Soest and T. lacistrum Sahlin), in order to find differences between antioxidant and cytotoxic activities among them. Dissimilarities between species in LC/MS patterns, in in vitro and intracellular antioxidant activity and also in the cytotoxicity assay were found. T. marginellum was the most efficient extract reducing intracellular ROS levels although in in vitro assays, T. obovatum was the best free radical scavenger. A relevant cytotoxic effect was found in T. lacistrum extract over HeLa and HepG2 cell lines.

Activation of Nrf2 target enzymes conferring protection against oxidative stress in PC12 cells by ginger principal constituent 6-shogaol.

Natural compounds containing phenoxyl groups and/or Michael acceptor units appear to possess antioxidant and cytoprotective properties. The ginger principal constituent 6-shogaol (6-S) represents one of such compounds. In this study, we reported that 6-S efficiently scavenges various free radicals in vitro, and displays remarkable cytoprotection against oxidative stress-induced cell damage in the neuron-like rat pheochromocytoma cell line, PC12 cells. Pretreatment of PC12 cells with 6-S significantly upregulates a series of phase II antioxidant molecules, such as glutathione, heme oxygenase 1, NAD(P)H: quinone oxidoreductase 1, thioredoxin reductase 1, and thioredoxin 1. A mechanistic study revealed that 6-S enhanced the translocation of Nrf2 from the cytosol to the nucleus and knockdown of Nrf2 abolished such protection, indicating that this cytoprotection is mediated by the activation of the transcription factor Nrf2. Another ginger constituent 6-gingerol (6-G), having a similar structure of 6-S but lacking the alpha,beta-unsaturated ketone structure (Michael acceptor moiety), failed to shelter PC12 cells from oxidative stress. Our results demonstrate that 6-S is a novel small molecule activator of Nrf2 in PC12 cells, and suggest that 6-S might be a potential candidate for the prevention of oxidative stress-mediated neurodegenerative disorders.

A simple and high-throughput in-cell Western assay using HepG2 cell line for investigating the potential hypocholesterolemic effects of food components and nutraceutics.

Since saving time and money are critical issues while developing innovative functional foods and nutraceutics, the use of specific and high-throughput assays for the fast screening of potentially bioactive ingredients is crucial. In this context, the aim of the present investigation was the development of an in-cell Western (ICW) assay, a quantitative colorimetric cell-based technique, at the HepG2 cell line for screening and evaluating the effects of potentially bioactive compounds on the low density lipoprotein (LDL) receptor (LDLR). It is known that LDLR plays a pivotal role in the binding and endocytosis of circulating LDL, increasing its plasma clearance. The ICW was optimised and validated using monacolin K, the main hypocholesterolemic component of red yeast rice. This provided a robust and reproducible assay useful for characterising the cholesterol-lowering properties of bioactive food components. To our knowledge, this is the first application of the ICW technique in the field of functional foods and nutraceutics.

Cell-based peptide screening to access the undruggable target space.

Only 20-30% of drug target proteins can be accessed by common drug classes, like small molecules or therapeutic antibodies. The vast majority of the remaining proteins are considered "undruggable" and include drug target proteins, like transcription factors, scaffold or adapter proteins, which play important roles in disease. However over the last years innovative compound classes including nucleotide derived drugs (e.g. siRNA, antisense), macrocyclic compounds and cell-permeable peptides matured significantly and hold now the potential to modulate these hard to access target proteins for therapeutic use. This article will focus on the discovery of cell-permeable peptides and discuss intracellular screening systems for peptides, which yield highly relevant peptides, because peptide selection takes place in eukaryotic cells, under conditions, which are very similar to the later therapeutic use.

Antioxidant potential of Sutherlandia frutescens and its protective effects against oxidative stress in various cell cultures.

BACKGROUND: Sutherlandia frutescens (L.) R.Br. (SF) is a South African plant that is widely used to treat stress, infections, cancer, and chronic diseases, many of which involve oxidative stress. The aim of the study was to quantitatively assess the antioxidant potential of SF extracts in cell-free system as well as in cell lines. METHODS: Dried SF vegetative parts were extracted using six different solvents, and the extracts were assessed for total phenolic and flavonoid contents, total reducing power, iron chelating capacity, and free radical scavenging power, including, scavenging of hydroxyl radicals, superoxide anions, nitric oxide, and hydrogen peroxide. We further investigated the freeze-dried hot water extract of SF (SFE) to assess its effect against oxidative stress induced by tert-butyl hydroperoxide (t-BHP), an organic peroxide. Three different cell lines: Chinese hamster ovary (CHO), human hepatoma (HepaRG), and human pulmonary alveolar carcinoma (A549) cells, were employed to determine cell viability, intracellular reactive oxygen species (ROS) levels, and reduced to oxidized glutathione levels (GSH/GSSG). RESULTS: The results indicated that: (1) SF extracts have significant antioxidant potential that is dependent upon the nature of the extraction solvent and (2) SFE protects against tBHP-induced oxidative stress in cells by scavenging ROS and preserving intracellular GSH/GSSG. CONCLUSION: Oxidative stress is implicated in a number of disorders, and due to the public's concerns about synthetic antioxidants, various natural antioxidants are being explored for their therapeutic potential. Our findings support claims for S. frutescens being a promising adjunctive therapeutic for oxidative stress-related health problems.

Mapping the cellular response to small molecules using chemogenomic fitness signatures.

Genome-wide characterization of the in vivo cellular response to perturbation is fundamental to understanding how cells survive stress. Identifying the proteins and pathways perturbed by small molecules affects biology and medicine by revealing the mechanisms of drug action. We used a yeast chemogenomics platform that quantifies the requirement for each gene for resistance to a compound in vivo to profile 3250 small molecules in a systematic and unbiased manner. We identified 317 compounds that specifically perturb the function of 121 genes and characterized the mechanism of specific compounds. Global analysis revealed that the cellular response to small molecules is limited and described by a network of 45 major chemogenomic signatures. Our results provide a resource for the discovery of functional interactions among genes, chemicals, and biological processes.

Alumina-zirconia composites functionalized with laminin-1 and laminin-5 for dentistry: effect of protein adsorption on cellular response.

The present paper describes a study on laminin interaction with the surface of two alumina-zirconia composites with different percentages of ZrO2, both with submicrometric grain size. As major molecules within the basement membrane (BM), laminins are important protein fragments for epithelial cell adhesion and migration. On the other hand, alumina-zirconia composites are very attractive materials for dental applications due to their esthetic and mechanical properties. X-Ray photoelectron spectroscopy and atomic force microscopy were used to study the adsorption of two types of laminin, laminin-1 (Ln-1) and laminin-5 (Ln-5), onto the ceramics surfaces. The in vitro cell response was determined by intracellular phosphorylation of major kinases. Ceramics samples functionalized with laminins showed better cellular activation than untreated specimens; furthermore, cellular activation was found to be greater for the composite with higher percentage in zirconia when functionalized with Ln-5, whereas the adsorption of Ln-1 resulted in a greater activation for the alumina-rich oxide.

Evaluation of antioxidant, immunomodulatory, and cytotoxic action of fractions from Eugenia uniflora L. and Eugenia malaccensis L.: correlation with polyphenol and flavanoid content.

An increasing number of biological activities presented by medicinal plants has been investigated over the years, and they are used in the search for new substances with lower side effects. Eugenia uniflora L. and Eugenia malaccensis L. (Myrtaceae) have many folk uses in various countries. This current study was designed to quantify the polyphenols and flavonoids contents and evaluate the immunomodulatory, antioxidant, and cytotoxic potentials of fractions from E. uniflora L. and E. malaccensis L. It was observed that the polyphenol content was higher in ethyl acetate fractions. These fractions have high antioxidant potential. E. malaccensis L. seeds showed the largest DPPH radical scavenger capacity (EC50 = 22.62). The fractions of E. malaccensis L. leaves showed lower antioxidant capacity. The samples did not alter the profile of proinflammatory cytokines and nitric oxide release. The results indicate that species of the family Myrtaceae are rich in compounds with antioxidant capacity, which can help reduce the inflammatory response.

Cytoprotective effect of white tea against H2O2-induced oxidative stress in vitro.

The protective effect of water extracts of white tea (WEWT) on oxidative stress in vitro is investigated. WEWT, like water extracts of green tea (WEGT) and water extracts of Pu-erh tea (WEPT), demonstrates a marked inhibition of the oxidation of liposome, albumin and LDLmodel systems. WEWT protects against H2O2-induced cytotoxicity, in a dose-dependent manner. The inhibition of ROS generation and MDA formation by WEWT in H2O2-induced Clone 9 cells parallels the effects on cell viability. Moreover, GSH and antioxidant enzymes may play an important role in the protective effect that is associated with H2O2-induced oxidative stress. The HPLC-DAD and HPLC-MS/MS analysis, shows that sixteen bioactive compounds are present in WEWT, which may partially account for its protective effect against oxidative insult. These results suggest that the mechanism of the protective actions of WEWT is related to its antioxidant potential and the maintenance of the normal redox status of the cell.