Competitive metal-binding stoichiometry between calcium and strontium by cell wall proteins of Neurospora crassa.

Cell wall proteins from Neurospora crassa were isolated and evaluated to demonstrate their metal ability to bind Ca2+ /Sr2+ by loading the solubilized protein fraction on to immobilized metal affinity chromatography (IMAC) column pre-equilibrated with Ca2+ /Sr2+ . The sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis IMAC eluent, revealed ∼18 proteins with a similarity in the proteome pattern of Ca2+ /Sr2+ fractions. Diethyl aminoethyl chromatography showed five proteins in common in binding to Ca2+ and Sr2+ , were subjected to N-terminal sequencing. The sequence analysis was studied for the determination of metal-binding site prediction by CHED software indicating that all five were found to have a high affinity toward Ca2+ . From these five, two were randomly selected and denoted as CWP-A (possess five Ca binding sites of six metal-binding sites) and CWP-B (possess six binding sites of eight metal-binding sites). They were selected for further characterization studies to determine their Ca2+ bound Sr2+ binding properties. Surprisingly, these proteins were able to bind Sr2+ ions (29 µmol) with equal affinity as to Ca2+ ions (42 µmol) by means of direct binding, and/or by displacing calcium as observed in metal-dependent proteolytic protection, fluorescence-based metal exchange assays, and molecular simulation studies. From the results, we demonstrate for the first time, that there is a stoichiometry between Ca2+ (an essential macro elemental metal ion) and Sr2+ ions (a nonessential element for which no reported metabolic activity is reported) for the metal-binding sites on cell wall proteins. This stoichiometry could be due to similar atomic dimensions and metal-protein structure stabilizing properties of Sr2+ compared to Ca2+ .

In vitro and in silico characterization of angiogenic inhibitors from Sophora interrupta.

Sophora interrupta Bedd, (Fabaceae) is used in Indian folk medicine to treat cancer. Angiogenesis is one of the crucial characteristics of cancer metastasis and is regulated by vascular endothelial growth factor (VEGF). In this study, we examined the antiangiogenic properties of the root ethyl acetate extract of Sophora interrupta by various methods. In vitro antioxidant activity (100-600 µg/ml) of S. interrupta ethyl acetate (SEA) extract was evaluated by DPPH and ABTS, anti-inflammatory activity (50, 100 and 150 µg/ml) by estimating nitric oxide (NO) levels, anti-angiogenic activity (200 and 500 µg/ml) was validated by chorio allantoic membrane (CAM) assay and in silico molecular dynamic (MD) simulations analyses (25 ns) were performed to identify the anti-angiogenic compounds extracted from root extract. The antioxidative activity of SEA extract at IC50 (200 ± 0.6 µg/mL) is equal to that of ascorbic acid at IC50 (50 ± 0.6 µg/mL), and the anti-inflammatory activity of SEA extract at IC50 (150 ± 0.2 µg/mL) was inhibited significantly by nitric oxide (NO) production. The SEA extract significantly reduced the sprouting of new blood vessels at ID50 500 ± 0.13 µg/mL in the CAM assay. Gas chromatography-mass spectrometry analysis of the SEA extract detected 34 secondary metabolites, of which 6a,12a-dihydro-6H-(1,3)dioxolo(5,6)benzofuro(3,2-c)chromen-3-ol (maackiain) and funiculosin formed strong hydrogen bond interactions with Lys 920, Thr 916 and Cys 919 (2H), as well as Glu 917 of VEGFR2, and these interactions were similar to those of the anti-angiogenic compound axitinib. Significant findings in all the assays performed indicate that SEA extract has potential anti-angiogenic compounds that may interfere with VEGF-induced cancer malignancy.

Isolation and Characterization of the Anticancer Compound Piceatannol from Sophora Interrupta Bedd.

BACKGROUND: Sophora belongs to the family of Fabaceae and the species in this genus are currently used as a folklore medicine for preventing a variety of ailments including cancer. Our aim was to identify and validate an anticancer compound from Sophora interrupta using multi-spectroscopic, anticancer screening, and molecular docking approach. METHODS: The cytotoxicity of the various solvent extracts, petroleum ether, n-butanol, and ethyl acetate (EtOAc) of the S. interrupta root powder was evaluated in a breast cancer cell lines (MCF-7). The extract that had anticancer activity was subjected to column chromatography based on the polarity of the solvents. The anticancer activity of the elution fractions was validated using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The isolated metabolite fraction with anticancer activity was run through a C18 column isocratic and gradient high-performance liquid chromatography (HPLC). The structure of the isolated compound was characterized using (1)H nuclear magnetic resonance (NMR), (13)C-NMR, Fourier transform infrared spectroscopy, and liquid chromatography-mass spectrometer methods. RESULTS: The crude EtAOc extract effectively inhibited the proliferation of MCF-7 cells. The column eluted chloroform and EtOAc (4:6) fraction of the EtOAc extract showed significant anticancer activity in the MCF-7 cells compared with normal mesenchymal stem cells. This fraction showed three major peaks in the HPLC chromatogram and the first major peak with a retention time (RT) of 7.153 was purified using preparative-HPLC. The structure of the compound is a piceatannol, which is a metabolic product of resveratrol. Piceatannol formed direct two hydrogen bond interactions between Cys912 (2H), and Glu878 of vascular endothelial growth factor receptor 1 (VEGFR1) with a glide-score (G-score) of -10.193, and two hydrogen bond interactions between Cys919, and Asp1046 of VEGFR2, with a G-score of -8.359. The structure is similar to that of the crystallized protein for VEGFR1 and R2. CONCLUSIONS: Piceatannol is a secondary metabolite of S. interrupta that has anticancer activity. Moreover, piceatannol has been isolated for the first time from S. interrupta.

Characterization of vitamin-cisplatin-loaded chitosan nano-particles for chemoprevention and cancer fatigue.

CONTEXT: Vitamins have been shown to reduce chemotherapy-related fatigue (CRF) by conserving energy loss both during and after cancer treatment. However, it remains unknown whether this reduction of fatigue interferes with the cancer drugs or alters the effectiveness of these agents. OBJECTIVES: The objective was to synthesize vitamin-cisplatin-loaded chitosan nano-particles for chemoprevention and cancer fatigue. MATERIALS AND METHODS: Multi-vitamin (C, D3, and B12)-cisplatin composite nano-formulation called NanoCisVital (NCV) to overcome CRF. The interactions between vitamins and NCV were characterized using scanning electron microscopy (SEM), Fourier transform infrared (FT-IR) spectroscopy, and a particle size analyser. The chemo-preventive activity was performed by in vitro bio assays. RESULTS: SEM analysis showed spherical shape and the size is < 225 nm. NCV inhibited the proliferating yeast cells as well as denaturation of bovine serum albumin, and it also reduced the sprouting of new blood vessels in dose-dependent manner. CONCLUSION: Collectively, these results demonstrate that the NCV particles can be used to reduce CRF without much affecting the anti-cancer properties of cisplatin.

Designing of potential inhibitors against Staphylococcus aureus sortase A: Combined analogue and structure based approach with in vitro validation.

Staphylococcus aureus sortase A is an attractive target of Gram-positive bacteria that plays a crucial role in anchoring of surface proteins to peptidoglycan present in bacterial cell wall. Inhibiting sortase A is an elementary and essential effort in preventing the pathogenesis. In this context, in silico virtual screening of in-house database was performed using ligand based pharmacophore model as a filter. The developed pharmacophore model AAHR 11 consists of two acceptors, one hydrophobic and one ring aromatic feature. Top ranked molecule KKR1 was docked into the active site of the target. After profound analysis, it was analyzed and optimized based on the observations from its binding pose orientation. Upgraded version of KKR1 was KKR2 and has improved docking score, binding interactions and best fit in the binding pocket. KKR1 along with KKR2 were further validated using 100 ns molecular dynamic studies. Both KKR1 and KKR2 contain Indole-thiazolidine moiety and were synthesized. The disk diffusion assay has good initial results (ZI of KKR1, KKR2 were 24, 38 mm at 10 µg/mL and ZI of Ampicillin was 22 at 10 µg/mL) and calculated MICs of the molecules (KKR1 5.56±0.28 µg/mL, KKR2 1.32±0.12 µg/mL, Ampicillin 8±1.1 µg/mL) were in good agreement with standard drug Ampicillin. KKR1 has shown IC50 of 1.23±0.14 µM whereas the optimized lead molecule KKR2 show IC50 of 0.008±0.07 µM. Results from in silico were validated by in vitro studies and proved that indole-thiazolidine molecules would be useful for future development as lead molecules against S. aureus sortase A.

Identification of human cyclooxegenase-2 inhibitors from Cyperus scariosus (R.Br) rhizomes.

Cyperus scariosus (R.Br) belongs to the family Cyperaceae and it has a diverse medicinal importance. To identify human cyclooxegenase-2 (COX-2) inhibitors from C. scariosus, the rhizome powder was exhaustively extracted with various solvents based on the increasing polarity. Based on the presence and absence of secondary metabolites, we have selected the methanolic extract to evaluate the anti-oxidant and anti-inflammatory activity. The same extract was further subjected to gas chromatography-mass spectroscopy (GC-MS) analysis to identify the active compounds. Binding affinities of these compounds towards anti-inflammatory protein COX-2 were analyzed using molecular docking interaction studies. Phytochemical analysis showed that methanol extract is positive for all secondary metabolites. The antioxidant activity of the C. scariosus rhizomes methanolic extract (CSRME) is half to that of ascorbic acid at 50 µg/ml. The anti-inflammatory activity of CSRME is higher than that of diclofenac sodium salt at high concentration, which is evident from the dose dependent inhibition of bovine serum albumin denaturation at 40 µg/ml-5 mg/ml. GC-MS analysis showed the presence of nine compounds, among all N-methyl-1-adamantaneacetamide and 1,5,diphenyl-2H-1,2,4- triazine form a hydrogen bond interactions with Ser-530 and Tyr-385 respectively and found similar interactions with crystal structure of diclofenac bound COX-2 protein. Benzene-1, 2-diol, 4-(4-bromo-3 chlorophenyl iminomethyl forms hydrogen bond interactions with Thr-199 and Thr-200 as similar to crystallized COX-2 protein with valdecoxib. Collectively our results suggest that CSRME contains medicinally important anti-inflammatory compounds and this justifies the use of this plant as a folklore medicine for preventing inflammation associated disorders.

In-Vitro and in-Silico characterization of Sophora interrupta plant extract as an anticancer activity.

Sophora interrupta belongs to the family of Fabaceae and the species in this genus have a diverse medicinal importance as a folk medicine for preventing many ailments including cancer. In order to evaluate the anticancer activity of S.interrupta, we have performed in vitro anti-oxidant, anti-inflammatory, anti-proliferative, and cell based anticancer activity in MCF-7 and PC-3 cell lines. Secondary metabolites of S.interrupta were used to identify anticancer compounds using Open Eye software. The antioxidant activity of the S.interrupta root ethylacetate (SEA) extract at 100 µg/ml is equal to that of ascorbic acid at 50 µg/ml. The antiinflammatory activity of SEA is half of that of diclofenac at 50 µg/ml. Anticancer activity was detected by measuring the mitochondrial dehydrogenase activity (MTT assay). The half maximal inhibitory concentrations (IC50) for MCF-7 and PC-3 cell lines are 250 and 700 µg/ml respectively. This was supported by the morphological changes such as membrane blebbing, cell detachment and rounded cell morphology when compared to the parental cells. In addition, we observed few green cells (live) over red cells (dead) based on the uptake of acridine orange and ethidium bromide dyes. Kaempferol-3-O-b-D-glucopyranoside, a Secondary metabolite of S.interrupta form 6 hydrogen bond interactions with Arg 202, Gln 207, Gly 227, Gly 229, Thr 231 and Ala 232 human DEAD box RNA helicase, DDX3 protein and is equivalent to crystal structure of adenosine mono phosphate to DDX3. Overall, it suggests that the SEA extract has anticancer compounds, and it can be used to enhance death receptor mediated cancer cell death.

Inferences from the ADMET analysis of predicted inhibitors to Follicle Stimulating Hormone in the context of infertility.

Follicle stimulating hormone (FSH) is a glycoprotein secreted by gonadotrophs of the anterior pituitary gland that regulates reproduction in mammals. FSH targets its receptor (FSHR) expressed only on grannulosa cells and induce the maturation of ovarian follicles in females. The levels of both FSH and FSHR rise until the middle of estrus cycle and then falls on level at the time of ovulation. It is associated with stimulated sertoli cell proliferation in testes and supports spermatogenesis in males. The interaction between the polypeptide FSH hormone and its corresponding receptor is highly selective. Therefore, it is of interest to inhibit FSH in the context of infertility. The structure of FSH (PDB ID: 1XWD) is screened using molecular docking techniques against the ZINC database (a database of 2.7 million compounds) with reference to known standard compounds. This exercise identifies compounds with better binding and ADMET (Absorption, Digestion, Metabolism, Excretion and Toxicity) properties compared to known standard compounds. These observations find application for the consideration of such compounds for further validation towards inhibiting the FSH.