Discovery of VEGFR inhibitors through virtual screening and energy assessment

Vascular endothelial growth factor receptor-2 (VEGFR-2) is crucial in promoting tumor angiogenesis and cancer metastasis. Thus, inhibition of VEGFR-2 has appeared as a good tactic for cancer treatment. To find out novel VEGFR-2 inhibitors, first, the PDB structure of VEGFR-2, 6GQO, was selected based on atomic nonlocal environment assessment (ANOLEA) and PROCHECK assessment. 6GQO was then further used for structure-based virtual screening (SBVS) of different molecular databases, including US-FDA approved drugs, US-FDA withdrawn drugs, may bridge, MDPI, and Specs databases using Glide. Based on SBVS, receptor fit, drug-like filters, and absorption, distribution, metabolism, excretion, and toxicity (ADMET) analysis of 427877 compounds, the best 22 hits were selected. From the 22 hits, hit 5 complex with 6GQO was put through molecular mechanics/generalized born surface area (MM/GBSA) study and hERG binding. The MM/GBSA study revealed that hit 5 possesses lesser binding free energy with more inferior stability in the receptor pocket than the reference compound. The VEGFR-2 inhibition assay of hit 5 disclosed an IC₅₀ of 165.23 nM against VEGFR-2, which can be possibly enhanced through structural modifications.     

Water relations and nitrogen fixation in potassium fedVigna radiata nodules

Drought created by withholding the irrigation at 30 and 45 d after sowing significantly decreased relative water content (RWC) and osmotic potential (ψ_s) ofVigna radiata (L.) Wilczek cv. MH-83-30 nodules. Potassium fed plants showed higher RWC, whereas ψ_s was further declined irrespective of soil moisture levels. The nitrogenase activity and leghemoglobin content of nodules markedly decreased under drought and nodules of potassium fed plants showed better recovery after rehydration. The proline content significantly increased under drought but declined upon reirrigation. Also, the C, N and K contents of nodules significantly declined under drought.     

Guggulsterone enhances glycosylated low density lipoprotein binding in rat liver

Modification of low density lipoprotein by nonenzymic glycosylation resulted in decreased receptor-mediated lipoprotein catabolism. Guggulsterone treatment caused significant increase in binding of [¹²⁵I] low density lipoprotein as well as [¹²⁵I] glycosylated low density lipoprotein. Scatchard plot analysis of the binding activity revealed that under the influence of guggulsterone, the liver membrane contains increased amounts of a functional lipoprotein receptor that binds more low density lipoprotein particles.     

Combining results from lectin affinity chromatography and glycocapture approaches substantially improves the coverage of the glycoproteome

Identification of glycosylated proteins, especially those in the plasma membrane, has the potential of defining diagnostic biomarkers and therapeutic targets as well as increasing our understanding of changes occurring in the glycoproteome during normal differentiation and disease processes. Although many cellular proteins are glycosylated they are rarely identified by mass spectrometric analysis (e.g. shotgun proteomics) of total cell lysates. Therefore, methods that specifically target glycoproteins are necessary to facilitate their isolation from total cell lysates prior to their identification by mass spectrometry-based analysis. To enrich for plasma membrane glycoproteins the methods must selectively target characteristics associated with proteins within this compartment. We demonstrate that the application of two methods, one that uses periodate to label glycoproteins of intact cells and a hydrazide resin to capture the labeled glycoproteins and another that targets glycoproteins with sialic acid residues using lectin affinity chromatography, in conjunction with liquid chromatography-tandem mass spectrometry is effective for plasma membrane glycoprotein identification. We demonstrate that this combination of methods dramatically increases coverage of the plasma membrane proteome (more than one-half of the membrane glycoproteins were identified by the two methods uniquely) and also results in the identification of a large number of secreted glycoproteins. Our approach avoids the need for subcellular fractionation and utilizes a simple detergent lysis step that effectively solubilizes membrane glycoproteins. The plasma membrane localization of a subset of proteins identified was validated, and the dynamics of their expression in HeLa cells was evaluated during the cell cycle. Results obtained from the cell cycle studies demonstrate that plasma membrane protein expression can change up to 4-fold as cells transit the cell cycle and demonstrate the need to consider such changes when carrying out quantitative proteomics comparison of cell lines.     

Dynamic contrast-enhanced magnetic resonance imaging of sunitinib-induced vascular changes to schedule chemotherapy in renal cell carcinoma xenograft tumors

In an attempt to develop better therapeutic approaches for metastatic renal cell carcinoma (RCC), the combination of the antiangiogenic drug sunitinib with gemcitabine was studied. Using dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI), we have previously determined that a sunitinib dosage of 20 mg/kg per day increased kidney tumor perfusion and decreased vascular permeability in a preclinical murine RCC model. This sunitinib dosage causing regularization of tumor vessels was selected to improve delivery of gemcitabine to the tumor. DCE-MRI was used to monitor regularization of vasculature with sunitinib in kidney tumors to schedule gemcitabine. We established an effective and nontoxic schedule of sunitinib combined with gemcitabine consisting of pretreatment with sunitinib for 3 days followed by four treatments of gemcitabine at 20 mg/kg given 3 days apart while continuing daily sunitinib treatment. This treatment caused significant tumor growth inhibition resulting in small residual tumor nodules exhibiting giant tumor cells with degenerative changes, which were observed both in kidney tumors and in spontaneous lung metastases, suggesting a systemic antitumor response. The combined therapy caused a significant increase in mouse survival. DCE-MRI monitoring of vascular changes induced by sunitinib, gemcitabine, and both combined showed increased tumor perfusion and decreased vascular permeability in kidney tumors. These findings, confirmed histologically by thinning of tumor blood vessels, suggest that both sunitinib and gemcitabine exert antiangiogenic effects in addition to cytotoxic antitumor activity. These studies show that DCE-MRI can be used to select the dose and schedule of antiangiogenic drugs to schedule chemotherapy and improve its efficacy.     

Nonstarch Polysaccharides Modulate Bacterial Microbiota,Pathways for Butyrate Production,and Abundance of Pathogenic Escherichia coli in the Pig Gastrointestinal Tract

The impact of nonstarch polysaccharides (NSP) differing in their functional properties on intestinal bacterial community composition, prevalence of butyrate production pathway genes, and occurrence of Escherichia coli virulence factors was studied for eight ileum-cannulated growing pigs by use of terminal restriction fragment length polymorphism (TRFLP) and quantitative PCR. A cornstarch- and casein-based diet was supplemented with low-viscosity, low-fermentability cellulose (CEL), with high-viscosity, low-fermentability carboxymethylcellulose (CMC), with low-viscosity, high-fermentability oat β-glucan (LG), and with high-viscosity, high-fermentability oat β-glucan (HG). Only minor effects of NSP fractions on the ileal bacterial community were observed, but NSP clearly changed the digestion in the small intestine. Compared to what was observed for CMC, more fermentable substrate was transferred into the large intestine with CEL, LG, and HG, resulting in higher levels of postileal dry-matter disappearance. Linear discriminant analysis of NSP and TRFLP profiles and 16S rRNA gene copy numbers for major bacterial groups revealed that CMC resulted in a distinctive bacterial community in comparison to the other NSP, which was characterized by higher gene copy numbers for total bacteria, Bacteroides-Prevotella-Porphyromonas, Clostridium cluster XIVa, and Enterobacteriaceae and increased prevalences of E. coli virulence factors in feces. The numbers of butyryl-coenzyme A (CoA) CoA transferase gene copies were higher than those of butyrate kinase gene copies in feces, and these quantities were affected by NSP. The present results suggest that the NSP fractions clearly and distinctly affected the taxonomic composition and metabolic features of the fecal microbiota. However, the effects were more linked to the individual NSP and to their effect on nutrient flow into the large intestine than to their shared functional properties.The porcine intestinal microbiota change in response to dietary carbohydrate composition due to specific substrate preferences of bacteria (6). Therefore, inclusion of specific nonstarch polysaccharides (NSP) in the diet of pigs allows manipulation of the composition of the intestinal microbiota. The NSP can also reduce digestibility of nutrients in the small intestine (8). The resulting changes in nutrient flow alter the availability of fermentable substrate in the different sections of the gut and thus may modify the bacterial community structure. Differences in the fermentability levels of individual NSP may not only affect the kinetics of their degradation by intestinal bacteria but may also change the composition of the fermentation end products (49). Particularly, butyrate is an important metabolite because of its potential to affect gene expression and to improve cellular development in enterocytes (38). The ability of gut microbiota to produce butyrate can vary considerably in response to environmental factors, such as diet composition (3). However, the number of butyrate-producing bacteria in complex fecal samples has been difficult to estimate by targeting the 16S rRNA gene, because these bacteria do not form a homogeneous phylogenetic group, and both butyrate producers and non-butyrate producers are found within the same phylogenetic clusters belonging to Clostridium clusters I, III, IV, XI, XIVa, XV, and XVI (27). Two alternative pathways for butyrate formation in bacteria harboring the rumen and human colon have been described (7, 26). The majority of human colonic butyrate producers use butyryl-coenzyme A (CoA) CoA transferase, whereas soil bacteria mostly utilize the butyrate kinase for the last step of butyrate formation (26, 27). However, information about the butyrate pathways used by intestinal bacteria in pigs is not available.In addition to the effects of the functional properties of NSP on intestinal physiology and fermentation processes, selection of specific NSP fractions may also prevent or stimulate overgrowth of pathogenic bacteria. For instance, dietary inclusion of highly viscous carboxymethylcellulose (CMC) has been shown to increase fecal shedding of enterotoxigenic Escherichia coli in weaned pigs (15). There is a need to identify those dietary NSP fractions that may either increase or reduce the numbers of potential pathogenic bacteria to formulate diets exerting beneficial effects on gut health, which is particularly important in antibiotic-free feeding regimens.Most studies pertaining on the effect of diet composition on the bacterial community in pigs have employed natural NSP sources and cereal-based diets, thereby resulting in a mixture of different soluble and insoluble NSP showing considerable interactions and modification of intestinal bacterial ecophysiology (6, 36, 37). Purified NSP fractions are increasingly available from the bioprocessing industry for use in food preparation and potentially in animal feeds, where economics and possible health benefits warrant this use. However, less is known about the fermentative properties of purified NSP fractions than about those of NSP in the grain matrix (37), which may also differ according to their origins.The aim of the present study was to examine the effects of four purified NSP fractions differing in their functional properties, i.e., viscosity and fermentability, on the ileal and fecal bacterial community, butyrate production pathway genes, and the occurrence of virulence factor genes of swine-pathogenic E. coli, including enterotoxigenic and enteroaggregative E. coli (11, 13).(This study was presented in part at the 11th Digestive Physiology in Pigs Symposium, Reus, Spain, 19 to 22 May 2009.)     

NPP-16/Nup50 Function and CDK-1 Inactivation Are Associated with Anoxia-induced Prophase Arrest in Caenorhabditis elegans

Oxygen, an essential nutrient, is sensed by a multiple of cellular pathways that facilitate the responses to and survival of oxygen deprivation. The Caenorhabditis elegans embryo exposed to severe oxygen deprivation (anoxia) enters a state of suspended animation in which cell cycle progression reversibly arrests at specific stages. The mechanisms regulating interphase, prophase, or metaphase arrest in response to anoxia are not completely understood. Characteristics of arrested prophase blastomeres and oocytes are the alignment of condensed chromosomes at the nuclear periphery and an arrest of nuclear envelope breakdown. Notably, anoxia-induced prophase arrest is suppressed in mutant embryos lacking nucleoporin NPP-16/NUP50 function, indicating that this nucleoporin plays an important role in prophase arrest in wild-type embryos. Although the inactive form of cyclin-dependent kinase (CDK-1) is detected in wild-type–arrested prophase blastomeres, the inactive state is not detected in the anoxia exposed npp-16 mutant. Furthermore, we found that CDK-1 localizes near chromosomes in anoxia-exposed embryos. These data support the notion that NPP-16 and CDK-1 function to arrest prophase blastomeres in C. elegans embryos. The anoxia-induced shift of cells from an actively dividing state to an arrested state reveals a previously uncharacterized prophase checkpoint in the C. elegans embryo.     

Lipid-induced extension of apolipoprotein E helix 4 correlates with low density lipoprotein receptor binding ability

Apolipoprotein E (apoE) serves as a ligand for the low density lipoprotein receptor (LDLR) only when bound to lipid. The N-terminal domain of lipid-free apoE exists as globular 4-helix bundle that is conferred with LDLR recognition ability after undergoing a lipid binding-induced conformational change. To investigate the structural basis for this phenomenon, site-directed spin label electron paramagnetic resonance spectroscopy experiments were conducted, focusing on the region near the C-terminal end of helix 4 (Ala-164). Using C112S apoE-N-terminal as template, a series of single cysteine substitution variants (at sequence positions 161, 165, 169, 173, 176, and 181) were produced, isolated, and labeled with the nitroxide probe, methane thiosulfonate. Electron paramagnetic resonance analysis revealed that lipid association induced fixed secondary structure in a region of the molecule known to exist as random coil in the lipid-free state. In a complementary approach, site-directed fluorescence analysis using an environmentally sensitive probe indicated that the lipid-induced transition of this region of the protein to alpha helix was accompanied by relocation to a more hydrophobic environment. In studies with full-length apoE single Cys variants, a similar random coil to stable backbone transition was observed, consistent with the concept that lipid interaction induced an extension of helix 4 beyond the boundary defining its lipid-free conformation. This structural transition likely represents a key conformational change necessary for manifestation of the LDLR recognition properties of apoE.     

Binding characteristics of a panel of monoclonal antibodies against the ligand binding domain of the human LDLr

To obtain a panel of monoclonal antibodies (MAbs) to study the folding and conformation of the low density lipoprotein receptor (LDLr), we have generated hybridomas from LDLr-deficient mice that had been immunized with the extracellular domain of the human LDLr. The 12 MAbs were specific for the ligand binding domain of the LDLr, with individual MAbs recognizing epitopes in ligand binding repeats 1, 2, 3, 5, and 7. A subset of the MAbs failed to react with the LDLr when disulfide bonds were reduced, and one MAb, specific for an epitope that spans ligand binding repeats 1 and 2, recognized two conformational forms of the LDLr with different affinities. Antibodies specific for ligand binding repeats 3, 5, and 7 completely blocked the binding of LDL particles to the LDLr on cultured human fibroblasts, whereas MAbs with epitopes in ligand binding repeats 1 and 2 partially blocked the binding of LDL to the LDLr. These anti-LDLr MAbs will serve as useful probes for further analysis of LDLr conformation and LDLr-mediated lipoprotein binding.     

Substituted hydrazinecarbothioamide as potent antitubercular agents: Synthesis and quantitative structure–activity relationship (QSAR)

A series of novel substituted hydrazinecarbothioamides was synthesized and evaluated for anti-TB activity. Three most active compounds viz. 1, 6 and 12 were found to exhibit minimum inhibitory concentration (MIC) of 0.4 μg/mL, whereas four compounds viz. 3, 5, 10 and 11 showed comparatively lesser activity with MIC value of 0.8 μg/mL against Mycobacterium tuberculosis strain. A highly significant QSAR equation explaining 81.8% variance is described.