Synthesis and biological evaluation of estradiol linked pyrrolo[2,1-c][1,4]benzodiazepine (PBD) conjugates as potential anticancer agents

A series of new estradiol linked pyrrolo[2,1-c][1,4]benzodiazepine (E(2)-PBD) conjugates (3a-f, 4a-f and 5a-f) with different linker architectures including a triazole moiety have been designed and synthesized. All the 18 compounds have been evaluated for their anticancer activity and it is observed that some of the compounds particularly 4c-e and 5c,d exhibited significant anticancer activity. The detailed biological aspects relating to the cell cycle effects and tubulin depolymerization activity have been examined with a view to understand the mechanism of action of these conjugates. Among all these conjugates, one of the compound 5c could be considered as the most effective compound particularly against MCF-7 breast cancer cell line.     

Phosphoproteomic screen identifies potential therapeutic targets in melanoma

Therapies directed against receptor tyrosine kinases are effective in many cancer subtypes, including lung and breast cancer. We used a phosphoproteomic platform to identify active receptor tyrosine kinases that might represent therapeutic targets in a panel of 25 melanoma cell strains. We detected activated receptors including TYRO3, AXL, MERTK, EPHB2, MET, IGF1R, EGFR, KIT, HER3, and HER4. Statistical analysis of receptor tyrosine kinase activation as well as ligand and receptor expression indicates that some receptors, such as FGFR3, may be activated via autocrine circuits. Short hairpin RNA knockdown targeting three of the active kinases identified in the screen, AXL, HER3, and IGF1R, inhibited the proliferation of melanoma cells and knockdown of active AXL also reduced melanoma cell migration. The changes in cellular phenotype observed on AXL knockdown seem to be modulated via the STAT3 signaling pathway, whereas the IGF1R-dependent alterations seem to be regulated by the AKT signaling pathway. Ultimately, this study identifies several novel targets for therapeutic intervention in melanoma.     

Solid Phase and Solution Phase Synthesis of Gamma Amino Acid Homo-Oligomers and Mixed Oligomers

Abstract  

An efficient stepwise synthesis of homo-oligomers and mixed oligomers of gabapentin and pregabalin on solid support using Fmoc-protected derivatives and HBTU/HOBt/DIEA as coupling agent is described. The synthesis was also carried out using solution phase methodology. The Gpn/Pgn homo oligomers and mixed oligomers forms C₉ helix in solution as determined by NMR study. Chiral as well as achiral gamma amino acids were used for the synthesis of oligomers in order to investigate the secondary structural preferences.     

Intracellular S1P generation is essential for S1P-induced motility of human lung endothelial cells: role of sphingosine kinase 1 and S1P lyase

Background

Earlier we have shown that extracellular sphingosine-1-phosphate (S1P) induces migration of human pulmonary artery endothelial cells (HPAECs) through the activation of S1P₁ receptor, PKCε, and PLD2-PKCζ-Rac1 signaling cascade. As endothelial cells generate intracellular S1P, here we have investigated the role of sphingosine kinases (SphKs) and S1P lyase (S1PL), that regulate intracellular S1P accumulation, in HPAEC motility.

Methodology/Principal Findings

Inhibition of SphK activity with a SphK inhibitor 2-(p-Hydroxyanilino)-4-(p-Chlorophenyl) Thiazole or down-regulation of Sphk1, but not SphK2, with siRNA decreased S1P_int, and attenuated S1P_ext or serum-induced motility of HPAECs. On the contrary, inhibition of S1PL with 4-deoxypyridoxine or knockdown of S1PL with siRNA increased S1P_int and potentiated motility of HPAECs to S1P_ext or serum. S1P_ext mediates cell motility through activation of Rac1 and IQGAP1 signal transduction in HPAECs. Silencing of SphK1 by siRNA attenuated Rac1 and IQGAP1 translocation to the cell periphery; however, knockdown of S1PL with siRNA or 4-deoxypyridoxine augmented activated Rac1 and stimulated Rac1 and IQGAP1 translocation to cell periphery. The increased cell motility mediated by down-regulation was S1PL was pertussis toxin sensitive suggesting “inside-out” signaling of intracellularly generated S1P. Although S1P did not accumulate significantly in media under basal or S1PL knockdown conditions, addition of sodium vanadate increased S1P levels in the medium and inside the cells most likely by blocking phosphatases including lipid phosphate phosphatases (LPPs). Furthermore, addition of anti-S1P mAb to the incubation medium blocked S1P_ext or 4-deoxypyridoxine-dependent endothelial cell motility.

Conclusions/Significance

These results suggest S1P_ext mediated endothelial cell motility is dependent on intracellular S1P production, which is regulated, in part, by SphK1 and S1PL.     

Up-regulation of MUC2 and IL-1β expression in human colonic epithelial cells by Shigella and its interaction with mucins

Background

The entire gastrointestinal tract is protected by a mucous layer, which contains complex glycoproteins called mucins. MUC2 is one such mucin that protects the colonic mucosa from invading microbes. The initial interaction between microbes and mucins is an important step for microbial pathogenesis. Hence, it was of interest to investigate the relationship between host (mucin) and pathogen interaction, including Shigella induced expression of MUC2 and IL-1β during shigellosis.

Methods

The mucin-Shigella interaction was revealed by an in vitro mucin-binding assay. Invasion of Shigella dysenteriae into HT-29 cells was analyzed by Transmission electron microscopy. Shigella induced mucin and IL-1β expression were analyzed by RT-PCR and Immunofluorescence.

Results

The clinical isolates of Shigella were found to be virulent by a congo-red binding assay. The in vitro mucin-binding assay revealed both Shigella dysenteriae and Shigella flexneri have binding affinity in the increasing order of: guinea pig small intestinal mucinShigella dysenteriae into HT-29 cells occurs within 2 hours. Interestingly, in Shigella dysenteriae infected conditions, significant increases in mRNA expression of MUC2 and IL-1β were observed in a time dependent manner. Further, immunofluorescence analysis of MUC2 shows more positive cells in Shigella dysenteriae treated cells than untreated cells.

Conclusions

Our study concludes that the Shigella species specifically binds to guinea pig colonic mucin, but not to guinea pig small intestinal mucin. The guinea pig colonic mucin showed a greater binding parameter (R), and more saturable binding, suggesting the presence of a finite number of receptor binding sites in the colonic mucin of the host. In addition, modification of mucins with TFMS and sodium metaperiodate significantly reduced mucin-bacterial binding; suggesting that the mucin-Shigella interaction occurs through carbohydrate epitopes on the mucin backbones. Overproduction of MUC2 may alter adherence and invasion of Shigella dysenteriae into human colonic epithelial cells.     

Using the biological taxonomy to access biological literature with PathBinderH

PathBinderH allows users to make queries that retrieve sentences and the abstracts containing them from PubMed. Another aspect of PathBinderH is that users can specify biological taxa in order to limit searches by mentioning either the specified taxa, or their subordinate taxa, in the biological taxonomy. Although the current project requires this function only for plant taxa, the principle is extensible to the entire taxonomy. AVAILABILITY: www.plantgenomics.iastate.edu/PathBinderH. Source code and databases on request.     

Attempts to delineate the relative contributions of changes in hydrophobicity and packing to changes in stability of ribonuclease S mutants

While the hydrophobic driving force is thought to be a major contributor to protein stability, it is difficult to experimentally dissect out its contribution to the overall free energy of folding. We have made large to small substitutions of buried hydrophobic residues at positions 8 and 13 in the peptide/protein complex, RNase-S, and have characterized the structures by X-ray crystallography. The thermodynamics of association of these mutant S peptides with S protein was measured in the presence of different concentrations of methanol and ethanol. The reduction in the strength of the hydrophobic driving force in the presence of these organic solvents was estimated from surface-tension data as well as from the dependence of the DeltaC(p) of protein/peptide binding on the alcohol concentration. The data indicated a decrease in the strength of the hydrophobic driving force of about 30-40% over a 0-30% range of the alcohol concentration. We observe that large to small substitutions destabilize the protein. However, the amount of destabilization, relative to the wild type, is independent of the alcohol concentration over the range of alcohol concentrations studied. The data clearly indicate that decreased stability of the mutants is primarily due to the loss of packing interactions rather than a reduced hydrophobic driving force and suggest a value of the hydrophobic driving force of less than 18 cal mol(-)(1) A(2).