Inhibition of Ribonuclease A by polyphenols present in green tea

We report the effect of the natural polyphenolic compounds from green tea on the catalytic activity of Ribonuclease A (RNase A). The compounds behave as noncompetitive inhibitors of the protein with inhibition constants ranging from 80-1300 microM. The dissociation constants range from 50-150 microM for the RNase A-polyphenol complexes as determined by ultraviolet (UV) and circular dichroism (CD) studies. We have also investigated the changes in the secondary structure of RNase A on complex formation by CD and Fourier transformed infrared (FTIR) spectroscopy. The presence of the gallate moiety has been shown to be important for the inhibition of enzymatic activity. Docking studies for these compounds indicate that the preferred site of binding is the region encompassing residues 34-39 with possible hydrogen bonding with Lys 7 and Arg 10. Finally we have also looked at changes in the accessible surface area of the interacting residues on complex formation for an insight into the residues involved in the interaction.     

Design of HIV-1 integrase inhibitors targeting the catalytic domain as well as its interaction with LEDGF/p75: a scaffold hopping approach using salicylate and catechol groups

HIV-1 integrase (IN) is a validated therapeutic target for antiviral drug design. However, the emergence of viral strains resistant to clinically studied IN inhibitors demands the discovery of novel inhibitors that are structurally as well mechanistically different. Herein, we describe the design and discovery of novel IN inhibitors targeting the catalytic domain as well as its interaction with LEDGF/p75, which is essential for the HIV-1 integration as an IN cofactor. By merging the pharmacophores of salicylate and catechol, the 2,3-dihydroxybenzamide (5a) was identified as a new scaffold to inhibit the strand transfer reaction efficiently. Further structural modifications on the 2,3-dihydroxybenzamide scaffold revealed that the heteroaromatic functionality attached on the carboxamide portion and the piperidin-1-ylsulfonyl substituted at the phenyl ring are beneficial for the activity, resulting in a low micromolar IN inhibitor (5p, IC(50)=5 μM) with more than 40-fold selectivity for the strand transfer over the 3'-processing reaction. More significantly, this active scaffold remarkably inhibited the interaction between IN and LEDGF/p75 cofactor. The prototype example, N-(cyclohexylmethyl)-2,3-dihydroxy-5-(piperidin-1-ylsulfonyl) benzamide (5u) inhibited the IN-LEDGF/p75 interaction with an IC(50) value of 8 μM. Using molecular modeling, the mechanism of action was hypothesized to involve the chelation of the divalent metal ions inside the IN active site. Furthermore, the inhibitor of IN-LEDGF/p75 interaction was properly bound to the LEDGF/p75 binding site on IN. This work provides a new and efficient approach to evolve novel HIV-1 IN inhibitors from rational integration and optimization of previously reported inhibitors.     

Autophagy facilitates glycolysis during Ras-mediated oncogenic transformation

The protumorigenic functions for autophagy are largely attributed to its ability to promote cancer cell survival in response to diverse stresses. Here we demonstrate an unexpected connection between autophagy and glucose metabolism that facilitates adhesion-independent transformation driven by a strong oncogenic insult-mutationally active Ras. In cells ectopically expressing oncogenic H-Ras as well as human cancer cell lines harboring endogenous K-Ras mutations, autophagy is induced following extracellular matrix detachment. Inhibiting autophagy due to the genetic deletion or RNA interference-mediated depletion of multiple autophagy regulators attenuates Ras-mediated adhesion-independent transformation and proliferation as well as reduces glycolytic capacity. Furthermore, in contrast to autophagy-competent cells, both proliferation and transformation in autophagy-deficient cells expressing oncogenic Ras are insensitive to reductions in glucose availability. Overall, increased glycolysis in autophagy-competent cells facilitates Ras-mediated adhesion-independent transformation, suggesting a unique mechanism by which autophagy may promote Ras-driven tumor growth in specific metabolic contexts.     

Direct effects of lithium chloride on the activities of delta 5-3 beta and 17 beta-hydroxysteroid dehydrogenase in the testis and Bidder's organ of the adult toad (Bufo melanostictus)--in vitro study

Steroidogenic key enzymes, i.e. delta 5-3 beta and 17 beta-hydroxysteroid dehydrogenase (delta 5-3 beta and 17 beta-HSD) activities, in the testis and Bidder's organ of the toad were inhibited and ascorbic acid synthesis in these organs was decreased by a wide range of lithium concentration in in vitro study. A significant inhibition was noted at a concentration of 2.0 mM, which is easily achieved in the blood during the treatment of manic patients by lithium chloride. This experiment reflected that lithium exerts a direct inhibitory effect on hydroxysteroid dehydrogenase activities in the testis and Bidder's organ--a rudimentary ovary in Bufo.     

The RETINOBLASTOMA-RELATED gene regulates stem cell maintenance in Arabidopsis roots

The maintenance of stem cells in defined locations is crucial for all multicellular organisms. Although intrinsic factors and signals for stem cell fate have been identified in several species, it has remained unclear how these connect to the ability to reenter the cell cycle that is one of the defining properties of stem cells. We show that local reduction of expression of the RETINOBLASTOMA-RELATED (RBR) gene in Arabidopsis roots increases the amount of stem cells without affecting cell cycle duration in mitotically active cells. Conversely, induced RBR overexpression dissipates stem cells prior to arresting other mitotic cells. Overexpression of D cyclins, KIP-related proteins, and E2F factors also affects root stem cell pool size, and genetic interactions suggest that these factors function in a canonical RBR pathway to regulate somatic stem cells. Expression analysis and genetic interactions position RBR-mediated regulation of the stem cell state downstream of the patterning gene SCARECROW.     

Maximization of production of secreted recombinant proteins in Pichia pastoris fed-batch fermentation

Pontryagin's Maximum Principle has been applied for optimization of secreted proteins from Pichia pastoris fed-batch fermentation. The objective of this work is to maximize the total accumulated product per unit operation time under different given conditions and system constraints. To obtain optimal solutions, an automated curve-fitting software, Table Curve 2D, was employed to construct the necessary mathematical models and solve the complicated functions. In the solution processes, the end of the glycerol batch phase was defined as the initial state of the system, the end of the methanol fed-batch phase as the final state, the cell mass produced along with product accumulated as state variables, and the specific growth rate (mu) as the control variable. Initially, a relationship between the specific production rate (rho) and mu was established. Then, according to Pontryagin's Maximum Principle, the admissible range of mu and its trajectories for the optimal operations were determined. Four representative cases with different combinations of the operation time along with the initial and final states were evaluated. A close correlation was obtained between the predicted values of the model equation with the experimental results from the Pichia pastoris fed-batch fermentations producing secreted alpha-galactosidase. The approaches proposed here greatly simplify the computational processes and validate the optimization strategy as a generalized approach to maximize the yield from fed-batch fermentations.     

Theaflavin derivatives in black tea and catechin derivatives in green tea inhibit HIV-1 entry by targeting gp41

Theaflavin derivatives and catechin derivatives are the major polyphenols in black tea and green tea, respectively. Several tea polyphenols, especially those with galloyl moiety, can inhibit HIV-1 replication with multiple mechanisms of action. Here we showed that the theaflavin derivatives had more potent anti-HIV-1 activity than catechin derivatives. These tea polyphenols could inhibit HIV-1 entry into target cells by blocking HIV-1 envelope glycoprotein-mediated membrane fusion. The fusion inhibitory activity of the tea polyphenols was correlated with their ability to block the formation of the gp41 six-helix bundle, a fusion-active core conformation. Computer-aided molecular docking analyses indicate that these tea polyphenols, theaflavin-3,3'-digallate (TF3) as an example, may bind to the highly conserved hydrophobic pocket on the surface of the central trimeric coiled coil formed by the N-terminal heptad repeats of gp41. These results indicate that tea, especially black tea, may be used as a source of anti-HIV agents and theaflavin derivatives may be applied as lead compounds for developing HIV-1 entry inhibitors targeting gp41.