Novel Semisynthetic Derivatives of Bile Acids as Effective Tyrosyl-DNA Phosphodiesterase 1 Inhibitors.

An Important task in the treatment of oncological and neurodegenerative diseases is the search for new inhibitors of DNA repair system enzymes. Tyrosyl-DNA phosphodiesterase 1 (Tdp1) is one of the DNA repair system enzymes involved in the removal of DNA damages caused by topoisomerase I inhibitors. Thus, reducing the activity of Tdp1 can increase the effectiveness of currently used anticancer drugs. We describe here a new class of semisynthetic small molecule Tdp1 inhibitors based on the bile acid scaffold that were originally identified by virtual screening. The influence of functional groups of bile acids (hydroxy and acetoxy groups in the steroid framework and amide fragment in the side chain) on inhibitory activity was investigated. In vitro studies demonstrate the ability of the semisynthetic derivatives to effectively inhibit Tdp1 with IC50 up to 0.29 µM. Furthermore, an excellent fit is realized for the ligands when docked into the active site of the Tdp1 enzyme

Agrobacterium tumefaciens-Induced Bacteraemia Does Not Lead to Reporter Gene Expression in Mouse Organs

Agrobacterium tumefaciens is the main plant biotechnology gene transfer tool with host range which can be extended to non-plant eukaryotic organisms under laboratory conditions. Known medical cases of Agrobacterium species isolation from bloodstream infections necessitate the assessment of biosafety-related risks of A. tumefaciens encounters with mammalian organisms. Here, we studied the survival of A. tumefaciens in bloodstream of mice injected with bacterial cultures. Bacterial titers of 108 CFU were detected in the blood of the injected animals up to two weeks after intravenous injection. Agrobacteria carrying Cauliflower mosaic virus (CaMV) 35S promoter-based constructs and isolated from the injected mice retained their capacity to promote green fluorescent protein (GFP) synthesis in Nicotiana benthamiana leaves. To examine whether or not the injected agrobacteria are able to express in mouse organs, we used an intron-containing GFP (GFPi) reporter driven either by a cytomegalovirus (CMV) promoter or by a CaMV 35S promoter. Western and northern blot analyses as well as RT-PCR analysis of liver, spleen and lung of mice injected with A. tumefaciens detected neither GFP protein nor its transcripts. Thus, bacteraemia induced in mice by A. tumefaciens does not lead to detectible levels of genetic transformation of mouse organs

Protein Scaffolds Can Enhance the Bistability of Multisite Phosphorylation Systems

The phosphorylation of a substrate at multiple sites is a common protein modification that can give rise to important structural and electrostatic changes. Scaffold proteins can enhance protein phosphorylation by facilitating an interaction between a protein kinase enzyme and its target substrate. In this work we consider a simple mathematical model of a scaffold protein and show that under specific conditions, the presence of the scaffold can substantially raise the likelihood that the resulting system will exhibit bistable behavior. This phenomenon is especially pronounced when the enzymatic reactions have sufficiently large KM, compared to the concentration of the target substrate. We also find for a closely related model that bistable systems tend to have a specific kinetic conformation. Using deficiency theory and other methods, we provide a number of necessary conditions for bistability, such as the presence of multiple phosphorylation sites and the dependence of the scaffold binding/unbinding rates on the number of phosphorylated sites

Membrane Recruitment of Scaffold Proteins Drives Specific Signaling

Cells must give the right response to each stimulus they receive. Scaffolding, a signaling process mediated by scaffold proteins, participates in the decoding of the cues by specifically directing signal transduction. The aim of this paper is to describe the molecular mechanisms of scaffolding, i.e. the principles by which scaffold proteins drive a specific response of the cell. Since similar scaffold proteins are found in many species, they evolved according to the purpose of each organism. This means they require adaptability. In the usual description of the mechanisms of scaffolding, scaffold proteins are considered as reactors where molecules involved in a cascade of reactions are simultaneously bound with the right orientation to meet and interact. This description is not realistic: (i) it is not verified by experiments and (ii) timing and orientation constraints make it complex which seems to contradict the required adaptability. A scaffold protein, Ste5, is used in the MAPK pathway of Saccharomyces Cerevisiae for the cell to provide a specific response to stimuli. The massive amount of data available for this pathway makes it ideal to investigate the actual mechanisms of scaffolding. Here, a complete treatment of the chemical reactions allows the computation of the distributions of all the proteins involved in the MAPK pathway when the cell receives various cues. These distributions are compared to several experimental results. It turns out that the molecular mechanisms of scaffolding are much simpler and more adaptable than previously thought in the reactor model. Scaffold proteins bind only one molecule at a time. Then, their membrane recruitment automatically drives specific, amplified and localized signal transductions. The mechanisms presented here, which explain how the membrane recruitment of a protein can produce a drastic change in the activity of cells, are generic and may be commonly used in many biological processes