Aryl Bis‐Sulfonamide Inhibitors of IspF from Arabidopsis thaliana and Plasmodium falciparum

2‐Methylerythritol 2,4‐cyclodiphosphate synthase (IspF) is an essential enzyme for the biosynthesis of isoprenoid precursors in plants and many human pathogens. The protein is an attractive target for the development of anti‐infectives and herbicides. Using a photometric assay, a screen of 40 000 compounds on IspF from Arabidopsis thaliana afforded symmetrical aryl bis‐sulfonamides that inhibit IspF from A. thaliana (AtIspF) and Plasmodium falciparum (PfIspF) with IC₅₀ values in the micromolar range. The ortho‐bis‐sulfonamide structural motif is essential for inhibitory activity. The best derivatives obtained by parallel synthesis showed IC₅₀ values of 1.4 μm against PfIspF and 240 nm against AtIspF. Substantial herbicidal activity was observed at a dose of 2 kg ha^?1. Molecular modeling studies served as the basis for an in silico search targeted at the discovery of novel, non‐symmetrical sulfonamide IspF inhibitors. The designed compounds were found to exhibit inhibitory activities in the double‐digit micromolar IC₅₀ range.     

Radical Scavenging Potential of the Phenothiazine Scaffold: A Computational Analysis

The reactivity of phenothiazine ( PS ), phenoselenazine ( PSE ), and phenotellurazine ( PTE ) with different reactive oxygen species (ROS) has been studied using density functional theory (DFT) in combination with the QM-ORSA (Quantum Mechanics-based Test for Overall Free Radical Scavenging Activity) protocol for an accurate kinetic rate calculation. Four radical scavenging mechanisms have been screened, namely hydrogen atom transfer (HAT), radical adduct formation (RAF), single electron transfer (SET), and the direct oxidation of the chalcogen atom. The chosen ROS are HO^., HOO^., and CH₃OO^.. PS , PSE , and PTE exhibit an excellent antioxidant activity in water regardless of the ROS due to their characteristic diffusion-controlled regime processes. For the HO^. radical, the primary active reaction mechanism is, for all antioxidants, RAF. But, for HOO^. and CH₃OO^., the dominant mechanism strongly depends on the antioxidant: HAT for PS and PSE , and SET for PTE . The scavenging efficiency decreases dramatically in lipid environment and remains only significant (via RAF) for the most reactive radical (HO^.). Therefore, PS , PSE , and PTE are excellent antioxidant molecules, especially in aqueous, physiological environments where they are active against a broad spectrum of harmful radicals. There is no advantage or significant difference in the scavenging efficiency when changing the chalcogen since the reactivity mainly derives from the amino hydrogen and the aromatic sites.     

Insights into the Genetic Variations of Human Cytochrome P450 2C9: Structural Analysis,Characterization and Comparison

Cytochromes P450 (CYP) are one of the major xenobiotic metabolizing enzymes with increasing importance in pharmacogenetics. The CYP2C9 enzyme is responsible for the metabolism of a wide range of clinical drugs. More than sixty genetic variations have been identified in CYP2C9 with many demonstrating reduced activity compared to the wild-type (WT) enzyme. The CYP2C9*8 allele is predominantly found in persons of African ancestry and results in altered clearance of several drug substrates of CYP2C9. The X-ray crystal structure of CYP2C9*8, which represents an amino acid variation from arginine to histidine at position 150 (R150H), was solved in complex with losartan. The overall conformation of the CYP2C9*8-losartan complex was similar to the previously solved complex with wild type (WT) protein, but it differs in the occupancy of losartan. One molecule of losartan was bound in the active site and another on the surface in an identical orientation to that observed in the WT complex. However, unlike the WT structure, the losartan in the access channel was not observed in the *8 complex. Furthermore, isothermal titration calorimetry studies illustrated weaker binding of losartan to *8 compared to WT. Interestingly, the CYP2C9*8 interaction with losartan was not as weak as the CYP2C9*3 variant, which showed up to three-fold weaker average dissociation constant compared to the WT. Taken together, the structural and solution characterization yields insights into the similarities and differences of losartan binding to CYP2C9 variants and provides a useful framework for probing the role of amino acid substitution and substrate dependent activity.     

Analysis of the Destabilization of Bacterial Membranes by Quaternary Ammonium Compounds: A Combined Experimental and Computational Study

The mechanism of action of quaternary ammonium compound (QAC) antiseptics has long been assumed to be straightforward membrane disruption, although the process of approaching and entering the membrane has little modeling precedent. Furthermore, questions have more recently arisen regarding bacterial resistance mechanisms, and why select classes of QACs (specifically, multicationic QACs) are less prone to resistance. In order to better understand such subtleties, a series of molecular dynamics simulations were utilized to help identify these molecular determinants, directly comparing mono-, bis-, and triscationic QACs in simulated membrane intercalation models. Three distinct membranes were simulated, mimicking the surfaces of Escherichia coli and Staphylococcus aureus, as well as a neutral phospholipid control. By analyzing the resulting trajectories in the form of a timeseries analysis, insight was gleaned regarding the significant steps and interactions involved in the destabilization of phospholipid bilayers within the bacterial membranes. Finally, to more specifically probe the effect of the hydrophobic section of the amphiphile that presumably penetrates the membrane, a series of alkyl- and ester-based biscationic quaternary ammonium compounds were prepared, tested for antimicrobial activity against both Gram-positive and Gram-negative bacteria, and modeled.     

Conformational Analysis of the Mannosidase Inhibitor Kifunensine: A Quantum Mechanical and Structural Approach

The varied yet family‐specific conformational pathways used by individual glycoside hydrolases (GHs) offer a tantalising prospect for the design of tightly binding and specific enzyme inhibitors. A cardinal example of a GH‐family‐specific inhibitor, and one that finds widespread practical use, is the natural product kifunensine, which is a low‐nanomolar inhibitor that is selective for GH family 47 inverting α‐mannosidases. Here we show, through quantum‐mechanical approaches, that kifunensine is restrained to a “ring‐flipped” ¹C₄ conformation with another accessible, but higher‐energy, region around the ^1,4B conformation. The conformations of kifunensine in complex with a range of GH47 enzymes—including an atomic‐level resolution (1 Å) structure of kifunensine with Caulobacter sp. CkGH47 reported herein and with GH family 38 and 92 α‐mannosidases—were mapped onto the kifunensine free‐energy landscape. These studies revealed that kifunensine has the ability to mimic the product state of GH47 enzymes but cannot mimic any conformational states relevant to the reaction coordinate of mannosidases from other families.     

Analysis of Pseudomonas aeruginosa PAO1 Lipid A Changes During the Interaction with Model Organism,Caenorhabditis elegans

Lipopolysaccharide (LPS) is the main surface constituent of Gram-negative bacteria. Lipid A, the hydrophobic moiety, outer monolayer of the outer cell membrane forms the major component of LPS. Immunogenic Lipid A is recognized by the innate immune system through the TLR 4/MD-2 complex. Pseudomonas aeruginosa PAO1, a Gram-negative bacterium is known to cause nosocomial infection and known for its adaptation to adverse environmental conditions. Pseudomonas aeruginosa can infect a broad host spectrum including Caenorhabditis elegans, a simple free living soil nematode. Here, we reveal that PAO1 modifies its Lipid A during the host interaction with C. elegans. The penta-acylated form of Lipid A was identified by using matrix assisted laser desorption ionization–time of flight analysis and the β-(1,6)-linked disaccharide of glucosamine with phosphate groups, 2 and 2′ amide linked fatty acid chain and 3 and 3′ ester linked fatty acids were investigated for the modification using the non destructive ¹H NMR, spin–lattice (T ₁) relaxation measurement, differential scanning calorimetry. T ₁ relaxation measurements showed that the 2 and 2′ amide linked fatty acid chain, –CH in the glucosamine disaccharide of PAO1 lipid A, in an exposed host had a different spin lattice relaxation time compared to an unexposed host and the findings were reconfirmed using in vitro human corneal epithelial cells cell lines. Furthermore, scanning electron microscope and confocal laser scanning microscopy analysis revealed that the P. aeruginosa PAO1 biofilm formation was disturbed in the exposed host condition. The daf-12, daf-16, tol-1, pmk-1, ins-7 and ilys3 immune genes of C. elegans were examined with live bacterial and isolated lipid moiety infection and the expression was found to be highly specific. Overall, the present study revealed that PAO1 modified its 2 and 2′ amide linked fatty acid chain in the lipid A of PAO1 LPS during the exposed host condition.     

Phosphate fixing capacity of soils: A survey,using the isotopic exchange technique,or soils from north-eastern France

A large variety of soil samples was collected from farms located in two north-eastern regions of France. Their phosphate fertility was assessed by the isotopic exchange kinetics method which allows for the measurement of a r₁/R ratio, where r₁ is the quantity of radioactivity still present in solution one minute after the injection of a quantity of radioactivity R into a soil-solution system at steady-state. The r₁/R parameter gives an estimate of the soil fixing capacity for phosphate ions (PFC). Results of the analysis of 233 soil samples revealed that the great majority of soils from these areas exhibit a high PFC. Neither the soil type nor mode of utilization appeared sufficient for prediction of the PFC. Attempts to express the PFC as a function of other soil parameters (pH, clay, organic matter, calcium carbonate and exchangeable cations), resulted in a significant regression equation involving soil clay content and soluble phosphate. Due to high PFC of soils in the Lorraine area, phosphate fertilizer could be applied at a time as close as possible to the plant requirements.