Morphological profiling of environmental chemicals enables efficient and untargeted exploration of combination effects.

Environmental chemicals are commonly studied one at a time, and there is a need to advance our understanding of the effect of exposure to their combinations. Here we apply high-content microscopy imaging of cells stained with multiplexed dyes (Cell Painting) to profile the effects of Cetyltrimethylammonium bromide (CTAB), Bisphenol A (BPA), and Dibutyltin dilaurate (DBTDL) exposure on four human cell lines; both individually and in all combinations. We show that morphological features can be used with multivariate data analysis to discern between exposures from individual compounds, concentrations, and combinations. CTAB and DBTDL induced concentration-dependent morphological changes across the four cell lines, and BPA exacerbated morphological effects when combined with CTAB and DBTDL. Combined exposure to CTAB and BPA induced changes in the ER, Golgi apparatus, nucleoli and cytoplasmic RNA in one of the cell lines. Different responses between cell lines indicate that multiple cell types are needed when assessing combination effects. The rapid and relatively low-cost experiments combined with high information content make Cell Painting an attractive methodology for future studies of combination effects. All data in the study is made publicly available on Figshare.

Mechanism of mycolic acid cyclopropane synthase: a theoretical study.

The reaction mechanism of mycolic acid cyclopropane synthase is investigated using hybrid density functional theory. The direct methylation mechanism is examined with a large model of the active site constructed on the basis of the crystal structure of the native enzyme. The important active site residue Glu140 is modeled in both ionized and neutral forms. We demonstrate that the reaction starts via the transfer of a methyl to the substrate double bond, followed by the transfer of a proton from the methyl cation to the bicarbonate present in the active site. The first step is calculated to be rate-limiting, in agreement with experimental kinetic results. The protonation state of Glu140 has a rather weak influence on the reaction energetics. In addition to the natural reaction, a possible side reaction, namely a carbocation rearrangement, is also considered and is shown to have a low barrier. Finally, the energetics for the sulfur ylide proposal, which has already been ruled out, is also estimated, showing a large energetic penalty for ylide formation.

Quantum chemical modeling of enzymatic reactions: the case of histone lysine methyltransferase.

Quantum chemical cluster models of enzyme active sites are today an important and powerful tool in the study of various aspects of enzymatic reactivity. This methodology has been applied to a wide spectrum of reactions and many important mechanistic problems have been solved. Herein, we report a systematic study of the reaction mechanism of the histone lysine methyltransferase (HKMT) SET7/9 enzyme, which catalyzes the methylation of the N-terminal histone tail of the chromatin structure. In this study, HKMT SET7/9 serves as a representative case to examine the modeling approach for the important class of methyl transfer enzymes. Active site models of different sizes are used to evaluate the methodology. In particular, the dependence of the calculated energies on the model size, the influence of the dielectric medium, and the particular choice of the dielectric constant are discussed. In addition, we examine the validity of some technical aspects, such as geometry optimization in solvent or with a large basis set, and the use of different density functional methods.

The reaction mechanism of phenylethanolamine N-methyltransferase: a density functional theory study.

Hybrid density functional theory methods were used to investigate the reaction mechanism of human phenylethanolamine N-methyltransferase (hPNMT). This enzyme catalyzes the S-adenosyl-L-methionine-dependent conversion of norepinephrine to epinephrine, which constitutes the terminal step in the catecholamine biosynthesis. Several models of the active site were constructed based on the X-ray structure. Geometries of the stationary points along the reaction path were optimized and the reaction barrier and energy were calculated and compared to the experimental values. The calculations demonstrate that the reaction takes place via an SN2 mechanism with methyl transfer being rate-limiting, a suggestion supported by mutagenesis studies. Optimal agreement with experimental data is reached using a model in which both active site glutamates are protonated. Overall, the mechanism of hPNMT is more similar to those of catechol O-methyltransferase and glycine N-methyltransferase than to that of guanidinoacetate N-methyltransferase in which methyl transfer is coupled to proton transfer.