Systematic Design of Adenosine Analogs as Inhibitors of a Clostridioides difficile-Specific DNA Adenine Methyltransferase Required for Normal Sporulation and Persistence.

Antivirulence agents targeting endospore-transmitted Clostridioides difficile infections are urgently needed. C. difficile-specific DNA adenine methyltransferase (CamA) is required for efficient sporulation and affects persistence in the colon. The active site of CamA is conserved and closely resembles those of hundreds of related S-adenosyl-l-methionine (SAM)-dependent methyltransferases, which makes the design of selective inhibitors more challenging. We explored the solvent-exposed edge of the SAM adenosine moiety and systematically designed 42 analogs of adenosine carrying substituents at the C6-amino group (N6) of adenosine. We compare the inhibitory properties and binding affinity of these diverse compounds and present the crystal structures of CamA in complex with 14 of them in the presence of substrate DNA. The most potent of these inhibitors, compound 39 (IC50 ∼ 0.4 µM and KD ∼ 0.2 µM), is selective for CamA against closely related bacterial and mammalian DNA and RNA adenine methyltransferases, protein lysine and arginine methyltransferases, and human adenosine receptors.

Development of a Harmonized Protocol for Measurement of Foliar Wash-off Parameters: First Results.

The foliar wash-off coefficient is a parameter used by environmental fate models to estimate the amount of chemical removed from leaf surfaces by rainfall. In the European Union it is used by FOCUS surface water models to estimate soil loadings following rainfall after leaf surfaces have been treated with plant protection products. Currently, a default value of 0.5/cm is assumed for this parameter, although there is provision to provide experimental data to replace this default. The European Food Safety Authority proposed to increase the default parameter value to 1.0/cm. This increases the need for experimental refinement studies. However, no guidance for a harmonized protocol exists to estimate this parameter. We describe the results of a ring-test conducted to start a process of developing a harmonized experimental protocol to measure the foliar wash-off parameters, conducted by several laboratories across Europe. The proposed design uses whole plants (rather than individual leaves) to retain as much realism as possible. The extent of wash-off is then determined by comparison of compound residues in two sets of plants (with and without a defined rainfall event) measured using a fully validated crop residue method. This initial ring test used tebuconazole (Folicur EW 250) sprayed at 100 g ai/ha onto tomato plants at BBCH25. Each laboratory measured the residues before and after a rainfall event of 20 mm/h for 1 h and calculated the percentage of wash-off from these data. There was good agreement across the eight participating laboratories with a mean percentage of wash-off of 66.8% and a 95% confidence interval of ±11.8%. Determination of robust wash-off parameters was therefore considered feasible using the proposed test design. Environ Toxicol Chem 2023;42:535-541. © 2022 SETAC.

Occurrence of brominated disinfection by-products in thermal spas.

Thermal spas are gaining more and more popularity among the population because they are used for recreational purposes. Disinfecting these baths without losing the health benefits poses a challenge for swimming pool operators. Previous studies have mainly focused on regulated chlorinated DBPs in freshwater pools with no bromide or seawater pools with very high bromide content. Thermal water pools have a low bromide content and in combination with chlorine can lead to chlorinated, brominated and mixed halogenated DBP species. The occurrence of brominated and mixed halogenated DBPs in these types of pools is largely unexplored, with very few or limited studies published on regulated DBPs and even fewer on emerging DBP classes. In the field of swimming pool water disinfection, apart from extensive studies in the field of drinking water disinfection, only a few studies are known in which >39 halogenated and 16 non-halogenated disinfection by-products, including regulated trihalomethanes (THM) and haloacetic acids (HAA), were investigated in swimming pool water. Calculated bromine incorporation factor (BIF) demonstrated that even small amounts of bromide in swimming pool water can lead to a large shift in DBP species towards brominated and mixed halogenated DBPs. Dihaloacetonitriles (DHANs) accounted for >50% of the calculated cytotoxicity and genotoxicity on average. Comparison of the target analysis with the TOX showed that a major part of the measured TOX (69% on average) could be explained by the regulated classes THMs, HAAs, and the unregulated class of HANs. This study aims to help operators of swimming pools with bromide-containing water to gain a better understanding of DBP formation in future monitoring and to fill the knowledge gap that has existed so far on the occurrence of DBPs in thermal water pools.

Synthesis of S-adenosyl-L-homocysteine capture compounds for selective photoinduced isolation of methyltransferases.

Understanding the interplay of different cellular proteins and their substrates is of major interest in the postgenomic era. For this purpose, selective isolation and identification of proteins from complex biological samples is necessary and targeted isolation of enzyme families is a challenging task. Over the last years, methods like activity-based protein profiling (ABPP) and capture compound mass spectrometry (CCMS) have been developed to reduce the complexity of the proteome by means of protein function in contrast to standard approaches, which utilize differences in physical properties for protein separation. To isolate and identify the subproteome consisting of S-adenosyl-L-methionine (SAM or AdoMet)-dependent methyltransferases (methylome), we developed and synthesized trifunctional capture compounds containing the chemically stable cofactor product S-adenosyl-L-homocysteine (SAH or AdoHcy) as selectivity function. SAH analogues with amino linkers at the N6 or C8 positions were synthesized and attached to scaffolds containing different photocrosslinking groups for covalent protein modification and biotin for affinity isolation. The utility of these SAH capture compounds for selective photoinduced protein isolation is demonstrated for various methyltransferases (MTases) acting on DNA, RNA and proteins as well as with Escherichia coli cell lysate. In addition, they can be used to determine dissociation constants for MTase-cofactor complexes.

Enzyme-directed positioning of nanoparticles on large DNA templates.

A method to position nanoparticles onto DNA with high resolution using an enzyme-based approach is described. This provides a convenient route to assemble multiple nanoparticles (e.g., Au and CdSe) to specific positions with a high level of control and expandability to more complex assemblies. Atomic force microscopy is used to analyze the nanostructures, which have potential interest for biosensor, optical waveguide, molecular electronics, and energy transfer studies.

Molecular scale architecture: engineered three- and four-way junctions.

Biomolecular self-assembly provides a basis for the bottom-up construction of useful and diverse nanoscale architectures. DNA is commonly used to create these assemblies and is often exploited as a lattice or an array. Although geometrically rigid and highly predictable, these sheets of repetitive constructs often lack the ability to be enzymatically manipulated or elongated by standard biochemical techniques. Here, we describe two approaches for the construction of position-controlled, molecular-scale, discrete, three- and four-way DNA junctions. The first approach for constructing these junctions relies on the use of nonmigrating cruciforms generated from synthetic oligonucleotides to which large, biologically generated, double-stranded DNA segments are enzymatically ligated. The second approach utilitizes the DNA methyltransferase-based SMILing (sequence-specific methyltransferase-induced labeling of DNA) method to site-specifically incorporate a biotin within biologically derived DNA. Streptavidin is then used to form junctions between unique DNA strands. The resultant assemblies have precise and predetermined connections with lengths that can be varied by enzymatic or hybridization techniques, or geometrically controlled with standard DNA functionalization methods. These junctions are positioned with single nucleotide resolution on large, micrometer-length templates. Both approaches generate DNA assemblies which are fully compatible with standard recombinant methods and thus provide a novel basis for nanoengineering applications.

Sequence-specific Methyltransferase-Induced Labelling (SMILing) of plasmid DNA for studying cell transfection.

Plasmid DNA (pUC19 and pBR322) was sequence-specifically, covalently labelled with Cy3 fluorophores using a newly synthesised N-adenosylaziridine cofactor and the DNA methyltransferase M.TaqI. The fluorescently labelled plasmids were used for transfection of mammalian cells and their intracellular distribution was visualised by epifluorescence and confocal fluorescence microscopy. Although these prokaryotic plasmids do not contain nuclear import sequences, translocation into the nuclei was observed.