Soaking suggests "alternative facts": Only co-crystallization discloses major ligand-induced interface rearrangements of a homodimeric tRNA-binding protein indicating a novel mode-of-inhibition.

For the efficient pathogenesis of Shigella, the causative agent of bacillary dysentery, full functionality of tRNA-guanine transglycosylase (TGT) is mandatory. TGT performs post-transcriptional modifications of tRNAs in the anticodon loop taking impact on virulence development. This suggests TGT as a putative target for selective anti-shigellosis drug therapy.

Efficient synthesis of fluorescent alkynyl C-nucleosides via Sonogashira coupling for the preparation of DNA-based polyfluorophores.

A facile and general procedure for the preparation of alkynyl C-nucleosides with varied fluorophores is presented. Sonogashira coupling was used as a key reaction to conjugate the dyes to an easily accessible ethynyl functionalized deoxyribose derivative. The new C-nucleosides were used for the preparation of DNA-based polyfluorophores.

Organocatalytic removal of formaldehyde adducts from RNA and DNA bases.

Formaldehyde is universally used to fix tissue specimens, where it forms hemiaminal and aminal adducts with biomolecules, hindering the ability to retrieve molecular information. Common methods for removing these adducts involve extended heating, which can cause extensive degradation of nucleic acids, particularly RNA. Here, we show that water-soluble bifunctional catalysts (anthranilates and phosphanilates) speed the reversal of formaldehyde adducts of mononucleotides over standard buffers.

Replacement of water molecules in a phosphate binding site by furanoside-appended lin-benzoguanine ligands of tRNA-guanine transglycosylase (TGT).

The enzyme tRNA-guanine transglycosylase has been identified as a drug target for the foodborne illness shigellosis. A key challenge in structure-based design for this enzyme is the filling of the polar ribose-34 pocket. Herein, we describe a novel series of ligands consisting of furanoside-appended lin-benzoguanines.

Beyond affinity: enthalpy-entropy factorization unravels complexity of a flat structure-activity relationship for inhibition of a tRNA-modifying enzyme.

Lead optimization focuses on binding-affinity improvement. If a flat structure-activity relationship is detected, usually optimization strategies are abolished as unattractive. Nonetheless, as affinity is composed of an enthalpic and entropic contribution, factorization of both can unravel the complexity of a flat, on first sight tedious SAR.

Chasing protons: how isothermal titration calorimetry, mutagenesis, and pKa calculations trace the locus of charge in ligand binding to a tRNA-binding enzyme.

Drug molecules should remain uncharged while traveling through the body and crossing membranes and should only adopt charged state upon protein binding, particularly if charge-assisted interactions can be established in deeply buried binding pockets. Such strategy requires careful pKa design and methods to elucidate whether and where protonation-state changes occur. We investigated the protonation inventory in a series of lin-benzoguanines binding to tRNA-guanine transglycosylase, showing pronounced buffer dependency during ITC measurements.

Impact of protein and ligand impurities on ITC-derived protein-ligand thermodynamics.

Background: The thermodynamic characterization of protein-ligand interactions by isothermal titration calorimetry (ITC) is a powerful tool in drug design, giving valuable insight into the interaction driving forces. ITC is thought to require protein and ligand solutions of high quality, meaning both the absence of contaminants as well as accurately determined concentrations.

Methods: Ligands synthesized to deviating purity and protein of different pureness were titrated by ITC.

High-affinity inhibitors of Zymomonas mobilis tRNA-guanine transglycosylase through convergent optimization.

The tRNA-modifying enzyme tRNA-guanine transglycosylase (TGT) has been recognized as a drug target for the treatment of the foodborne illness shigellosis. The active site of TGT consists of three pockets: the central guanine/preQ1 recognition site and the ribose-33 and ribose-34 pockets. In previous work, lin-benzoguanines and lin-benzohypoxanthines, which differ by the presence of an exocyclic NH2 group in the former and its absence in the latter, were used as central scaffolds that bind to the guanine/preQ1 recognition site and allow suitable functionalization along exit vectors targeting the two ribose pockets.

Launching spiking ligands into a protein-protein interface: a promising strategy to destabilize and break interface formation in a tRNA modifying enzyme.

Apart from competitive active-site inhibition of protein function, perturbance of protein-protein interactions by small molecules in oligodomain enzymes opens new perspectives for innovative therapeutics. tRNA-guanine transglycosylase (TGT), a potential target to treat shigellosis, is active only as the homodimer. Consequently, disruption of the dimer interface by small molecules provides a novel inhibition mode.

Total synthesis and configurational assignment of the marine natural product haliclamide.

The marine natural product haliclamide has been synthesized based on macrocyclization by ring-closing olefin metathesis. Using either enantiomer of two of the four building blocks that were employed to assemble the diene precursor for the metathesis reaction, three non-natural isomers of haliclamide were also prepared. On the basis of the comparison of the (1)H and (13)C NMR spectra of the individual stereoisomers with literature data for the natural product, the configuration of the previously unassigned stereocenters at C9 and C20 of haliclamide could be determined to be S for both carbons.

Free enthalpies of replacing water molecules in protein binding pockets.

Water molecules in the binding pocket of a protein and their role in ligand binding have increasingly raised interest in recent years. Displacement of such water molecules by ligand atoms can be either favourable or unfavourable for ligand binding depending on the change in free enthalpy. In this study, we investigate the displacement of water molecules by an apolar probe in the binding pocket of two proteins, cyclin-dependent kinase 2 and tRNA-guanine transglycosylase, using the method of enveloping distribution sampling (EDS) to obtain free enthalpy differences.

From lin-benzoguanines to lin-benzohypoxanthines as ligands for Zymomonas mobilis tRNA-guanine transglycosylase: replacement of protein-ligand hydrogen bonding by importing water clusters.

The foodborne illness shigellosis is caused by Shigella bacteria that secrete the highly cytotoxic Shiga toxin, which is also formed by the closely related enterohemorrhagic Escherichia coli (EHEC). It has been shown that tRNA-guanine transglycosylase (TGT) is essential for the pathogenicity of Shigella flexneri. Herein, the molecular recognition properties of a guanine binding pocket in Zymomonas mobilis TGT are investigated with a series of lin-benzohypoxanthine- and lin-benzoguanine-based inhibitors that bear substituents to occupy either the ribose-33 or the ribose-34 pocket.

Inhibitors of the kinase IspE: structure-activity relationships and co-crystal structure analysis.

Enzymes of the non-mevalonate pathway for isoprenoid biosynthesis are therapeutic targets for the treatment of important infectious diseases. Whereas this pathway is absent in humans, it is used by plants, many eubacteria and apicomplexan protozoa, including major human pathogens such as Plasmodium falciparum and Mycobacterium tuberculosis. Herein, we report on the design, preparation and biological evaluation of a new series of ligands for IspE protein, a kinase from this pathway.