Novel Chemotypes for Inhibition of Bacterial and Mammalian Carbohydrate-Binding Proteins

The carbohydrate-binding proteins (lectins) emerged as viable targets to combat viral as well as bacterial pathogens. Therefore, drugs targeting lectins are desired; however their identification and development is challenging and is currently primarily focused on carbohydrate−based inhibitors. Therefore, new strategies and sensitive methods are required. Fragment-based drug design (FBDD) has proven to be a promising strategy for approaching difficult targets such as lectins. To address the current limitations in design of drug-like inhibitors for lectins, non- and metal-dependent bacterial or mammalian lectins are used. First, bacterial lectins from the opportunistic human pathogens Pseudomonas aeruginosa (LecA (PA-IL) and LecB (PA-IIL)) and Burkholderia ambifaria (BambL) were employed as models to establish ligand- (F- glycan) and protein-observed 19F NMR (PrOF) methods for drug discovery. To demonstrate the utility of these methods for fragment-based drug discovery (FBDD), a druggable pocket in BambL was uncovered as a potential target site for allosteric inhibitors. Finally, these methods were employed as well as other biophysical (X-ray, SPR), computational and biochemical techniques to discover a novel class of drug-like molecules for targeting the carbohydrate-binding site of metal-dependent bacterial and mammalian lectins. Together, the 19F NMR-based methods and discovery of metal- binding pharmacophores (MBPs) as novel chemotypes will support the development of small molecule inhibitors for metal-dependent lectins and bacterial lectins as new therapeutic approaches against antibiotic-resistant pathogens

Druggable Allosteric Sites in β-Propeller Lectins

Carbohydrate-binding proteins (lectins) are auspicious targets in drug discovery to combat antimicrobial resistance; however, their non-carbohydrate drug-like inhibitors are still unavailable. Here, we present a druggable pocket in a β-propeller lectin BambL from Burkholderia ambifaria as a potential target for allosteric inhibitors. This site was identified employing 19F NMR fragment screening and a computational pocket prediction algorithm SiteMap. The structure–activity relationship study revealed the most promising fragment with a dissociation constant of 0.3±0.1 mM and a ligand efficiency of 0.3 kcal mol−1 HA−1 that affected the orthosteric site. This effect was substantiated by site-directed mutagenesis in the orthosteric and secondary pockets. Future drug-discovery campaigns that aim to develop small molecule inhibitors can benefit from allosteric sites in lectins as a new therapeutic approach against antibiotic-resistant pathogens

Protein-observed 19F NMR of LecA from Pseudomonas aeruginosa

The carbohydrate-binding protein LecA (PA-IL) from Pseudomonas aeruginosa plays an important role in the formation of biofilms in chronic infections. Development of inhibitors to disrupt LecAmediated biofilms is desired but it is limited to carbohydrate-based ligands. Moreover, discovery of drug-like ligands for LecA is challenging because of its weak affinities. Therefore, we established a protein-observed 19F (PrOF) nuclear magnetic resonance (NMR) to probe ligand binding to LecA. LecA was labeled with 5-fluoroindole to incorporate 5-fluorotryptophanes and the resonances were assigned by site-directed mutagenesis. This incorporation did not disrupt LecA preference for natural ligands, Ca2+ and D-galactose (D-Gal). Following NMR perturbation of W42, which is located in the carbohydrate-binding region of LecA, allowed to monitor binding of low-affinity ligands such as N-acetyl D-galactosamine (D-GalNAc, Kd = 780 ± 97 μM). Moreover, PrOF NMR titration with glycomimetic of LecA p-nitrophenyl β-D-galactoside (pNPGal, Kd = 54 ± 6 μM) demonstrated a 6-fold improved binding of D-Gal proving this approach to be valuable for ligand design in future drug discovery campaigns that aim to generate inhibitors of LecA

Stereoselective synthesis of fluorinated galactopyranosides as potential molecular probes for galactophilic proteins : assessment of monofluorogalactoside–LecA interactions

The replacement of hydroxyl groups by fluorine atoms on hexopyranoside scaffolds may allow access to invaluable tools for studying various biochemical processes. As part of ongoing activities toward the preparation of fluorinated carbohydrates, a systematic investigation involving the synthesis and biological evaluation of a series of mono‐ and polyfluorinated galactopyranosides is described. Various monofluorogalactopyranosides, a trifluorinated, and a tetrafluorinated galactopyranoside have been prepared using a Chiron approach. Given the scarcity of these compounds in the literature, in addition to their synthesis, their biological profiles were evaluated. Firstly, the fluorinated compounds were investigated as antiproliferative agents using normal human and mouse cells in comparison with cancerous cells. Most of the fluorinated compounds showed no antiproliferative activity. Secondly, these carbohydrate probes were used as potential inhibitors of galactophilic lectins. The first transverse relaxation‐optimized spectroscopy (TROSY) NMR experiments were performed on these interactions, examining chemical shift perturbations of the backbone resonances of LecA, a virulence factor from Pseudomonas aeruginosa. Moreover, taking advantage of the fluorine atom, the 19F NMR resonances of the monofluorogalactopyranosides were directly monitored in the presence and absence of LecA to assess ligand binding. Lastly, these results were corroborated with the binding potencies of the monofluorinated galactopyranoside derivatives by isothermal titration calorimetry experiments. Analogues with fluorine atoms at C‐3 and C‐4 showed weaker affinities with LecA as compared to those with the fluorine atom at C‐2 or C‐6. This research has focused on the chemical synthesis of “drug‐like” low‐molecular‐weight inhibitors that circumvent drawbacks typically associated with natural oligosaccharides

Targeting the Central Pocket of the Pseudomonas aeruginosa Lectin LecA

Pseudomonas aeruginosa is an opportunistic ESKAPE pathogen that produces two lectins, LecA and LecB, as part of its large arsenal of virulence factors. Both carbohydrate-binding proteins are central to the initial and later persistent infection processes, i. e. bacterial adhesion and biofilm formation. The biofilm matrix is a major resistance determinant and protects the bacteria against external threats such as the host immune system or antibiotic treatment. Therefore, the development of drugs against the P. aeruginosa biofilm is of particular interest to restore efficacy of antimicrobials. Carbohydrate-based inhibitors for LecA and LecB were previously shown to efficiently reduce biofilm formations. Here, we report a new approach for inhibiting LecA with synthetic molecules bridging the established carbohydrate-binding site and a central cavity located between two LecA protomers of the lectin tetramer. Inspired by in silico design, we synthesized various galactosidic LecA inhibitors with aromatic moieties targeting this central pocket. These compounds reached low micromolar affinities, validated in different biophysical assays. Finally, X-ray diffraction analysis revealed the interactions of this compound class with LecA. This new mode of action paves the way to a novel route towards inhibition of P. aeruginosa biofilms

Targeting the Central Pocket of the Pseudomonas aeruginosa Lectin LecA

Pseudomonas aeruginosa is an opportunistic ESKAPE pathogen that produces two lectins, LecA and LecB, as part of its large arsenal of virulence factors. Both carbohydrate-binding proteins are central to the initial and later persistent infection processes, i. e. bacterial adhesion and biofilm formation. The biofilm matrix is a major resistance determinant and protects the bacteria against external threats such as the host immune system or antibiotic treatment. Therefore, the development of drugs against the P. aeruginosa biofilm is of particular interest to restore efficacy of antimicrobials. Carbohydrate-based inhibitors for LecA and LecB were previously shown to efficiently reduce biofilm formations. Here, we report a new approach for inhibiting LecA with synthetic molecules bridging the established carbohydrate-binding site and a central cavity located between two LecA protomers of the lectin tetramer. Inspired by in silico design, we synthesized various galactosidic LecA inhibitors with aromatic moieties targeting this central pocket. These compounds reached low micromolar affinities, validated in different biophysical assays. Finally, X-ray diffraction analysis revealed the interactions of this compound class with LecA. This new mode of action paves the way to a novel route towards inhibition of P. aeruginosa biofilms

Targeting undruggable carbohydrate recognition sites through focused fragment library design

Carbohydrate-protein interactions are key for cell-cell and host-pathogen recognition and thus, emerged as viable therapeutic targets. However, their hydrophilic nature poses major limitations to the conventional development of drug-like inhibitors. To address this shortcoming, four fragment libraries were screened to identify metal-binding pharmacophores (MBPs) as novel scaffolds for inhibition of Ca2+-dependent carbohydrate-protein interactions. Here, we show the effect of MBPs on the clinically relevant lectins DC-SIGN, Langerin, LecA and LecB. Detailed structural and biochemical investigations revealed the specificity of MBPs for different Ca2+-dependent lectins. Exploring the structure-activity relationships of several fragments uncovered the functional groups in the MBPs suitable for modification to further improve lectin binding and selectivity. Selected inhibitors bound efficiently to DCSIGN-expressing cells. Altogether, the discovery of MBPs as a promising class of Ca2+- dependent lectin inhibitors creates a foundation for fragment-based ligand design for future drug discovery campaigns

YB-1 promotes microtubule assembly in vitro through interaction with tubulin and microtubules

<p>Abstract</p> <p>Background</p> <p>YB-1 is a major regulator of gene expression in eukaryotic cells. In addition to its role in transcription, YB-1 plays a key role in translation and stabilization of mRNAs.</p> <p>Results</p> <p>We show here that YB-1 interacts with tubulin and microtubules and stimulates microtubule assembly <it>in vitro</it>. High resolution imaging via electron and atomic force microscopy revealed that microtubules assembled in the presence of YB-1 exhibited a normal single wall ultrastructure and indicated that YB-1 most probably coats the outer microtubule wall. Furthermore, we found that YB-1 also promotes the assembly of MAPs-tubulin and subtilisin-treated tubulin. Finally, we demonstrated that tubulin interferes with RNA:YB-1 complexes.</p> <p>Conclusion</p> <p>These results suggest that YB-1 may regulate microtubule assembly <it>in vivo </it>and that its interaction with tubulin may contribute to the control of mRNA translation.</p

The Use of Long-Read Sequencing to Study the Phylogenetic Diversity of the Potato Varieties Plastome of the Ural Selection

Plastid DNA holds a substantial amount of plant genetic information, including maternal ancestry information. It helps to uncover interrelations between a wide variety of tuberous species of the genus Solanum to search for promising sources of high-yielding potato varieties resistant to bio- and abiotic stressors. This paper demonstrated the opportunities of de novo assembly of potato plastid DNA and its phylogenetic and genome type identification based only on Oxford Nanopore Technologies (ONT) long reads. According to our results, of 28 potato varieties developed at the Ural Research Institute of Agriculture, 16 varieties had one of the most primitive W-type plastomes. Ten varieties&rsquo; plastomes belonged to the T-type of cultivated Solanum tuberosum subsp. tuberosum. The varieties Legenda and 15-27-1 were the closest to the wild species Solanum chacoense plastome. Using long-sequencing reads, we confirmed the presence of two isoforms of the plastid genome differing in the orientation of SSC region. We should note that irrespective of sequencing depth and improvements in software for working with ONT reads, a correct de novo plastome assembly and its annotation using only long-reads is impossible. The most problematic regions are homopolymers longer than 5 bp&mdash;they account for all detected indels, leading to a change in the reading frame or the deletion of entire genes

The Use of Long-Read Sequencing to Study the Phylogenetic Diversity of the Potato Varieties Plastome of the Ural Selection

Plastid DNA holds a substantial amount of plant genetic information, including maternal ancestry information. It helps to uncover interrelations between a wide variety of tuberous species of the genus Solanum to search for promising sources of high-yielding potato varieties resistant to bio- and abiotic stressors. This paper demonstrated the opportunities of de novo assembly of potato plastid DNA and its phylogenetic and genome type identification based only on Oxford Nanopore Technologies (ONT) long reads. According to our results, of 28 potato varieties developed at the Ural Research Institute of Agriculture, 16 varieties had one of the most primitive W-type plastomes. Ten varieties’ plastomes belonged to the T-type of cultivated Solanum tuberosum subsp. tuberosum. The varieties Legenda and 15-27-1 were the closest to the wild species Solanum chacoense plastome. Using long-sequencing reads, we confirmed the presence of two isoforms of the plastid genome differing in the orientation of SSC region. We should note that irrespective of sequencing depth and improvements in software for working with ONT reads, a correct de novo plastome assembly and its annotation using only long-reads is impossible. The most problematic regions are homopolymers longer than 5 bp—they account for all detected indels, leading to a change in the reading frame or the deletion of entire genes