Multifrequency STD NMR Unveils the Interactions of Antibiotics With Burkholderia multivorans Biofilm Exopolysaccharide

Biofilms confine bacterial cells within self-produced matrices, offering advantages such as protection from antibiotics and entrapment of nutrients. Polysaccharides are major components in these macromolecular assemblies, and their interactions with other chemicals are of high relevance for the benefits provided by the biofilm 3D molecular matrix. NMR is a powerful technique for the study and characterization of the interactions between molecules of biological relevance. In this study, we have applied multifrequency saturation transfer difference (STD) NMR and DOSY NMR approaches to elucidate the interactions between the exopolysaccharide produced by Burkholderia multivorans C1576 (EpolC1576) and the antibiotics kanamycin and ceftadizime. The NMR strategies presented here allowed for an extensive characterization at an atomic level of the mechanisms behind the implication of the EpolC1576 in the recalcitrance phenomena, which is the ability of bacteria in biofilms to survive in the presence of antibiotics. Our results suggest an active role for EpolC1576 in the recalcitrance mechanisms toward kanamycin and ceftadizime, though through two different mechanisms.

A COVID moonshot: assessment of ligand binding to the SARS-CoV-2 main protease by saturation transfer difference NMR spectroscopy

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiological cause of the coronavirus disease 2019, for which no effective antiviral therapeutics are available. The SARS-CoV-2 main protease (Mpro) is essential for viral replication and constitutes a promising therapeutic target. Many efforts aimed at deriving effective Mpro inhibitors are currently underway, including an international open-science discovery project, codenamed COVID Moonshot. As part of COVID Moonshot, we used saturation transfer difference nuclear magnetic resonance (STD-NMR) spectroscopy to assess the binding of putative Mpro ligands to the viral protease, including molecules identified by crystallographic fragment screening and novel compounds designed as Mpro inhibitors. In this manner, we aimed to complement enzymatic activity assays of Mpro performed by other groups with information on ligand affinity. We have made the Mpro STD-NMR data publicly available. Here, we provide detailed information on the NMR protocols used and challenges faced, thereby placing these data into context. Our goal is to assist the interpretation of Mpro STD-NMR data, thereby accelerating ongoing drug design efforts.

Synthesis of Tetravalent Thio- and Selenogalactoside-Presenting Galactoclusters and Their Interactions with Bacterial Lectin PA-IL from Pseudomonas aeruginosa

Synthesis of tetravalent thio- and selenogalactopyranoside-containing glycoclusters using azide-alkyne click strategy is presented. Prepared compounds are potential ligands of Pseudomonas aeruginosa lectin PA-IL. P. aeruginosa is an opportunistic human pathogen associated with cystic fibrosis, and PA-IL is one of its virulence factors. The interactions of PA-IL and tetravalent glycoconjugates were investigated using hemagglutination inhibition assay and compared with mono- and divalent galactosides (propargyl 1-thio- and 1-seleno-β-d-galactopyranoside, digalactosyl diselenide and digalactosyl disulfide). The lectin-carbohydrate interactions were also studied by saturation transfer difference NMR technique. Both thio- and seleno-tetravalent glycoconjugates were able to inhibit PA-IL significantly better than simple d-galactose or their intermediate compounds from the synthesis.

Detection of ligand binding to glycopolymers using saturation transfer difference NMR

Saturation transfer from glycopolymers to bound small molecules enables detection of ligand binding to polymers.

Saturation Transfer Difference NMR Spectroscopy Using Glycopolymers

<p>We report the use of Saturation Transfer Difference (STD) NMR spectroscopy to observe the interaction of various phenylboronic acids (PBAs) with synthetic glycopolymers presenting galactose and glucose. After optimizing experimental parameters to maximize spin diffusion, the binding of boronic acids to the glycopolymers was examined using STD NMR. Efficient amplification factor build-up curves which were used to generate an epitope map for the boronic acid binding to the glycopolymers. STD-NMR was also used to detect the interaction between indole and a galactosylated glycopolymer, providing an indole-based view of this C-H – π interaction.</p>

Saturation Transfer Difference NMR Spectroscopy Using Glycopolymers

<p>We report the use of Saturation Transfer Difference (STD) NMR spectroscopy to observe the interaction of various phenylboronic acids (PBAs) with synthetic glycopolymers presenting galactose and glucose. After optimizing experimental parameters to maximize spin diffusion, the binding of boronic acids to the glycopolymers was examined using STD NMR. Efficient amplification factor build-up curves which were used to generate an epitope map for the boronic acid binding to the glycopolymers. STD-NMR was also used to detect the interaction between indole and a galactosylated glycopolymer, providing an indole-based view of this C-H – π interaction.</p>

Saturation transfer difference NMR on the integral trimeric membrane transport protein GltPh determines cooperative substrate binding

Saturation-transfer difference (STD) NMR spectroscopy is a fast and versatile method which can be applied for drug-screening purposes, allowing the determination of essential ligand binding affinities (KD). Although widely employed to study soluble proteins, its use remains negligible for membrane proteins. Here the use of STD NMR for KD determination is demonstrated for two competing substrates with very different binding affinities (low nanomolar to millimolar) for an integral membrane transport protein in both detergent-solubilised micelles and reconstituted proteoliposomes. GltPh, a homotrimeric aspartate transporter from Pyrococcus horikoshii, is an archaeal homolog of mammalian membrane transport proteins—known as excitatory amino acid transporters (EAATs). They are found within the central nervous system and are responsible for fast uptake of the neurotransmitter glutamate, essential for neuronal function. Differences in both KD’s and cooperativity are observed between detergent micelles and proteoliposomes, the physiological implications of which are discussed.