Search Results (42)

Cell surface sialylation is utilized by a number of pathogenic bacteria to evade the host immune system through molecular mimicry of host sialoglycoconjugates. Human pathogen Neisseria meningitidis serotype B (NmB) expresses both sialylated capsule and surface lipooligosaccharides as pivotal virulence factors. An essential enzyme in the sialylation pathway of NmB is CMP-sialic acid synthetase (CSS), which produces the activated nucleotide sugar necessary for sialic acid transfer. In this work, novel C-4, -5, -7, and -9 functionalized derivatives of neuraminic acid β-methyl glycoside (Neuβ2Me) were synthesized as candidate CSS inhibitors. A number of these were found to reduce the activity of NmB CSS in vitro. The highest inhibition of NmB CSS, in a mixed mode manner, was observed with a Neu5Acβ2Me C-9 serine carboxamide. Direct interaction with the enzyme was confirmed by Saturation Transfer Difference (STD) NMR. Supplementation of growth media with this compound reduced lipooligosaccharide (LOS) sialylation of living N. meningitidis, thus providing an interesting starting point for the development of specific NmB CSS inhibitors as an alternative treatment strategy to fight bacterial infections. Full article

Heparan sulfate proteoglycans serve as initial attachment sites for several viruses and bacteria. Recent studies suggest that SARS-CoV-2 similarly exploits these glycosaminoglycans, facilitating conformational changes in the spike protein that promote the interaction between the receptor-binding domain (S1-RBD) and the cellular angiotensin-converting enzyme 2 receptor (ACE2), thereby triggering the virus internalization process. The molecular details that drive this process, particularly the co-receptor role of heparan sulfate (HS), remain incompletely understood. The interaction between an HS hexasaccharide (hexa) and the N343 glycosylated S1-RBD of the wild-type (WT) and Omicron variant of SARS-CoV-2 was investigated. The conformational properties of hexa with these S1-RBDs in unbound and bound states are explored using multiple independent MD simulations; the protein binding epitope of hexa, as well as the details of its interaction with S1-RBD of the Omicron variant, are characterized by comparing experimental and theoretical ¹H STD NMR signals. This investigation identifies the role played by the glycosyl moiety at N343 in potentially affecting this interaction in both WT and Omicron S1-RBD, explaining the observed low specificity and multi-modal nature of the interaction between HS oligosaccharides and these S1-RBDs. Full article

The synthesis of bioplastics from renewable resources is essential for green living. PHBV (poly(3-hydroxybutyrate-co-3-hydroxyvalerate)) is a biodegradable and biocompatible material ideal for various industrial applications. The impact of levulinic (LA), valeric acids (VA), and sodium propionate (SPr) as co-substrates in biomass and the synthesis of 3-hydroxy valerate (3HV) and co-polymerization of PHBV by Burkholderia thailandensis E264 (BtE264) was assessed. Thermogravimetric, XRD, NMR, and mechanical characterization were performed on the homopolymer (PHB) and co-polymer (PHBV), and compared to the PHBV-STD. BtE264 produced the co-polymer PHBV when adding any of the three co-substrates. LA showed a higher positive effect on microbial growth (8.4 g∙L⁻¹) and PHBV production (3.91 g∙L⁻¹), representing 78 and 22 mol % of 3HB and 3HV, respectively. The PHBV obtained with LA had a melting temperature (Tm) lower than the PHB homopolymer and presented lower values for melting enthalpies (ΔHf); the degree of crystallization and TGA values indicated that PHBV had better thermal stability. Additionally, FTIR and NMR revealed that BtE264 synthesizes PHBV with an organization in monomeric units (3HB-3HV), suggesting differentiated incorporation of the monomers, improving 3.4 times the break elongation the co-polymer’s tensile properties. This study highlights the co-substrates’ relevance in PHBV synthesis using BtE264 for the first time. Full article

Understanding the binding interactions between protein-bound uremic toxins (PBUTs) and human serum albumin (HSA) is critical for advancing treatments for chronic kidney disease (CKD). While previous studies have suggested that putrescine, a diamine PBUT, exhibits moderate binding affinity to HSA, this study provides evidence of the contrary. Using isothermal titration calorimetry and saturation transfer difference nuclear magnetic resonance , we demonstrate that putrescine’s interaction with HSA is weak, non-specific, and thermodynamically negligible in the range of conditions studied. Unlike earlier studies relying on spectroscopy techniques such as UV–visible absorption and fluorescence, which may overestimate binding strength, the results presented here highlight the limitations of indirect methodologies and underscore the importance of more sensitive approaches for accurate energy characterization. Our findings suggest that putrescine only weakly interacts non-specifically with HSA and may bind more preferentially to other plasma proteins, contributing to its accumulation in CKD patients. Full article

Background: In this study, two chalcone analogs were synthesized through in silico and experimental methods, and their potential to inhibit the lipoxygenase enzyme, which plays a role in the inflammation pathway, was assessed. Specifically, this study is a continuation of previous research in which chalcone derivatives were synthesized and characterized. Objectives/Methods: In the current work, we present the re-synthesis of two chalcones, with a focus on their docking studies, NMR analysis, and dynamic simulations. The structure of each chalcone was elucidated through a combination of Nuclear Magnetic Resonance (NMR) and Density Functional Theory (DFT). The substituent effect on the absorption spectrum of the two chalcone derivatives was studied. Results: A “LOX–chalcone” complex, predicted by docking studies, was further examined using molecular dynamics (MD) simulations to evaluate the stability of the complex. After fully characterizing the “LOX–chalcone” complexes in silico, the atomic details of each chalcone’s interaction with LOX-1 and 5-LOX were revealed through Saturation Transfer Difference (STD) NMR (Nuclear Magnetic Resonance). Finally, their selectivity profile was investigated against human 15-LOX-1 and general Lipoxidase activity. Conclusions: The in silico methods suggest that chalcones could be promising lead compounds for drug designs targeting the LOX enzyme. Full article

Combining the pharmacological properties of the 1,2,3-triazole and dihydropyrimidinone classes of compounds, two small families of mono- and di(1,2,3-triazole)-dihydropyrimidinone hybrids, A and B, were previously synthesized. The main objective of this work was to investigate the potential anti-Alzheimer effects of these hybrids. The inhibitory activities of cholinesterases (AChE and BuChE), antioxidant activity, and the inhibitory mechanism through in silico (molecular docking) and in solution (STD-NMR) experiments were evaluated. The 1,2,3-triazole-dihydropyrimidinone hybrids (A and B) showed moderate in vitro inhibitory activity on eqBuChE (IC₅₀ values between 1 and 58.4 μM). The best inhibitor was the hybrid B4, featuring two 1,2,3-triazole cores, which exhibited stronger inhibition than galantamine, with an IC₅₀ of 1 ± 0.1 μM for eqBuChE, through a mixed inhibition mechanism. Among the hybrids A, the most promising inhibitor was A1, exhibiting an IC₅₀ of 12 ± 2 µM, similar to that of galantamine. Molecular docking and STD-NMR experiments revealed the key binding interactions of these promising inhibitors with BuChE. Hybrids A and B did not display Artemia salina toxicity below 100 μM. Full article

Ketoprofen (KTF) and ketorolac (KTL) are among the most primarily used non-steroidal anti-inflammatory drugs (NSAIDs) in humans to alleviate moderate pain and to treat inflammation. Their binding affinity with albumin (the main globular protein responsible for the biodistribution of drugs in the bloodstream) was previously determined by spectroscopy without considering some conventional pitfalls. Thus, the present work updates the biophysical characterization of the interactions of HSA:KTF and HSA:KTL by ¹H saturation-transfer difference nuclear magnetic resonance (¹H STD-NMR), ultraviolet (UV) absorption, circular dichroism (CD), steady-state, and time-resolved fluorescence spectroscopies combined with in silico calculations. The binding of HSA:NSAIDs is spontaneous, endothermic, and entropically driven, leading to a conformational rearrangement of HSA with a slight decrease in the α-helix content (7.1% to 7.6%). The predominance of the static quenching mechanism (ground-state association) was identified. Thus, both Stern–Volmer quenching constant (K_SV) and binding constant (K_b) values enabled the determination of the binding affinity. In this sense, the K_SV and K_b values were found in the order of 10⁴ M⁻¹ at human body temperature, indicating moderate binding affinity with differences in the range of 0.7- and 3.4-fold between KTF and KTL, which agree with the previously reported experimental pharmacokinetic profile. According to ¹H STD-NMR data combined with in silico calculations, the aromatic groups in relation to the aliphatic moiety of the drugs interact preferentially with HSA into subdomain IIIA (site II) and are stabilized by interactions via hydrogen bonding and hydrophobic forces. In general, the data obtained in this study have been revised and updated in comparison to those previously reported by other authors who did not account for inner filter corrections, spectral backgrounds, or the identification of the primary mathematical approach for determining the binding affinity of HSA:KTF and HSA:KTL. Full article

Cardiovascular diseases (CVDs), especially arterial hypertension, stand as prominent contributors to global mortality. Regrettably, individuals with diabetes encounter a two-fold increase in the risk of mortality associated with CVDs. Hydrochlorothiazide (HCTZ) represents a primary intervention for hypertension, particularly in diabetic patients. Nevertheless, there has not yet been a comprehensive assessment of the biophysical characteristics regarding the impact of glucose levels on its binding affinity with human serum albumin (HSA). Thus, the present work reports the interactive profile of HSA/HCTZ in nonglycemic, normoglycemic (80 mg/dL), and hyperglycemic (320 mg/dL) conditions by time-resolved fluorescence, saturation transfer difference–nuclear magnetic resonance (STD-NMR), and surface plasmon resonance (SPR). There was a moderate ground state association of HSA/HCTZ with subdomain IIA that was affected in the presence of different glucose levels. The hyperglycemic condition decreased the binding affinity of HCTZ to subdomain IIA and increased the possibility of subdomain IB also being considered as a secondary binding site due to cooperativity and/or alterations in the protein’s structure. Overall, the glucose level under hyperglycemic conditions led to the cavities being more likely to receive more ligands, offering insights into the necessity of glucose control in the human bloodstream to not impact the residence time (pharmacokinetic profile) and pharmacotherapeutic potential of HCTZ. Full article

The interaction of heparin with antithrombin (AT) involves a specific sequence corresponding to the pentasaccharide GlcNAc/NS6S-GlcA-GlcNS3S6S-IdoA2S-GlcNS6S (AGA*IA). Recent studies have revealed that two AGA*IA-containing hexasaccharides, which differ in the sulfation degree of the iduronic acid unit, exhibit similar binding to AT, albeit with different affinities. However, the lack of experimental data concerning the molecular contacts between these ligands and the amino acids within the protein-binding site prevents a detailed description of the complexes. Differential epitope mapping (DEEP)-STD NMR, in combination with MD simulations, enables the experimental observation and comparison of two heparin pentasaccharides interacting with AT, revealing slightly different bound orientations and distinct affinities of both glycans for AT. We demonstrate the effectiveness of the differential solvent DEEP-STD NMR approach in determining the presence of polar residues in the recognition sites of glycosaminoglycan-binding proteins. Full article

Obesity is a risk factor for many diseases, such as type 2 diabetes and cardiovascular diseases. In line with the need for precision medicine, the search for biomarkers reporting the progression of obesity- and diet-associated disorders is urgent. We used NMR to determine the metabolomics profile of key organs (lung, liver, heart, skeletal muscle, kidney, and brain) and serum from male C57Bl/6J mice (5 weeks old) fed for 6, 10, and 14 weeks on a high-fat and high-sucrose diet (HFHSD) vs. a standard diet (STD). We determined metabolite concentrations in the organs at each time point, which allowed us to discriminate age- and diet-related effects as well as the interactions between both, highlighting the need to evaluate the influence of age as a confounding factor on metabolic signatures. Notably, the analysis revealed the influence of time on metabolite concentrations in the STD condition, probably reflecting the juvenile-to-adult transition. Variations impacted the liver and lung metabolites, revealing the strong influence of the HFHS diet on normal metabolism maturation during youth. Full article

Human Galectin-3 (hGal-3) is a protein that selectively binds to β-galactosides and holds diverse roles in both normal and pathological circumstances. Therefore, targeting hGal-3 has become a vibrant area of research in the pharmaceutical chemistry. As a step towards the development of novel hGal-3 inhibitors, we synthesized and investigated derivatives of thiodigalactoside (TDG) modified with different aromatic substituents. Specifically, we describe a high-yielding synthetic route of thiodigalactoside (TDG); an optimized procedure for the synthesis of the novel 3,3′-di-O-(quinoline-2-yl)methyl)-TDG and three other known, symmetric 3,3′-di-O-TDG derivatives ((naphthalene-2yl)methyl, benzyl, (7-methoxy-2H-1-benzopyran-2-on-4-yl)methyl). In the present study, using competition Saturation Transfer Difference (STD) NMR spectroscopy, we determined the dissociation constant (K_d) of the former three TDG derivatives produced to characterize the strength of the interaction with the target protein (hGal-3). Based on the K_d values determined, the (naphthalen-2-yl)methyl, the (quinolin-2-yl)methyl and the benzyl derivatives bind to hGal-3 94, 30 and 24 times more strongly than TDG. Then, we studied the binding modes of the derivatives in silico by molecular docking calculations. Docking poses similar to the canonical binding modes of well-known hGal-3 inhibitors have been found. However, additional binding forces, cation–π interactions between the arginine residues in the binding pocket of the protein and the aromatic groups of the ligands, have been established as significant features. Our results offer a molecular-level understanding of the varying affinities observed among the synthesized thiodigalactoside derivatives, which can be a key aspect in the future development of more effective ligands of hGal-3. Full article

Saturation transfer difference (STD), inter-ligand NOEs (INPHARMA NMR), and docking calculations are reported for investigating specific binding sites of the high-affinity synthetic 7-nitrobenz-2-oxa-1,3-diazoyl-4-C₁₂ fatty acid (NBD-C₁₂ FA) with non-labeled human serum albumin (HSA) and in competition with the drugs warfarin and ibuprofen. A limited number of negative interligand NOEs between NBD-C₁₂ FA and warfarin were interpreted in terms of a short-range allosteric competitive binding in the wide Sudlow’s binding site II (FA7) of NBD-C₁₂ FA with Ser-202, Lys-199, and Trp-214 and warfarin with Arg-218 and Arg-222. In contrast, the significant number of interligand NOEs between NBD-C₁₂ FA and ibuprofen were interpreted in terms of a competitive binding mode in Sudlow’s binding site I (FA3 and FA4) with Ser-342, Arg-348, Arg-485, Arg-410, and Tyr-411. NBD-C₁₂ FA has the unique structural properties, compared to short-, medium-, and long-chain saturated and unsaturated natural free fatty acids, of interacting with well-defined structures with amino acids of both the internal and external polar anchor sites in Sudlow’s binding site I and with amino acids in both FA3 and FA4 in Sudlow’s binding site II. The NBD-C₁₂ FA, therefore, interacts with novel structural characteristics in the drug binding sites I and II and can be regarded as a prototype molecule for drug development. Full article

Medium- and long-chain saturated and unsaturated free fatty acids (FFAs) are known to bind to human serum albumin (HSA), the main plasma carrier protein. Atomic-level structural data regarding the binding mode in Sudlow’s sites I (FA7) and II (FA4, FA3) of the polyunsaturated ω-3 fatty acids docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), however, are largely unknown. Herein, we report the combined use of saturation transfer difference (STD) and Interligand NOEs for Pharmacophore Mapping (INPHARMA) NMR techniques and molecular docking calculations to investigate the binding mode of DHA and EPA in Sudlow’s sites Ι and ΙΙ of HSA. The docking calculations and the significant number of interligand NOEs between DHA and EPA and the drugs warfarin and ibuprofen, which are stereotypical ligands for Sudlow’s sites I and II, respectively, were interpreted in terms of competitive binding modes and the presence of two orientations of DHA and EPA at the binding sites FA7 and FA4. The exceptional flexibility of the long-chain DHA and EPA and the formation of strongly folded structural motives are the key properties of HSA–PUFA complexes. Full article

The inhibition of carbohydrate-lectin interactions is being explored as an efficient approach to anti adhesion therapy and biofilm destabilization, two alternative antimicrobial strategies that are being explored against resistant pathogens. BC2L-C is a new type of lectin from Burkholderia cenocepacia that binds (mammalian) fucosides at the N-terminal domain and (bacterial) mannosides at the C-terminal domain. This double carbohydrate specificity allows the lectin to crosslink host cells and bacterial cells. We have recently reported the design and generation of the first glycomimetic antagonists of BC2L-C, β-C- or β-N-fucosides that target the fucose-specific N-terminal domain (BC2L-C-Nt). The low water solubility of the designed N-fucosides prevented a full examination of this promising series of ligands. In this work, we describe the synthesis and biophysical evaluation of new L-fucosyl and L-galactosyl amides, designed to be water soluble and to interact with BC2L-C-Nt. The protein–ligand interaction was investigated by Saturation Transfer Difference NMR, Isothermal Titration Calorimetry and crystallographic studies. STD-NMR experiments showed that both fucosyl and galactosyl amides compete with α-methyl fucoside for lectin binding. A new hit compound was identified with good water solubility and an affinity for BC2L-C-Nt of 159 μM (ITC), which represents a one order of magnitude gain over α-methyl fucoside. The x-ray structure of its complex with BC2L-C-Nt was solved at 1.55 Å resolution. Full article

Fragment-based lead discovery (FBLD) is a powerful application for developing ligands as modulators of disease targets. This approach strategy involves identification of interactions between low-molecular weight compounds (100–300 Da) and their putative targets, often with low affinity (K_D ~0.1–1 mM) interactions. The focus of this screening methodology is to optimize and streamline identification of fragments with higher ligand efficiency (LE) than typical high-throughput screening. The focus of this review is on the last half decade of fragment-based drug discovery strategies that have been used for antimicrobial drug discovery. Full article