Dolores Díaz

Enthalpic Nature of the CH/π Interaction Involved in the Recognition of Carbohydrates by Aromatic Compounds, Confirmed by a Novel Interplay of NMR, Calorimetry, and Theoretical Calculations

Specific interactions between molecules, including those produced by a given solute, and the surrounding solvent are essential to drive molecular recognition processes. A simple molecule such as benzene is capable of recognizing and differentiating among very similar entities, such as methyl 2,3,4,6-tetra-O-methyl-alpha-D-galactopyranoside (alpha-Me(5)Gal), methyl 2,3,4,6-tetra-O-methyl-beta-D-galactopyranoside (beta-Me(5)Gal), 1,2,3,4,6-penta-O-acetyl-beta-D-galactopyranose (beta-Ac(5)Gal), and methyl 2,3,4,6-tetra-O-methyl-alpha-D-mannopyranoside (alpha-Me(5)Man). In order to determine if these complexes are formed, the interaction energy between benzene and the different carbohydrates was determined, using Calvet microcalorimetry, as the enthalpy of solvation. These enthalpy values were -89.0 +/- 2.0, -88.7 +/- 5.5, -132.5 +/- 6.2, and -78.8 +/- 3.9 kJ mol(-1) for the four complexes, respectively. Characterization of the different complexes was completed by establishing the molecular region where the interaction takes place using NMR. It was determined that beta-Me(5)Gal is stabilized by the CH/pi interaction produced by the nonpolar region of the carbohydrate on the alpha face. In contrast, alpha-Me(5)Man is not specifically solvated by benzene and does not present any stacking interaction. Although alpha-Me(5)Gal has a geometry similar to that of its epimer, the obtained NMR data seem to indicate that the axial methoxy group at the anomeric position increases the distance of the benzene molecules from the pyranose ring. Substitution of the methoxy groups by acetate moieties, as in beta-Ac(5)Gal, precludes the approach of benzene to produce the CH/pi interaction. In fact, the elevated stabilization energy of beta-Ac(5)Gal is probably due to the interaction between benzene and the methyl groups of the acetyls. Therefore, methoxy and acetyl substituents have different effects on the protons of the pyranose ring.

Aromatic–Carbohydrate Interactions: An NMR and Computational Study of Model Systems

The interactions of simple carbohydrates with aromatic moieties have been investigated experimentally by NMR spectroscopy. The analysis of the changes in the chemical shifts of the sugar proton signals induced upon addition of aromatic entities has been interpreted in terms of interaction geometries. Phenol and aromatic amino acids (phenylalanine, tyrosine, tryptophan) have been used. The observed sugar-aromatic interactions depend on the chemical nature of the sugar, and thus on the stereochemistries of the different carbon atoms, and also on the solvent. A preliminary study of the solvation state of a model monosaccharide (methyl beta-galactopyranoside) in aqueous solution, both alone and in the presence of benzene and phenol, has also been carried out by monitoring of intermolecular homonuclear solvent-sugar and aromatic-sugar NOEs. These experimental results have been compared with those obtained by density functional theory methods and molecular mechanics calculations.

Protein-carbohydrate interactions studied by NMR: from molecular recognition to drug design.

Diseases that result from infection are, in general, a consequence of specific interactions between a pathogenic organism and the cells. The study of host-pathogen interactions has provided insights for the design of drugs with therapeutic properties. One area that has proved to be promising for such studies is the constituted by carbohydrates which participate in biological processes of paramount importance. On the one hand, carbohydrates have shown to be information carriers with similar, if not higher, importance than traditionally considered carriers as amino acids and nucleic acids. On the other hand, the knowledge on molecular recognition of sugars by lectins and other carbohydrate-binding proteins has been employed for the development of new biomedical strategies. Biophysical techniques such as X-Ray crystallography and NMR spectroscopy lead currently the investigation on this field. In this review, a description of traditional and novel NMR methodologies employed in the study of sugar-protein interactions is briefly presented in combination with a palette of NMR-based studies related to biologically and/or pharmaceutically relevant applications.

NMR and molecular recognition. The application of ligand-based NMR methods to monitor molecular interactions

NMR spectroscopy allows the monitoring of molecular recognition processes in solution. Nowadays, a plethora of NMR methods are available to deduce the key features of the interaction from both the ligand or the receptor points of view (receptor-observed or ligand-observed NMR methods, respectively). Herein, we present the most used ligand-observed NMR experiments for drug discovery making special emphasis on their experimental aspects and on their applications to the study of protein–carbohydrate interactions.

Discovery of (S)-1-(1-(4-Chloro-3-fluorophenyl)-2-hydroxyethyl)-4-(2-((1-methyl-1H-pyrazol-5-yl)amino)pyrimidin-4-yl)pyridin-2(1H)-one (GDC-0994), an Extracellular Signal-Regulated Kinase 1/2 (ERK1/2) Inhibitor in Early Clinical Development.

The extracellular signal-regulated kinases ERK1/2 represent an essential node within the RAS/RAF/MEK/ERK signaling cascade that is commonly activated by oncogenic mutations in BRAF or RAS or by upstream oncogenic signaling. While targeting upstream nodes with RAF and MEK inhibitors has proven effective clinically, resistance frequently develops through reactivation of the pathway. Simultaneous targeting of multiple nodes in the pathway, such as MEK and ERK, offers the prospect of enhanced efficacy as well as reduced potential for acquired resistance. Described herein is the discovery and characterization of GDC-0994 (22), an orally bioavailable small molecule inhibitor selective for ERK kinase activity.

Insights into the Glycosaminoglycan-Mediated Cytotoxic Mechanism of Eosinophil Cationic Protein Revealed by NMR

Protein-glycosaminoglycan interactions are essential in many biological processes and human diseases, yet how their recognition occurs is poorly understood. Eosinophil cationic protein (ECP) is a cytotoxic ribonuclease that interacts with glycosaminoglycans at the cell surface; this promotes the destabilization of the cellular membrane and triggers ECPs toxic activity. To understand this membrane destabilization event and the differences in the toxicity of ECP and its homologues, the high resolution solution structure of the complex between full length folded ECP and a heparin-derived trisaccharide (O-iPr-α-D-GlcNS6S-α(1-4)-L-IdoA2S-α(1-4)-D-GlcNS6S) has been solved by NMR methods and molecular dynamics simulations. The bound protein retains the tertiary structure of the free protein. The (2)S(0) conformation of the IdoA ring is preferably recognized by the protein. We have identified the precise location of the heparin binding site, dissected the specific interactions responsible for molecular recognition, and defined the structural requirements for this interaction. The structure reveals the contribution of Arg7, Gln14, and His15 in helix α1, Gln40 in strand β1, His64 in loop 4, and His128 in strand β6 in the recognition event and corroborates the previously reported participation of residues Arg34-Asn39. The participation of the catalytic triad (His15, Lys38, His128) in recognizing the heparin mimetic reveals, at atomic resolution, the mechanism of heparins inhibition of ECPs ribonucleolytic activity. We have integrated all the available data to propose a molecular model for the membrane interaction process. The solved NMR complex provides the structural model necessary to design inhibitors to block ECPs toxicity implicated in eosinophil pathologies.

The L-Leu Hexamer, a Short and Highly Enantioselective Peptide Catalyst for the Juliá–Colonna Epoxidation: Stabilization of a Helical Conformation in DMSO

Short peptides are intriguing chiral catalysts (“synthetic enzymes”) that combine features typical to organocatalysts (e.g. , catalytically active functional groups of low molecular mass) with structural properties of larger biomolecules. Among these, synthetic homopolypeptides introduced in the 1980s by Juli , Colonna et al. rank as the first ones to catalyze highly enantioselective reactions in the absence of metals, in particular the asymmetric epoxidation of chalcones. The transformation of chalcone (1) to its highly enantio-enriched epoxide (2) catalyzed by poly-l-leucine (PLL) is shown in Scheme 1.

Lysine Nε-trimethylation, a tool for improving the selectivity of antimicrobial peptides.

The effects of lysine N(epsilon)-trimethylation at selected positions of the antimicrobial cecropin A-melittin hybrid peptide KWKLFKKIGAVLKVL-amide have been studied. All five monotrimethylated, four bis-trimethylated plus the per-trimethylated analogues have been synthesized and tested for antimicrobial activity on Leishmania parasites and on Gram-positive and -negative bacteria, as well as for hemolysis of sheep erythrocytes as a measure of cytotoxicity. The impact of trimethylation on the solution conformation of selected analogues has been evaluated by NMR, which indicates a slight decrease in the alpha-helical content of the modified peptides, particularly in the N-terminal region. Trimethylation also enhances the proteolytic stability of mono- and bis-trimethylated analogues by 2-3-fold. Although it tends to lower antimicrobial activity in absolute terms, trimethylation causes an even higher decrease in hemolytic activity and therefore results in improved selectivity for several analogues. The monotrimethylated analogue at position 6 shows the overall best selectivity against both the Leishmania donovani protozoan and Acinetobacter baumannii, a Gram-negative bacterium of increasing clinical concern.

gem‐Difluorocarbadisaccharides: Restoring the exo‐Anomeric Effect

Molecular mimicry is an essential part of the development of drugs and molecular probes. In the chemical glycobiology field, although many glycomimetics have been developed in the past years, it has been considered that many failures in their use are related to the lack of the anomeric effects in these analogues. Additionally, the origin of the anomeric effects is still the subject of virulent scientific debates. Herein, by combining chemical synthesis, NMR methods, and theoretical calculations, we show that it is possible to restore the anomeric effect for an acetal when replacing one of the oxygen atoms by a CF2 group. This result provides key findings in chemical sciences. On the one hand, it strongly suggests the key relevance of the stereoelectronic component of the anomeric effect. On the other hand, the CF2 analogue adopts the natural glycoside conformation, which might provide new avenues for sugar-based drug design.

Structural framework for the modulation of the activity of the hybrid antibiotic peptide cecropin A-melittin [CA(1-7)M(2-9)] by Nε-lysine trimethylation.

The 3D structures of six linear pentadecapeptides derived from the cecropin A–melittin antimicrobial peptide CA(1–7)M(2–9) [KWKLFKKIGAVLKVL‐NH2] have been studied. These analogues are modified by ε‐NH2 trimethylation of one or more lysine residues and showed variation in both antimicrobial and cytotoxic activities, depending on the number and position of modified lysines. Since it is expected that these peptides will display a strong conformational ordering when in contact with membranes, we have investigated their structure on the basis of the data extracted from NMR experiments performed in membrane‐mimetic environments. We show that inclusion of Nε‐trimethylated lysine residues induces a certain degree of structural flexibility, while preserving to a variable extent a largely α‐helical structure. In addition, peptide orientation with respect to SDS micelles has been explored by detection of the intensity changes of peptide NMR signals upon addition of a paramagnetic probe (Mn2+ ions).