Pedro M. Nieto

Tachykinins and Tachykinin Receptors: Structure and Activity Relationships

In addition to the classical neurotransmitters, acetylcholine and noradrenaline, a wide number of peptides with neurotransmitter activity have been identified in the past few years. Among them, the tachykinins substance P (SP), neurokinin A (NKA) and neurokinin B (NKB) appear to act as mediators of nonadrenergic, noncholinergic (NANC) excitatory neurotransmission. Tachykinins interact with specific membrane proteins, belonging to the family of G protein-coupling cell membrane receptors. Until now, three tachykinin receptors termed NK1 (NK1R), NK2 (NK2R) and NK3 (NK3R) have been cloned in different species. A large amount of reports suggests that these peptides are involved in nociception and neuroimmunomodulation, and in the development of different diseases such as bronchial asthma, inflammatory bowel syndrome and psychiatric disorders. Tachykinin receptor antagonists are therefore promising, therapeutically relevant agents. However, and in spite of extensive research, the obtention of selective antagonists of tachykinin receptors have revealed very difficult. An understanding of how ligands interact with their receptors is essential to permit a rational design of compounds acting selectively at the tachykinin receptor level. The major aim of the present article is to review the structure-activity data that exist for tachykinins and their receptors, with the purpose of getting insight into basic structural requirements that determine ligand/receptor interaction.

Ligand–Receptor Binding Affinities from Saturation Transfer Difference (STD) NMR Spectroscopy: The Binding Isotherm of STD Initial Growth Rates

The direct evaluation of dissociation constants (K(D)) from the variation of saturation transfer difference (STD) NMR spectroscopy values with the receptor-ligand ratio is not feasible due to the complex dependence of STD intensities on the spectral properties of the observed signals. Indirect evaluation, by competition experiments, allows the determination of K(D), as long as a ligand of known affinity is available for the protein under study. Herein, we present a novel protocol based on STD NMR spectroscopy for the direct measurements of receptor-ligand dissociation constants (K(D)) from single-ligand titration experiments. The influence of several experimental factors on STD values has been studied in detail, confirming the marked impact on standard determinations of protein-ligand affinities by STD NMR spectroscopy. These factors, namely, STD saturation time, ligand residence time in the complex, and the intensity of the signal, affect the accumulation of saturation in the free ligand by processes closely related to fast protein-ligand rebinding and longitudinal relaxation of the ligand signals. The proposed method avoids the dependence of the magnitudes of ligand STD signals at a given saturation time on spurious factors by constructing the binding isotherms using the initial growth rates of the STD amplification factors, in a similar way to the use of NOE growing rates to estimate cross relaxation rates for distance evaluations. Herein, it is demonstrated that the effects of these factors are cancelled out by analyzing the protein-ligand association curve using STD values at the limit of zero saturation time, when virtually no ligand rebinding or relaxation takes place. The approach is validated for two well-studied protein-ligand systems: the binding of the saccharides GlcNAc and GlcNAcbeta1,4GlcNAc (chitobiose) to the wheat germ agglutinin (WGA) lectin, and the interaction of the amino acid L-tryptophan to bovine serum albumin (BSA). In all cases, the experimental K(D) measured under different experimental conditions converged to the thermodynamic values. The proposed protocol allows accurate determinations of protein-ligand dissociation constants, extending the applicability of the STD NMR spectroscopy for affinity measurements, which is of particular relevance for those proteins for which a ligand of known affinity is not available.

The Activation of Fibroblast Growth Factors by Heparin: Synthesis, Structure, and Biological Activity of Heparin‐Like Oligosaccharides

An effective strategy has been designed for the synthesis of oligosaccharides of different sizes structurally related to the regular region of heparin; this is illustrated by the preparation of hexasaccharide 1 and octasaccharide 2. This synthetic strategy provides the oligosaccharide sequence containing a D‐glucosamine unit at the nonreducing end that is not available either by enzymatic or chemical degradation of heparin. It may permit, after slight modifications, the preparation of oligosaccharide fragments with different charge distribution as well. NMR spectroscopy and molecular dynamics simulations have shown that the overall structure of 1 in solution is a stable right‐hand helix with four residues per turn. Hexasaccharide 1 and, most likely, octasaccharide 2 are, therefore, chemically well‐defined structural models of naturally occurring heparin‐like oligosaccharides for use in binding and biological activity studies. Both compounds 1 and 2 induce the mitogenic activity of acid fibroblast growth factor (FGF1), with the half‐maximum activating concentration of 2 being equivalent to that of heparin. Sedimentation equilibrium analysis with compound 2 suggests that heparin‐induced FGF1 dimerization is not an absolute requirement for biological activity.

STD-NMR: application to transient interactions between biomolecules—a quantitative approach

Saturation transfer difference NMR (STD NMR) spectroscopy is one of the most powerful NMR techniques for detection and characterization of transient (fast) receptor–ligand interactions in solution. By observing the signals of a small molecule (ligand) with spectroscopic properties suitable for high-resolution studies, irrespective of receptor size, STD NMR enables quantitative structural and affinity information to be obtained about the molecular recognition process under study. Approximately one decade after its introduction, the technique has reached maturity, and is highly robust and useful. The objective of this article is to review the current status of this powerful technique, with particular emphasis on quantitative applications, within the framework of the (bio-)chemistry of molecular recognition.

Inositolphosphoglycan Mediators Structurally Related to Glycosyl Phosphatidylinositol Anchors: Synthesis, Structure and Biological Activity

The preparation of the pseudopentasaccharide 1a, an inositol-phosphoglycan (IPG) that contains the conserved linear structure of glycosyl phosphatidylinositol anchors (GPI anchors), was carried out by using a highly convergent 2+3-block synthesis approach which involves imidate and sulfoxide glycosylation reactions. The preferred solution conformation of this structure was determined by using NMR spectroscopy and molecular dynamics simulations prior to carrying out quantitative structure--activity relationship studies in connection with the insulin signalling process. The ability of 1a to stimulate lipogenesis in rat adipocytes as well as to inhibit cAMP dependent protein kinase and to activate pyruvate dehydrogenase phosphatase was investigated. Compound 1a did not show any significant activity, which may be taken as a strong indication that the GPI anchors are not the precursors of the IPG mediators.

1,2-mannobioside mimic: Synthesis, DC-SIGN interaction by NMR and docking, and antiviral activity

The design and preparation of carbohydrate ligands for DC‐SIGN is a topic of high interest because of the role played by this C‐type lectin in immunity and infection processes. The low chemical stability of carbohydrates against enzymatic hydrolysis by glycosylases has stimulated the search for new alternatives more stable in vivo. Herein, we present a good alternative for a DC‐SIGN ligand based on a mannobioside mimic with a higher enzymatic stability than the corresponding disaccharide. NMR and docking studies have been performed to study the interaction of this mimic with DC‐SIGN in solution demonstrating that this pseudomannobioside is a good ligand for this lectin. In vitro studies using an infection model with Ebola pseudotyped virus demonstrates that this compound presents an antiviral activity even better than the corresponding disaccharide and could be an interesting ligand to prepare multivalent systems with higher affinities for DC‐SIGN with potential biomedical applications.

Mutants of the Arabidopsis thaliana Cation/H+ Antiporter AtNHX1 Conferring Increased Salt Tolerance in Yeast . The Endosome/prevacuolar compartment is a target for salt toxicity

Mutants of the plant cation/H+ antiporter AtNHX1 that confer greater halotolerance were generated by random mutagenesis and selected in yeast by phenotypic complementation. The amino acid substitutions that were selected were conservative and occurred in the second half of the membrane-associated N terminus. AtNHX1 complemented the lack of endogenous ScNHX1 in endosomal protein trafficking assays. Growth enhancement on hygromycin B and vanadate media agreed with a generally improved endosomal/prevacuolar function of the mutated proteins. In vivo measurements by 31P NMR revealed that wild-type and mutant AtNHX1 transporters did not affect cytosolic or vacuolar pH. Surprisingly, when yeast cells were challenged with lithium, a tracer for sodium, the main effect of the mutations in AtNHX1 was a reduction in the amount of compartmentalized lithium. When purified and reconstituted into proteoliposomes or assayed in intact vacuoles isolated from yeast cells, a representative mutant transporter (V318I) showed a greater cation discrimination favoring potassium transport over that of sodium or lithium. Together, our data suggest that the endosome/prevacuolar compartment is a target for salt toxicity. Poisoning by toxic cations in the endosome/prevacuolar compartment is detrimental for cell functions, but it can be alleviated by improving the discrimination of transported alkali cations by the resident cation/H+ antiporter.

The Activation of Fibroblast Growth Factors (FGFs) by Glycosaminoglycans: Influence of the Sulfation Pattern on the Biological Activity of FGF-1

Six synthetic heparin‐like oligosaccharides have been used to investigate the effect of the oligosaccharide sulfation pattern on the stimulation of acidic fibroblast growth factor (FGF‐1) induced mitogenesis signaling and the biological significance of FGF‐1 trans dimerization in the FGF‐1 activation process. It has been found that some molecules with a sulfation pattern that does not contain the internal trisaccharide motif, which has been proposed for high affinity for FGF‐1, stimulate FGF‐1 more efficiently than those with the structure of the regular region of heparin. In contrast to regular region oligosaccharides, in which the sulfate groups are distributed on both sides of their helical three‐dimensional structures, the molecules containing this particular sulfation pattern display the sulfate groups only on one side of the helix. These results and the fact that these oligosaccharides do not promote FGF‐1 dimerization according to sedimentation‐equilibrium analysis, confirm the importance of negative‐charge distribution in the activation process and strongly suggest that FGF dimerization is not a general and absolute requirement for biological activity.

Nitration of tyrosine 74 prevents human cytochrome c to play a key role in apoptosis signaling by blocking caspase-9 activation

Tyrosine nitration is one of the most common post-transcriptional modifications of proteins, so affecting their structure and function. Human cytochrome c, with five tyrosine residues, is an excellent case study as it is a well-known protein playing a double physiological role in different cell compartments. On one hand, it acts as electron carrier within the mitochondrial respiratory electron transport chain, and on the other hand, it serves as a cytoplasmic apoptosis-triggering agent. In a previous paper, we reported the effect of nitration on physicochemical and kinetic features of monotyrosine cytochrome c mutants. Here, we analyse the nitration-induced changes in secondary structure, thermal stability, haem environment, alkaline transition and molecular dynamics of three of such monotyrosine mutants--the so-called h-Y67, h-Y74 and h-Y97--which have four tyrosines replaced by phenylalanines and just keep the tyrosine residue giving its number to the mutant. The resulting data, along with the functional analyses of the three mutants, indicate that it is the specific nitration of solvent-exposed Tyr74 which enhances the peroxidase activity and blocks the ability of Cc to activate caspase-9, thereby preventing the apoptosis signaling pathway.

Synthesis of Novel DC-SIGN Ligands with an α-Fucosylamide Anchor

The dendritic cell‐specific intercellular adhesion molecule (ICAM) 3‐grabbing nonintegrin (DC‐SIGN) is a C‐type lectin that appears to perform several different functions. Besides mediating adhesion between dendritic cells and T lymphocytes, DC‐SIGN recognizes several pathogens some of which, including HIV, appear to exploit it to invade host organisms. The intriguing diversity of the roles attributed to DC‐SIGN and their therapeutic implications have stimulated the search for new ligands that could be used as biological probes and possibly as lead compounds for drug development. The natural ligands of DC‐SIGN consist of mannose oligosaccharides or fucose‐containing Lewis‐type determinants. Using the known 3D structure of the Lewis‐x trisaccharide, we have identified some monovalent α‐fucosylamides that bind to DC‐SIGN with inhibitory constants 0.4–0.5 mM, as determined by SPR, and have characterized their interaction with the protein by STD NMR spectroscopy. This work establishes for the first time α‐fucosylamides as functional mimics of chemically and enzymatically unstable α‐fucosides and describes interesting candidates for the preparation of multivalent systems able to block the receptor DC‐SIGN with high affinity and with potential biomedical applications.