Development of ETB Selective Agonists: Solution Structure of a Linear Endothelin-1 Analogue,ET-1 [Cys(Acm)1,15, Ala3, Leu7, dAsp8, Aib11]

Abstract

The solution structure of a synthetic ET_B selective agonist, ET-l[Cys(Acm)^1,15, Ala³, Leu⁷, dAsp⁸, Aib¹¹] has been solved by ¹H NMR and molecular modelling studies. Such solution structures of linear modified peptides in aqueous methanol are being used in an ongoing program of research designed to assist in an understanding of the basic structural requirements for the biological activity of vasoconstrictors. The resulting structure of this peptide is characterised by an α-helical conformation between residues Leu⁶-His¹⁶ and by N- and C-termi- ni which assume no defined conformation. A knowledge of the solution structures of this and related peptides, which are ETB selective agonists, are proving to be important in the understanding of how they interact with the ETB receptor.     

13C and 1H NMR studies of ionizations and hydrogen bonding in chymotrypsin-glyoxal inhibitor complexes

Benzyloxycarbonyl (Z)-Ala-Pro-Phe-glyoxal and Z-Ala-Ala-Phe-glyoxal have both been shown to be inhibitors of alpha-chymotrypsin with minimal Ki values of 19 and 344 nM, respectively, at neutral pH. These Ki values increased at low and high pH with pKa values of approximately 4.0 and approximately 10.5, respectively. By using surface plasmon resonance, we show that the apparent association rate constant for Z-Ala-Pro-Phe-glyoxal is much lower than the value expected for a diffusion-controlled reaction. 13C NMR has been used to show that at low pH the glyoxal keto carbon is sp3-hybridized with a chemical shift of approximately 100.7 ppm and that the aldehyde carbon is hydrated with a chemical shift of approximately 91.6 ppm. The signal at approximately 100.7 ppm is assigned to the hemiketal formed between the hydroxy group of serine 195 and the keto carbon of the glyoxal. In a slow exchange process controlled by a pKa of approximately 4.5, the aldehyde carbon dehydrates to give a signal at approximately 205.5 ppm and the hemiketal forms an oxyanion at approximately 107.0 ppm. At higher pH, the re-hydration of the glyoxal aldehyde carbon leads to the signal at 107 ppm being replaced by a signal at 104 ppm (pKa approximately 9.2). On binding either Z-Ala-Pro-Phe-glyoxal or Z-Ala-Ala-Phe-glyoxal to alpha-chymotrypsin at 4 and 25 degrees C, 1H NMR is used to show that the binding of these glyoxal inhibitors raises the pKa value of the imidazolium ion of histidine 57 to a value of >11 at both 4 and 25 degrees C. We discuss the mechanistic significance of these results, and we propose that it is ligand binding that raises the pKa value of the imidazolium ring of histidine 57 allowing it to enhance the nucleophilicity of the hydroxy group of the active site serine 195 and lower the pKa value of the oxyanion forming a zwitterionic tetrahedral intermediate during catalysis.     

Conformational analysis of the broad-spectrum antibacterial peptide, ranatuerin-2CSa: identification of a full length helix-turn-helix motif

Design of clinically valuable antibacterial agents based upon naturally occurring peptides requires the use of spectroscopic methods, particularly NMR, to determine the three-dimensional structure of the native peptide so that analogues with improved therapeutic properties can be made. Ranatuerin-2CSa (GILSSFKGVAKGVAKDLAG KLLETLKCKITGC), first isolated from skin secretions of the Cascades frog, Rana cascadae, represents a promising candidate for drug development. The peptide shows potent growth inhibitory activity against Escherichia coli (MIC=5 microM) and Staphylococcus aureus (MIC=10 microM) but displays haemolytic activity against human erythrocytes (LC(50)=160 microM). The solution structure of ranatuerin-2CSa was investigated by proton NMR spectroscopy and molecular modelling. In aqueous solution, the peptide lacks secondary structure but, in a 2,2,2-trifluoroethanol (TFE-d(3))-H(2)O solvent mixture, the structure is characterised by a full length helix-turn-helix conformation between residues I(2)-L(21), L(22)-L(25) and K(26)-T(30) respectively. This structural information will facilitate the design of novel therapeutic agents based upon the ranatuerin-2CSa structure with improved antimicrobial potencies but decreased cytolytic activities against mammalian cells.     

Conformational analysis of an EP24.15 inhibitor by NMR and molecular modelling

The enzyme thimet oligopeptidase (EC3.4.24.15, EP24.15) is responsible for the hydrolysis of a number of neuropeptides. Despite much research examining its substrate specificity, little is known about the conformational requirements of its active site. We have used 1D ¹H and 2D TOCSY NMR experiments to assign the proton resonances of the EP24.15 inhibitor, N-[1-(R,S)-carboxy-3-phenylpropyl]-Ala-Ala-Tyr-p-aminobenzoate (cFP), and 2D ROESY NMR to investigate whether cFP exhibits any conformational preferences in CD₃OD and in aqueous CD₃OD. Molecular modelling of charged cFP in the gaseous phase generated a number of conformations that were consistent with the NMR data obtained in CD₃OD. Analogous modelling on the uncharged cFP did not result in conformations consistent with any of the NMR data, but did suggest that, under non-polar conditions, cFP could adopt a hairpin conformation which would allow simultaneous coordination of the two carboxyl groups of cFP to the zinc ion in the active site of EP24.15.     

Structural and positional studies of the antimicrobial peptide brevinin‐1BYa in membrane‐mimetic environments

Brevinin‐1BYa (FLPILASLAAKFGPKLFCLVTKKC), first isolated from skin secretions of the foothill yellow‐legged frog Rana boylii, shows broad‐spectrum activity, being particularly effective against opportunistic yeast pathogens. The structure of brevinin‐1BYa was investigated in various solution and membrane‐mimicking environments by proton nuclear magnetic resonance (¹H‐NMR) spectroscopy and molecular modelling. The peptide does not possess a secondary structure in aqueous solution. In a 33% 2,2,2‐trifluoroethanol (TFE‐d₃)‐H₂O solvent mixture, as well as in membrane‐mimicking sodium dodecyl sulfate and dodecylphosphocholine micelles, the peptide's structure is characterised by a flexible helix‐hinge‐helix motif, with the hinge located at the Gly¹³/Pro¹⁴ residues, and the two α‐helices extending from Pro³ to Phe¹² and from Pro¹⁴ to Thr²¹. Positional studies involving the peptide in sodium dodecyl sulfate and dodecylphosphocholine micelles using 5‐doxyl‐labelled stearic acid and manganese chloride paramagnetic probes show that the peptide's helical segments lie parallel to the micellar surface, with the residues on the hydrophobic face of the amphipathic helices facing towards the micelle core and the hydrophilic residues pointing outwards, suggesting that the peptide exerts its biological activity by a non–pore‐forming mechanism.     

Development of potent anti-infective agents from Silurana tropicalis: Conformational analysis of the amphipathic,alpha-helical antimicrobial peptide XT-7 and its non-haemolytic analogue [G4K]XT-7

Peptide XT-7 (GLLGP⁵LLKIA¹⁰AKVGS¹⁵NLL.NH₂) is a cationic, leucine-rich peptide, first isolated from skin secretions of the frog, Silurana tropicalis (Pipidae). The peptide shows potent, broad-spectrum antimicrobial activity but its therapeutic potential is limited by haemolytic activity (LC₅₀ = 140 µM). The analogue [G4K]XT-7, however, retains potent antimicrobial activity but is non-haemolytic (LC₅₀ > 500 µM). In order to elucidate the molecular basis for this difference in properties, the three dimensional structures of XT-7 and the analogue have been investigated by proton NMR spectroscopy and molecular modelling. In aqueous solution, both peptides lack secondary structure. In a 2,2,2-trifluoroethanol (TFE-d₃)-H₂O mixed solvent system, XT-7 is characterised by a right handed α-helical conformation between residues Leu³ and Leu¹⁷ whereas [G4K]XT-7 adopts a more restricted α-helical conformation between residues Leu⁶ and Leu¹⁷. A similar conformation for XT-7 in 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC) micellular media was observed with a helical segment between Leu³ and Leu¹⁷. However, differences in side chain orientations restricting the hydrophilic residues to a smaller patch resulted in an increased hydrophobic surface relative to the conformation in TFE-H₂O. Molecular modelling of the structures obtained in our study demonstrates the amphipathic character of the helical segments. It is proposed that the marked decrease in haemolytic activity produced by the substitution Gly⁴ → Lys in XT-7 arises from a decrease in both helicity and hydrophobicity. These studies may facilitate the development of potent but non-toxic anti-infective agents based upon the structure of XT-7.     

An NMR study of alterations in [1-13C]glucose metabolism in C6 glioma cells by gliotoxic amino acids

A series of glutamate analogues, known as gliotoxins, are toxic to astrocytes in culture, and are inhibitors or substrates of high affinity sodium-dependent glutamate transporters. The mechanisms by which these gliotoxins cause toxicity are not fully understood. The effects of a series of gliotoxic amino acids (L-alpha-aminoadipate, L-serine-O-sulphate, D-aspartate and L-cysteate) on metabolism of [1-13C]glucose were examined in C6 glioma cells using 13C nuclear magnetic resonance (NMR) spectroscopy. The cells were preincubated in the presence of sub toxic concentrations of each gliotoxin (400 micromol/l) for 20 h. This was followed by incubation (4 h) with [1-13C]glucose (5.5 mmol/l) in the presence and absence of each gliotoxin. The incorporation of 13C label into the observed metabolites was analysed. Following preincubation with L-alpha-aminoadipate, D-aspartate, and L-serine-O-sulphate there was a significant decrease in the incorporation of 13C label into glutamate, alanine and lactate from [1-13C]glucose. In the presence of L-cysteate production of labelled glutamate was decreased, while there was no significant effect on the concentrations of labelled lactate and alanine. There was no change in the quantity of LDH released into the medium after incubation of the cells with any of the gliotoxins. Overall these results indicate that the presence of gliotoxins profoundly alters the flux of glucose to lactate, alanine, aspartate and glutamate.     

RGMa Regulates Cortical Interneuron Migration and Differentiation

The etiology of neuropsychiatric disorders, including schizophrenia and autism, has been linked to a failure to establish the intricate neural network comprising excitatory pyramidal and inhibitory interneurons during neocortex development. A large proportion of cortical inhibitory interneurons originate in the medial ganglionic eminence (MGE) of the ventral telencephalon and then migrate through the ventral subventricular zone, across the corticostriatal junction, into the embryonic cortex. Successful navigation of newborn interneurons through the complex environment of the ventral telencephalon is governed by spatiotemporally restricted deployment of both chemorepulsive and chemoattractive guidance cues which work in concert to create a migratory corridor. Despite the expanding list of interneuron guidance cues, cues responsible for preventing interneurons from re-entering the ventricular zone of the ganglionic eminences have not been well characterized. Here we provide evidence that the chemorepulsive axon guidance cue, RGMa (Repulsive Guidance Molecule a), may fulfill this function. The ventricular zone restricted expression of RGMa in the ganglionic eminences and the presence of its receptor, Neogenin, in the ventricular zone and on newborn and maturing MGE-derived interneurons implicates RGMa-Neogenin interactions in interneuron differentiation and migration. Using an in vitro approach, we show that RGMa promotes interneuron differentiation by potentiating neurite outgrowth. In addition, using in vitro explant and migration assays, we provide evidence that RGMa is a repulsive guidance cue for newborn interneurons migrating out of the ganglionic eminence ventricular zone. Intriguingly, the alternative Neogenin ligand, Netrin-1, had no effect on migration. However, we observed complete abrogation of RGMa-induced chemorepulsion when newborn interneurons were simultaneously exposed to RGMa and Netrin-1 gradients, suggesting a novel mechanism for the tight regulation of RGMa-guided interneuron migration. We propose that during peak neurogenesis, repulsive RGMa-Neogenin interactions drive interneurons into the migratory corridor and prevent re-entry into the ventricular zone of the ganglionic eminences.