NMR structure of neuromedin C, a neurotransmitter with an amino terminal CuII-, NiII-binding (ATCUN) motif

The structure of neuromedin C, a 10-residue bombesin-like neuropeptide with the sequence Gly-Asn-His-Trp-Ala-Val-Gly-His-Leu-Met-NH2, has been investigated. Like human serum albumin, neuromedin C contains the amino-terminal CuII-, NiII-binding (ATCUN) motif which has high affinity for CuII and NiII. The solution structure of the NiII-peptide complex has been calculated based on 2D ROESY data obtained at 25 degrees C, using a hybrid distance geometry-simulated annealing approach. Comparison of 1H, 13C and 15N chemical shifts and ROESY data in the presence and absence of NiII demonstrates that the metal binds at the N-terminus of the peptide, leading to a conformational change. The metal complex adopts a conformation comprising two connected turns including residues 1Gly to 3His and 5Ala to 8His. The first turn corresponds to the NiII coordination ligands in a square planar conformation, and the second reflects the interaction between 4Trp and 8His. The results may have important physiological implications in the phenomenon of neurotransmission.     

Development of a three-dimensional CysLT1 (LTD4) antagonist model with an incorporated amino acid residue from the receptor

This paper describes the molecular modeling of leukotriene CysLT1 (or LTD4) receptor antagonists. Several different structural classes of CysLT1 antagonists were superimposed onto the new and highly rigid CysLT1 antagonist 8-carboxy-3'-[2-(2-quinolinyl)ethenyl]flavone (1, VUF 5017) to generate a common pharmacophoric arrangement. On the basis of known structure-activity relationships of CysLT1 antagonists, the quinoline nitrogen (or a bioisosteric equivalent thereof) and an acidic function were taken as the matching points. In order to optimize the fitting of acidic moieties of all antagonists, an arginine residue from the receptor was proposed as the interaction site for the acidic moieties. Incorporation of this amino acid residue into the model revealed additional interactions between the guanidine group and the nitrogen atoms of quinoline-containing CysLT1 antagonists. In some cases, the arginine may even interact with pi-clouds of phenyl residues of CysLT1 antagonists. The alignment of Montelukast (MK-476) suggests the presence of an additional pocket in the binding site for CysLT1 antagonists. The derived model should be useful for a better understanding of the molecular recognition of the leukotriene CysLT1 receptor.     

Accommodation of insertions in helices: the mutation in hemoglobin Catonsville (Pro 37 alpha-Glu-Thr 38 alpha) generates a 3(10)-->alpha bulge

Hemoglobin Catonsville is a mutation of human hemoglobin (an alpha 2 beta 2 tetramer) in which a glutamate residue is inserted into the first turn of a highly conserved 3(10) helix (the C helix) of each alpha subunit. In theory, amino acid insertions (or deletions) in protein helices can be accommodated via two distinct mechanisms. One, termed the register shift mechanism, preserves the geometry of the helix while requiring all of the residues on one flank of the insertion site to rotate by 100 degrees in the case of an alpha helix or by 120 degrees in the case of a 3(10) helix. The other, termed the bulge (or indentation) mechanism, distorts the local geometry of the helix but does not alter the helix register. High-resolution X-ray diffraction analysis of deoxyhemoglobin Catonsville shows that the inserted residue is accommodated as a bulge, demonstrating that this is a viable mechanism. (In contrast, no such evidence is yet available for the register shift mechanism.) More specifically, the insertion converts one turn of the C helix from 3(10) geometry to alpha helix-like geometry, raising the possibility that a common mechanism for accommodating insertions and deletions within helices may involve localized interconversions between 3(10), alpha, and pi helical structures.     

Thermodynamic study of Fe2O3-Fe2(SO4)3 equilibrium using an oxyanionic electrolyte (Na2SO4-I)

The emf of the cell, Pt, Ar + O₂ + SO₂ + SO₃/Na₂SO₄-I/Fe₂O₂ + Fe₂(SO₄)₃, Pt, has been measured in the temperature range 800 to 1000 K, using a gas mixture of known input composition as the reference electrode. The equilibrium composition of the reference gas at the measuring temperatures was computed using the thermodynamic data on the gaseous species reported in the literature. A mixture of ferric oxide and sulfate was kept in a closed system to ensure establishment of equilibrium partial pressure at the electrode. The cell was designed to avoid physical contact between Fe₂(SO₄)₃ and Na₂SO₄ electrolyte. Uncertainties arising from the formation of sulfate solid solution were thus eliminated. The Gibbs’ energy of formation of ferric sulfate calculated from the emf is discussed in comparison with data reported in the literature. There is no evidence for the formation of oxysulfates in the Fe-S-0 system. Based on the results obtained in the present study for Fe₂(SO₄)₃ and literature data for other phases, chemical potential diagrams have been constructed for the Fe-S-O system at 900 and 1100 K.     

Excitatory amino acid receptor ligands: resolution, absolute stereochemistry, and enantiopharmacology of 2-amino-3-(4-butyl-3-hydroxyisoxazol-5-yl)propionic acid

(RS)-2-Amino-3-(4-butyl-3-hydroxyisoxazol-5-yl)propionic acid (Bu-HIBO, 6) has previously been shown to be an agonist at (RS)-2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propionic acid (AMPA) receptors and an inhibitor of CaCl2-dependent [3H]-(S)-glutamic acid binding (J. Med. Chem. 1992, 35, 3512-3519). To elucidate the pharmacological significance of this latter binding affinity, which is also shown by quisqualic acid (3) but not by AMPA, we have now resolved Bu-HIBO via diastereomeric salt formation using the diprotected Bu-HIBO derivative 11 and the enantiomers of 1-phenylethylamine (PEA). The absolute stereochemistry of (S)-Bu-HIBO (7) (ee = 99.0%) and (R)-Bu-HIBO (8) (ee > 99.6%) were established by an X-ray crystallographic analysis of compound 15, a salt of (R)-PEA, and diprotected 8. Circular dichroism spectra of 7 and 8 were recorded. Whereas 7 (IC50 = 0.64 microM) and 8 (IC50 = 0.57 microM) were equipotent as inhibitors of CaCl2-dependent [3H]-(S)-glutamic acid binding, neither enantiomer showed significant affinity for the synaptosomal (S)-glutamic acid uptake system(s). AMPA receptor affinity (IC50 = 0.48 microM) and agonism (EC50 = 17 microM) were shown to reside exclusively in the S-enantiomer, 7. Compounds 7 and 8 did not interact detectably with kainic acid or N-methyl-D-aspartic acid (NMDA) receptor sites. Neither 7 nor 8 affected the function of the metabotropic (S)-glutamic acid receptors mGlu2 and mGlu4a, expressed in CHO cells. Compound 8 was shown also to be inactive at mGlu1 alpha, whereas 7 was determined to be a moderately potent antagonist at mGlu1 alpha (Ki = 110 microM) and mGlu5a (Ki = 97 microM). Using the rat cortical wedge preparation, the AMPA receptor agonist effect of 7 was markedly potentiated by coadministration of 8 at 21 degrees C, but not at 2-4 degrees C. These observations together indicate that the potentiation of the AMPA receptor agonism of 7 by 8 is not mediated by metabotropic (S)-glutamate receptors but rather by the CaCl2-dependent (S)-glutamic acid binding system, which shows the characteristics of a transport mechanism. After intravenous administration in mice, 7 (ED50 = 44 mumol/kg) was slightly more potent than AMPA (1) (ED50 = 55 mumol/kg) and twice as potent as Bu-HIBO (6) (ED50 = 94 mumol/kg) as a convulsant, whereas 8 was inactive. After subcutaneous administration in mice, Bu-HIBO (ED50 = 110 mumol/kg) was twice as potent as AMPA (ED50 = 220 mumol/kg) as a convulsant. Since 7 and Bu-HIBO (EC50 = 37 microM) are much weaker than AMPA (EC50 = 3.5 microM) as AMPA receptor agonists in vitro, the presence of a butyl group in the molecules of Bu-HIBO and 7 seems to facilitate the penetration of these compounds through the blood-brain barrier.     

Designer DNA-binding drugs: the crystal structure of a meta-hydroxy analogue of Hoechst 33258 bound to d(CGCGAATTCGCG)2

An analogue of the DNA binding compound Hoechst 33258, which has the para hydroxyl group altered to be at the meta position, together with the replacement of one benzimidazole group by pyridylimidazole, has been cocrystallized with the dodecanucleotide sequence d(CGCGAATTCGCG)2. The X-ray structure has been determined at 2.2 A resolution and refined to an R factor of 20.1%. The ligand binds in the minor groove at the sequence 5'-AATTC with the bulky piperazine group extending over the CxG base pair. This binding is stabilised by hydrogen bonding and numerous close van der Waals contacts to the surface of the groove walls. The meta-hydroxyl group was found in two distinct orientations, neither of which participates in direct hydrogen bonds to the exocyclic amino group of a guanine base. The conformation of the drug differs from that found previously in other X-ray structures of Hoechst 33258-DNA complexes. There is significant variation between the minor groove widths in the complexes of Hoechst 33258 and the meta-hydroxyl derivative as a result of these conformational differences. Reasons are discussed for the inability of this derivative to actively recognise guanine.     

Excitatory amino acid receptor antagonists: resolution, absolute stereochemistry, and pharmacology of (S)- and (R)-2-amino-2-(5-tert-butyl-3-hydroxyisoxazol-4-yl)acetic acid (ATAA)

We have previously shown that (RS)-2-amino-2-(5-tert-butyl-3-hydroxyisoxazol-4-yl)acetic acid (ATAA) is an antagonist at N-methyl-D-aspartic acid (NMDA) and (RS)-2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propionic acid (AMPA) receptors. We have now resolved ATAA via diastereomeric salt formation using N-BOC protected ATAA and (R)- and (S)-phenylethylamine. Enantiomeric purities (ee > 98%) of (R)- and (S)-ATAA were determined using the Crownpak CR(-) and CR(+) columns, respectively. The absolute configuration of (R)-ATAA was established by an X-ray crystallographic analysis of the (R)-phenylethylamine salt of N-BOC-(R)-ATAA. Like ATAA, neither (R)- nor (S)-ATAA significantly affected (IC50 > 100 microM) the receptor binding of tritiated AMPA, kainic acid, or (RS)-3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid, the latter being a competitive NMDA antagonist. Electrophysiological experiments, using the rat cortical wedge preparation, showed the NMDA antagonist effect as well as the AMPA antagonist effect of ATAA to reside exclusively in the (R)-enantiomer (Ki = 75 +/- 5 microM and 57 +/- 1 microM, respectively). Neither (R)- nor (S)-ATAA significantly reduced kainic acid-induced excitation (Ki > 1,000 microM).     

Antagonist properties of a phosphono isoxazole amino acid at glutamate R1-4 (R,S)-2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid receptor subtypes

All three subtypes of beta-adrenoceptors are coupled to stimulation of adenylyl cyclase activity via the stimulatory guanine-nucleotide-binding protein. Nevertheless, the beta3 adrenoceptor (beta3-AR) differs significantly from the other subtypes in terms of pharmacology. Most strikingly, it recognizes as agonists several compounds acting as potent beta1-AR and beta2-AR antagonists. Furthermore, the human beta3-AR is quite different from the animal beta3-AR. Molecular modelling studies followed by site-directed mutagenesis was used here to identify some of the amino acid residues which may be implicated in ligand binding and signal transduction of the beta3-AR. Three contiguous residues, valine-leucine-alanine, which are present in the first transmembrane domain at positions 48-50 of the human receptor but are absent in all known rodent sequences, were thought to be important for species specificity. When these three residues were deleted from the human receptor, no 'rodent-like' pharmacological profile was obtained in terms of either binding or adenylyl cyclase activation. Glycine at position 53, also in the first transmembrane domain in the human beta3-AR, has been suggested to participate in beta2-/beta3-AR subtype selectivity. Replacement of this glycine residue by phenylalanine, which is the residue present at the homologous position in the human beta2-AR, left the beta3-AR pharmacological profile unaltered in terms of specificity and selectivity. Aspartate residue 117, in the third transmembrane domain, has been found to be essential for ligand binding and consequently adenylyl cyclase activation in several bioamine receptors. When this residue was replaced by a leucine residue in the beta3-AR, ligand binding and signal transduction were suppressed. Finally, replacement of asparagine at position 312 in the sixth transmembrane domain by an alanine residue, led to alterations in the signal-transduction pathway.     

Synthesis of an unsaturated fatty acid analogue (18-(4''-azido-2''-hydroxybenzoylamino)-oleic acid) and its interaction with lysophosphatidylcholine: acyl-CoA-O-acyltransferase

Acylation/deacylation reactions represent a basic requirement of triglyceride as well as phospholipid metabolism, and maintenance of membrane lipid composition. In order to examine enzymes participating in these pathways, we synthesized 18-(4'-azido-2'-hydroxybenzoylamino)-oleic acid, an iodinable photoaffinity analogue of oleic acid as a new tool for analyzing enzymes, especially those binding unsaturated fatty acids or acyl-CoAs. For the synthesis of omega-amino-oleic acid, coupling two bifunctional Cg-components was used. The described synthesis scheme is also suited for the specific generation of other fatty acid analogues with distinct positions of the double bond. The functionality of 18-(4'-azido-2'-hydroxybenzoylamino)-oleic acid was investigated with the enzyme lysophosphatidylcholine:acyl-CoA-O-acyltransferase (LAT) [EC 2.3.1.23], an enzyme that shows high specificity towards (poly)unsaturated fatty acyl-CoAs. It could be shown that the photolabel, esterified with coenzyme A, acts in the dark as a reversible inhibitor of the enzyme activity, but photolysis of the label results in irreversible inactivation of LAT. This inactivation could be prevented by addition of the native substrate arachidonyl-CoA during photolysis. Several proteins could be specifically visualized using the iodinated analogue. The data indicate that this new photoaffinity label may have application to identify and characterize lipid biosynthetic enzymes using unsaturated fatty acids as well as acyl-CoA binding proteins and the active site of these proteins.     

Luminescence properties of red phosphors Ca_（10）Li（PO_4）_7：Eu~（3＋）

A series of red phosphors Ca10Li (PO4)7:Eu3+ were synthesized by high temperature solid-state reaction method. Their luminescence properties were characterized by means of photoluminescence excitation and emission spectra,CIE chromaticity and quantum efficiency. Results indicated that the phosphors could be effectively excited by the near ultraviolet (NUV) light (393 nm). The main emission peaks of the phosphor were ascribed to the transition 5D0-7F2 (613 and 617 nm) of Eu3+ ion when samples were excited by...