Structural requirements of cholenamide derivatives as the LXR ligands.

A study of the structural requirements of cholic acid derivatives as liver X receptor (LXR) ligands was performed. A model of cholenamide derivative 1 complexed with LXR showed that the C24 carbonyl oxygen forms a hydrogen bond with His435 located close to Trp457. The N,N-dimethyl group is located in a hydrophobic pocket. Based on these data, we designed compounds with high affinity for LXRs. Cholenamide derivatives 1-11 were synthesized from 3ß-acetyl-Δ5-cholenic acid 20, and lactams 12-19 were synthesized from alcohol 25. Tertiary amides 3 and 4 showed higher activity in reporter assays, and compounds with hydrophobic residues exhibited the highest activity of all derivatives. The stereochemistry at C23 was found to be an important determinant of EC50 and gene transactivation, as each isomer exhibited different activity.

Synthesis of 5a,5a'-dicarba-d-glucobioses from conformationally restricted carbaglucosyl triflates using SN2-type inversion with carbaglucosyl nucleophiles.

Novel carbohydrate mimics were designed which contain two 5a-carba-d-glucose residues, one each at reducing and nonreducing end, and thus these mimics are 5a,5a'-dicarba-d-glucobioses. Dicarbadisaccharides have attractive features such as stability against endogenous degradative enzymes and being resistant to glycation reactions such as the Maillard reaction. For the synthesis of dicarba-ß-d-isomaltose derivatives, the carbaglucosyl triflate locked in 4C1 conformation was synthesized by protecting with butane-2,3-diacetal group or benzylidene group. Then, 5a,5a'-dicarba-ß-d-maltose and 5a,5a'-dicarba-α,ß-d-trehalose were synthesized by the SN2-type inversion reaction using 4,6-O-benzylidene carbaglucosyl triflate with 4-OH and 1-OH carba-ß-d-glucose derivatives, respectively, and similarly 5a,5a'-dicarba-α-d-isomaltose with 6-OH carba-α-d-glucose derivative.

Functional differentiation of the glycosyltransferases that contribute to the chemical diversity of bioactive flavonol glycosides in grapevines (Vitis vinifera).

We identified two glycosyltransferases that contribute to the structural diversification of flavonol glycosides in grapevine (Vitis vinifera): glycosyltransferase 5 (Vv GT5) and Vv GT6. Biochemical analyses showed that Vv GT5 is a UDP-glucuronic acid:flavonol-3-O-glucuronosyltransferase (GAT), and Vv GT6 is a bifunctional UDP-glucose/UDP-galactose:flavonol-3-O-glucosyltransferase/galactosyltransferase. The Vv GT5 and Vv GT6 genes have very high sequence similarity (91%) and are located in tandem on chromosome 11, suggesting that one of these genes arose from the other by gene duplication. Both of these enzymes were expressed in accordance with flavonol synthase gene expression and flavonoid distribution patterns in this plant, corroborating their significance in flavonol glycoside biosynthesis. The determinant of the specificity of Vv GT5 for UDP-glucuronic acid was found to be Arg-140, which corresponded to none of the determinants previously identified for other plant GATs in primary structures, providing another example of convergent evolution of plant GAT. We also analyzed the determinants of the sugar donor specificity of Vv GT6. Gln-373 and Pro-19 were found to play important roles in the bifunctional specificity of the enzyme. The results presented here suggest that the sugar donor specificities of these Vv GTs could be determined by a limited number of amino acid substitutions in the primary structures of protein duplicates, illustrating the plasticity of plant glycosyltransferases in acquiring new sugar donor specificities.

Reminiscences of research on the chemistry and biology of natural sterols in insects, plants and humans.

Natural sterols often occur as a heterogeneous mixture of homologs, which had disturbed the progress of steroid research. Development and application of GC methodology overcame this difficulty and enabled us to obtain detailed sterol profiles. Together, fine synthesis of stereo-defined isomers and homologs of steroids having oxygenated side chains allowed us to compare them with natural samples as well as to investigate structure-activity relationship. Advance of HPLC technology also facilitated the determination of the stereochemical structure of naturally occurring steroidal compounds, which were obtained only in minute amounts. This review highlights three topics out of our steroid research that have been performed mainly at Tokyo Institute of Technology around 1970-1990. These are sterol metabolism in insects focusing on the mechanism of the conversion of plant sterols to cholesterol and ecdysone biosynthesis, the synthesis and biochemical research of active forms of vitamin D3 derivatives, and the synthesis and microanalysis of plant hormone brassinosteroids.

A silicon transporter in rice.

Silicon is beneficial to plant growth and helps plants to overcome abiotic and biotic stresses by preventing lodging (falling over) and increasing resistance to pests and diseases, as well as other stresses. Silicon is essential for high and sustainable production of rice, but the molecular mechanism responsible for the uptake of silicon is unknown. Here we describe the Low silicon rice 1 (Lsi1) gene, which controls silicon accumulation in rice, a typical silicon-accumulating plant. This gene belongs to the aquaporin family and is constitutively expressed in the roots. Lsi1 is localized on the plasma membrane of the distal side of both exodermis and endodermis cells, where casparian strips are located. Suppression of Lsi1 expression resulted in reduced silicon uptake. Furthermore, expression of Lsi1 in Xenopus oocytes showed transport activity for silicon only. The identification of a silicon transporter provides both an insight into the silicon uptake system in plants, and a new strategy for producing crops with high resistance to multiple stresses by genetic modification of the root's silicon uptake capacity.

Mechanism of inverse agonist action of sarpogrelate at the constitutively active mutant of human 5-HT2A receptor revealed by molecular modeling.

We previously reported that sarpogrelate, a selective 5-HT2A antagonist, showed a potent inverse agonist activity to constitutively active mutant (C322K) of human 5-HT2A receptor (5-HT2AR). However, it remains to be unknown about the actual mechanism of this mutant for its constitutive activation as well as inverse agonist activity of sarpogrelate. Our model shows that mutation (C322K) of 5-HT2AR causes electronic repulsion between positively charged Arg173(3.50) and Lys322(6.34) residues resulting outward movement of the C-terminus of transmembrane helix (TMH) III. This motion of TMH III leads to a partially active structure of the receptor, which may be a key step in receptor activation. The structural model of the partially active receptor also indicates that the binding of sarpogrelate to the constitutively active receptor causes an inward swing of TMH III to an inactive receptor structure. Therefore, the present study may suggest that the electronic repulsion causing outward movement of the C-terminus of TMH III may be the key step for constitutive activation of mutant C322K of 5-HT2AR and the inward movement of TMH III causes the inverse agonist activity of sarpogrelate.

Chimeric yeast G-protein α subunit harboring a 37-residue C-terminal gustducin-specific sequence is functional in Saccharomyces cerevisiae.

Despite many recent studies of G-protein-coupled receptor (GPCR) structures, it is not yet well understood how these receptors activate G proteins. The GPCR assay using baker's yeast, Saccharomyces cerevisiae, is an effective experimental model for the characterization of GPCR-Gα interactions. Here, using the yeast endogenous Gα protein (Gpa1p) as template, we constructed various chimeric Gα proteins with a region that is considered to be necessary for interaction with mammalian receptors. The signaling assay using the yeast pheromone receptor revealed that the chimeric Gα protein harboring 37 gustducin-specific amino acid residues at its C-terminus (GPA1/gust37) maintained functionality in yeast. In contrast, GPA1/gust44, a variant routinely used in mammalian experimental systems, was not functional.

Characterization of the beta-D-glucopyranoside binding site of the human bitter taste receptor hTAS2R16.

G-protein-coupled receptors mediate the senses of taste, smell, and vision in mammals. Humans recognize thousands of compounds as bitter, and this response is mediated by the hTAS2R family, which is one of the G-protein-coupled receptors composed of only 25 receptors. However, structural information on these receptors is limited. To address the molecular basis of bitter tastant discrimination by the hTAS2Rs, we performed ligand docking simulation and functional analysis using a series of point mutants of hTAS2R16 to identify its binding sites. The docking simulation predicted two candidate binding structures for a salicin-hTAS2R16 complex, and at least seven amino acid residues in transmembrane 3 (TM3), TM5, and TM6 were shown to be involved in ligand recognition. We also identified the probable salicin-hTAS2R16 binding mode using a mutated receptor experiment. This study characterizes the molecular interaction between hTAS2R16 and beta-D-glucopyranoside and will also facilitate rational design of bitter blockers.

Bulky high-mannose-type N-glycan blocks the taste-modifying activity of miraculin.

BACKGROUND: Miraculin (MCL) is a taste-modifying protein that converts sourness into sweetness. The molecular mechanism underlying the taste-modifying action of MCL is unknown. METHODS: Here, a yeast expression system for MCL was constructed to accelerate analysis of its structure-function relationships. The Saccharomyces cerevisiae expression system has advantages as a high-throughput analysis system, but compared to other hosts it is characterized by a relatively low level of recombinant protein expression. To alleviate this weakness, in this study we optimized the codon usage and signal-sequence as the first step. Recombinant MCL (rMCL) was expressed and purified, and the sensory taste was analyzed. RESULTS: As a result, a 2 mg/l yield of rMCL was successfully obtained. Although sensory taste evaluation showed that rMCL was flat in taste under all the pH conditions employed, taste-modifying activity similar to that of native MCL was recovered after deglycosylation. Mutagenetic analysis revealed that the N-glycan attached to Asn42 was bulky in rMCL. CONCLUSIONS: The high-mannose-type N-glycan attached in yeast blocks the taste-modifying activity of rMCL. GENERAL SIGNIFICANCE: The bulky N-glycan attached to Asn42 may cause steric hindrance in the interaction between active residues and the sweet taste receptor hT1R2/hT1R3.

Metastable decomposition and hydrogen migration of ethane dication produced in an intense femtosecond near-infrared laser field.

We investigated a formation channel of triatomic molecular hydrogen ions from ethane dication induced by irradiation of intense laser fields (800 nm, 100 fs, ∼1 × 10(14) W∕cm(2)) by using time of flight mass spectrometry. Hydrogen ion and molecular hydrogen ion (H,D)(n)(+) (n = 1-3) ejected from ethane dications, produced by double ionization of three types of samples, CH(3)CH(3), CD(3)CD(3), and CH(3)CD(3), were measured. All fragments were found to comprise components with a kinetic energy of ∼3.5 eV originating from a two-body Coulomb explosion of ethane dications. Based on the signal intensities and the anisotropy of the ejection direction with respect to the laser polarization direction, the branching ratios, H(+):D(+) = 66:34, H(2)(+):HD(+):D(2)(+) = 63:6:31, and H(3)(+):H(2)D(+):HD(2)(+):D(3)(+) = 26:31:34:9 for the decomposition of C(2)H(3)D(3)(2+), were determined. The ratio of hydrogen molecules, H(2):HD:D(2) = 31:48:21, was also estimated from the signal intensities of the counter ion C(2)(H,D)(4)(2+). The similarity in the extent of H∕D mixture in (H,D)(3)(+) with that of (H,D)(2) suggests that these two dissociation channels have a common precursor with the C(2)H(4)(2+)...H(2) complex structure, as proposed theoretically in the case of H(3)(+) ejection from allene dication [A. M. Mebel and A. D. Bandrauk, J. Chem. Phys. 129, 224311 (2008)]. In contrast, the (H,D)(2)(+) ejection path with a lower extent of H∕D mixture and a large anisotropy is expected to proceed essentially via a different path with a much rapid decomposition rate. For the Coulomb explosion path of C-C bond breaking, the yield ratios of two channels, CH(3)CD(3)(2+)→ CH(3)(+) + CD(3)(+) and CH(2)D(+) + CHD(2)(+), were 81:19 and 92:8 for the perpendicular and parallel directions, respectively. This indicates that the process occurs at a rapid rate, which is comparable to hydrogen migration through the C-C bond, resulting in smaller anisotropy for the latter channel that needs H∕D exchange.

Structure-activity relationships of GPR120 agonists based on a docking simulation.

GPR120 is a G protein-coupled receptor expressed preferentially in the intestinal tract and adipose tissue, that has been implicated in mediating free fatty acid-stimulated glucagon-like peptide-1 (GLP-1) secretion. To develop GPR120-specific agonists, a series of compounds (denoted as NCG compounds) derived from a peroxisome proliferator-activated receptor γ agonist were synthesized, and their structure-activity relationships as GPR120 agonists were explored. To examine the agonistic activities of these newly synthesized NCG compounds, and of compounds already shown to have GPR120 agonistic activity (grifolic acid and MEDICA16), we conducted docking simulation in a GPR120 homology model that was developed on the basis of a photoactivated model derived from the crystal structure of bovine rhodopsin. We calculated the hydrogen bonding energies between the compounds and the GPR120 model. These energies correlated well with the GPR120 agonistic activity of the compounds (R(2) = 0.73). NCG21, the NCG compound with the lowest calculated hydrogen bonding energy, showed the most potent extracellular signal-regulated kinase (ERK) activation in a cloned GPR120 system. Furthermore, NCG21 potently activated ERK, intracellular calcium responses and GLP-1 secretion in murine enteroendocrine STC-1 cells that express GPR120 endogenously. Moreover, administration of NCG21 into the mouse colon caused an increase in plasma GLP-1 levels. Taken together, our present study showed that a docking simulation using a GPR120 homology model might be useful to predict the agonistic activity of compounds.

Anti-Clostridium difficile potential of tetramic acid derivatives from Pseudomonas aeruginosa quorum-sensing autoinducers.

We have examined the potential bactericidal activities of several tetramic acids derived from Pseudomonas autoinducers against Clostridium difficile, a cause of antibiotic-associated pseudomembranous colitis. Clinical isolates of C. difficile (n=4) were incubated in broth with a chemically synthesized Pseudomonas autoinducer and its tetramic acid derivatives. The structure-activity relationship and the mechanisms of action were examined by a time-killing assay and by determination of the morphological/staining characteristics. The use of some tetramic acids derived from N-3-oxododecanoyl L-homoserine lactone resulted in more than 3-log reductions in the viability of C. difficile within 30 min at 30 microM. The outer membrane was suggested to be one of the targets for the bactericidal activity of tetramic acid, because disturbance of the bacterial outer surface was demonstrated by alteration of the Gram-staining characteristic and electron microscopy. The data for the tetramic acid derivatives demonstrate that the keto-enol structure and the length of the acyl side chain of tetramic acid may be essential for the antibacterial activity of this molecule. These results suggest the potential for tetramic acid derivatives to be novel agents with activity against C. difficile.

The human bitter taste receptor, hTAS2R16, discriminates slight differences in the configuration of disaccharides.

Sweetness and bitterness are key determinants of food acceptance and rejection, respectively. Sugars, such as sucrose and fructose, are generally recognized as sweet. However, not all sugars are sweet, and even anomers may have quite different tastes. For example, gentiobiose is bitter, whereas its anomer, isomaltose, is sweet. Despite this unique sensory character, the molecular basis of the bitterness of gentiobiose remains to be clarified. In this study, we used calcium imaging analysis of human embryonic kidney 293T cells that heterologously expressed human taste receptors to demonstrate that gentiobiose activated hTAS2R16, a bitter taste receptor, but not hT1R2/hT1R3, a sweet taste receptor. In contrast, isomaltose activated hT1R2/hT1R3. As a result, these anomers elicit different taste sensations. Mutational analysis of hTAS2R16 also indicated that gentiobiose and ß-D-glucopyranosides, such as salicin share a common binding site of hTAS2R16.

Photoactive ligands probing the sweet taste receptor. Design and synthesis of highly potent diazirinyl D-phenylalanine derivatives.

Some D-amino acids such as d-tryptophan and D-phenylalanine are well known as naturally-occurring sweeteners. Photoreactive D-phenylalanine derivatives containing trifluoromethyldiazirinyl moiety at 3- or 4-position of phenylalanine, were designed as sweeteners for functional analysis with photoaffinity labeling. The trifluoromethyldiazirinyl D-phenylalanine derivatives were prepared effectively with chemo-enzymatic methods using L-amino acid oxidase and were found to have potent activity toward the human sweet taste receptor.

Engineered mutation of some important amino acids in angiotensin II type 1 (AT1) receptor increases the binding affinity of AT1-receptor antagonists.

The present study was designed to examine the binding affinity and functional potency of selective angiotensin II type 1 (AT(1))-receptor antagonists towards specific mutants of AT(1) receptor using site-directed mutagenesis. We also compared our results with the wild-type AT(1) receptor and investigated the possible reasons behind that. Both wild-type and mutant receptors were expressed in COS-7 cells and the binding affinities of the antagonists were determined by radioligand binding assay. Inhibition of agonist-stimulated inositol phosphate accumulation by the antagonists was also done. Substitution of asparagine(235) of intracellular loop 3 of the AT(1) receptor by arginine increased the binding affinity of the antagonists 5 - 34-fold, whereas the increase in the binding affinity of the antagonists in the phenylalanine(239) mutant by arginine and tryptophan (F239R and F239W) were 3 - 19-fold and 2 - 15-fold higher, respectively, compared to the wild-type AT(1) receptor. The results suggested that substitution by a positively charged or sterically hindered amino acid in the AT(1) receptor allows it to interact with the acidic tetrazole moiety and carboxylate groups of the antagonists more strongly compared to the wild-type receptor. These findings may play an important role to change the binding affinity of the antagonists to an effective level for the pharmacological function of the drugs.

Highly fucosylated N-glycan ligands for mannan-binding protein expressed specifically on CD26 (DPPVI) isolated from a human colorectal carcinoma cell line, SW1116.

The serum mannan-binding protein (MBP) is a host defense C-type lectin specific for mannose, N-acetylglucosamine, and fucose residues, and exhibits growth inhibitory activity toward human colorectal carcinoma cells. The MBP-ligand oligosaccharides (MLO) isolated from a human colorectal carcinoma cell line, SW1116, are large, multiantennary N-glycans with highly fucosylated polylactosamine-type structures having Le(b)-Le(a) or tandem repeats of the Le(a) structure at their nonreducing ends. In this study, we isolated the major MBP-ligand glycoproteins from SW1116 cell lysates with an MBP column and identified them as CD26/dipeptidyl peptidase IV (DPPIV) (110 kDa) and CD98 heavy chain (CD98hc)/4F2hc (82 kDa). Glycosidase digestion revealed that CD26 contained such complex-type N-glycans that appear to mediate the MBP binding. MALDI-MS of the N-glycans released from CD26 by PNGase F demonstrated conclusively that CD26 is the major MLO-carrying protein. More interestingly, a comparison of the N-glycans released from the MBP-binding and non-MBP-binding glycopeptides suggested that complex-type N-glycans carrying a minimum of 4 Le(a)/Le(b) epitopes arranged either as multimeric tandem repeats or terminal epitopes on multiantennary structures are critically important for the high affinity binding to MBP. Analysis of the N-glycan attachment sites demonstrated that the high affinity MLO was expressed preferentially at some N-glycosylation sites, but this site preference was not so stringent. Finally, hypothetical 3D models of tandem repeats of the Le(a) epitope and the MBP-Lewis oligosaccharide complex were presented.

A TNF receptor loop peptide mimic blocks RANK ligand-induced signaling, bone resorption, and bone loss.

Activating receptor activator of NF-kappaB (RANK) and TNF receptor (TNFR) promote osteoclast differentiation. A critical ligand contact site on the TNFR is partly conserved in RANK. Surface plasmon resonance studies showed that a peptide (WP9QY) that mimics this TNFR contact site and inhibits TNF-alpha-induced activity bound to RANK ligand (RANKL). Changing a single residue predicted to play an important role in the interaction reduced the binding significantly. WP9QY, but not the altered control peptide, inhibited the RANKL-induced activation of RANK-dependent signaling in RAW 264.7 cells but had no effect on M-CSF-induced activation of some of the same signaling events. WP9QY but not the control peptide also prevented RANKL-induced bone resorption and osteoclastogenesis, even when TNFRs were absent or blocked. In vivo, where both RANKL and TNF-alpha promote osteoclastogenesis, osteoclast activity, and bone loss, WP9QY prevented the increased osteoclastogenesis and bone loss induced in mice by ovariectomy or low dietary calcium, in the latter case in both wild-type and TNFR double-knockout mice. These results suggest that a peptide that mimics a TNFR ligand contact site blocks bone resorption by interfering with recruitment and activation of osteoclasts by both RANKL and TNF.

Structure-activity relationship study on alpha1 adrenergic receptor antagonists from beer.

Hordatine A and aperidine have been previously isolated from beer as active ingredients, which bind to muscarinic M3 receptor. In addition, these compounds have exhibited antagonist activity against the alpha1A adrenoceptor. Although the relative structures of these two molecules have previously been determined, the absolute stereochemistry was unclear. Hence, to elucidate the absolute stereochemistry of natural hordatine A, we synthesized each enantiomer of hordatine A and aperidine from optically pure dehydrodi-p-coumaric acid. Several additional related compounds were also synthesized for structure-activity relationship studies. Chiral column HPLC analysis demonstrated that the absolute stereochemistry of natural hordatine A is (2S,3S), while based on the isomerization mechanism, the stereochemistry of aperidine is (2R,3S). The alpha1A adrenoceptor binding activity of (2R,3R)-hordatine A is the most potent among the enantiomeric pairs of hordatines and aperidines. Furthermore, the related, synthetic compound, (2R,3R)-methyl benzofurancarboxylate exhibits antagonist activity against the alpha1A adrenoceptor at a lower concentration than that of hordatine A.

Microstructure of poly(gamma-glutamic acid) produced by Bacillus subtilis consisting of clusters of D- and L-glutamic acid repeating units.

Poly(gamma-glutamic acid) (PGA) produced by a strain of Bacillus subtilis was partially hydrolyzed into various oligopeptides so that the dipeptide fraction was isolated by the preparative thin-layer chromatography. HPLC analysis was applied to the detection of each of the four stereoisomers in this fraction using chemically synthesized authentic samples. The fraction consisted of N-gamma- d-glutamyl- d-glutamic acid, N-gamma- l-glutamyl- l-glutamic acid, N-gamma- d-glutamyl- l-glutamic acid, and N-gamma- l-glutamyl- d-glutamic acid at a ratio of 5.9:6.0:1.0:1.0. On the basis of this result, a model was proposed for the microstructure of the bacterial PGA, in which d- and l-glutamic acid repeating units are alternately linked in a single chain of the molecule.

Structural modelling and mutation analysis of a nociceptin receptor and its ligand complexes.

We recently modelled and proposed four ligand-bound conformations for a G-protein-coupled receptor, namely, forms I, II, III and IV, based on the 3D structure and functional evidences for rhodopsin. In this study, the same strategy was applied to a human nociceptin receptor (NR), in order to predict ligand-bound receptor structures. Additionally, site-directed mutagenesis studies were carried out to evaluate these structures. A Thr138Ala mutant demonstrated the same affinity for [Phe(1)Psi(CH(2)-NH)Gly(2)]nociceptin(1-13)NH(2) as the wild-type receptor; however, the affinity of this mutant for nociceptin was 20-fold lower than that of the wild type. A Ser223Ala mutation showed the same characteristics as those of the wild type. On the other hand, a Gln280Ala mutation reduced the affinity to nociceptin by more than 60-folds. These results suggested that a change in the conformation of NR following agonist binding did not accompany the rigid-body rotation of the sixth transmembrane segment that was reported for an adrenergic receptor and a kappa-opioid receptor. NR is potently activated not only by nociceptin but also a synthetic peptide, i.e. Ac-RYYRIK-NH(2), although the amino acid sequences of both these ligands are completely different. The model explains why both the ligands activate NR and shows that their receptor-bound conformations have similar 3D structures.