Propofol Binding to the Resting State of the Gloeobacter violaceus Ligand-gated Ion Channel (GLIC) Induces Structural Changes in the Inter- and Intrasubunit Transmembrane Domain (TMD) Cavities

General anesthetics exert many of their CNS actions by binding to and modulating membrane-embedded pentameric ligand-gated ion channels (pLGICs). The structural mechanisms underlying how anesthetics modulate pLGIC function remain largely unknown. GLIC, a prokaryotic pLGIC homologue, is inhibited by general anesthetics, suggesting anesthetics stabilize a closed channel state, but in anesthetic-bound GLIC crystal structures the channel appears open. Here, using functional GLIC channels expressed in oocytes, we examined whether propofol induces structural rearrangements in the GLIC transmembrane domain (TMD). Residues in the GLIC TMD that frame intrasubunit and intersubunit water-accessible cavities were individually mutated to cysteine. We measured and compared the rates of modification of the introduced cysteines by sulfhydryl-reactive reagents in the absence and presence of propofol. Propofol slowed the rate of modification of L240C (intersubunit) and increased the rate of modification of T254C (intrasubunit), indicating that propofol binding induces structural rearrangements in these cavities that alter the local environment near these residues. Propofol acceleration of T254C modification suggests that in the resting state propofol does not bind in the TMD intrasubunit cavity as observed in the crystal structure of GLIC with bound propofol (Nury, H., Van Renterghem, C., Weng, Y., Tran, A., Baaden, M., Dufresne, V., Changeux, J. P., Sonner, J. M., Delarue, M., and Corringer, P. J. (2011) Nature 469, 428–431). In silico docking using a GLIC closed channel homology model suggests propofol binds to intersubunit sites in the TMD in the resting state. Propofol-induced motions in the intersubunit cavity were distinct from motions associated with channel activation, indicating propofol stabilizes a novel closed state.     

Synthesis of 2-,4,-6-, and/or 7-substituted quinoline derivatives as human dihydroorotate dehydrogenase (hDHODH) inhibitors and anticancer agents: 3D QSAR-assisted design

Following our research for human dihydroorotate dehydrogenase (hDHODH) inhibitors as anticancer agents, herein we describe 3D QSAR-based design, synthesis and in vitro screening of 2-,4,-6-, and/or 7-substituted quinoline derivatives as hDHODH inhibitors and anticancer agents. We have designed 2-,4,-6-, and/or 7-substituted quinoline derivatives and predicted their hDHODH inhibitory activity based on 3D QSAR study on 45 substituted quinoline derivatives as hDHODH inhibitors, and also predicted toxicity. Designed compounds were docked into the binding site of hDHODH. Designed compounds which showed good predictive activity, no toxicity, and good docking score were selected for the synthesis, and in vitro screening as hDHODH inhibitors in an enzyme inhibition assay, and anticancer agents in MTT assay against cancer cell lines (HT-29 and MDA-MB-231). Synthesized compounds 7 and 14 demonstrated IC₅₀ value of 1.56?µM and 1.22?µM, against hDHODH, respectively, and these are our lead compounds for the development of new hDHODH inhibitors and anticancer agents.     

2-Amino-5-substituted-1,3,4-oxadiazole as chemosensor for Ni(II) ion detection: antifungal,antioxidant, DNA binding,and molecular docking studies

An oxadiazole derivative 2 was prepared by condensation reaction through cyclization of semicarbazone in the presence of bromine; the structural confirmation was supported by ¹H and ¹³C nuclear magnetic resonance (NMR) spectroscopy, Fourier transform-infrared spectroscopy, and liquid chromatography-mass spectrometry. Its sensing ability towards Ni²⁺ ion was examined showing a binding constant of 1.04 × 10⁵ compared with other suitable metal cations (Ca²⁺, Co²⁺, Cr³⁺, Ag⁺, Pb²⁺, Fe³⁺, Mg²⁺, and K⁺) using ultraviolet–visible (UV–vis) and fluorescence spectroscopic studies. The minimum concentration of Ni²⁺ ions and limit of detection was found to be 9.4 μM. A job's plot gave the binding stoichiometry ratio of oxadiazole derivative 2 vs Ni²⁺ ions as 2:1. Furthermore, the intercalative binding mode of oxadiazole derivative 2 with calf thymus DNA was supported by ultraviolet–visible (UV–vis) and fluorescent light, viscosity, cyclic voltammetry, time-resolved fluorescence, and circular dichroism measurements. The molecular docking result gave the binding score for oxadiazole derivative 2 as −6.5 kcal/mol, which further confirmed the intercalative interaction. In addition, the antifungal activity of oxadiazole derivative 2 was also screened against several fungal strains (C. albicans, C. glabrata, and C. tropicalis) by broth dilution and disc diffusion methods. In antioxidant studies, the oxadiazole derivative 2 showed potential scavenging activity against 2,2-diphenyl-1-picrylhydrazyl and H₂O₂ free radicals.     

Synthesis and anticonvulsant activity of new 2-substituted-5- [2-(2-fluorophenoxy)phenyl]-1,3,4-oxadiazoles and 1,2,4-triazoles

A series of new 2-substituted-5-[2-(2-fluorophenoxy)phenyl]-1,3,4-oxadiazoles has been synthesized and screened for their anticonvulsant activities. Compound 3 shows considerable anticonvulsant activity both in PTZ and MES models. It seems that this effect is mediated by benzodiazepine receptors and other unknown mechanism, respectively.     

Genetic selection for a highly functional cysteine-less membrane protein using site saturation mutagenesis

We describe an efficient method for generating highly functional membrane proteins with variant amino acids at defined positions that couples a modified site saturation strategy with functional genetic selection. We applied this method to the production of a cysteine-less variant of the Crithidia fasciculata inosine-guanosine permease CfNT2 to facilitate biochemical studies using thiol-specific modifying reagents. Of 10 endogenous cysteine residues in CfNT2, two cannot be replaced with serine or alanine without loss of function. High-quality single- and double-mutant libraries were produced by combining a previously reported site saturation mutagenesis scheme based on the Stratagene Quikchange method with a novel gel purification step that effectively eliminated template DNA from the products. Following selection for functional complementation in Saccharomyces cerevisiae cells auxotrophic for purines, several highly functional noncysteine substitutions were efficiently identified at each desired position, allowing the construction of cysteine-less variants of CfNT2 that retained wild-type affinity for inosine. This combination of an improved site saturation mutagenesis technique and positive genetic selection provides a simple and efficient means to identify functional and perhaps unexpected amino acid variants at a desired position.     

Anti-malarial activity of Baylis-Hillman adducts from substituted 2-chloronicotinaldehydes

New Baylis–Hillman adducts are synthesized based on substituted 2-chloronicotinaldehydes and screened for their in vitro anti-malarial activity against chloroquine sensitive and chloroquine resistant Plasmodium falciparum. Out of the six new compounds synthesized and screened, 2b, 2c and 2d compounds showed substantial anti-malarial activity.     

[S2], [S3], and [N3] coordination modes of hydrotris(thioxotriazolyl)borato. Zinc, nickel, cobalt, and bismuth complexes

The ligand hydrotris(1,4-dihydro-3-methyl-4-phenyl-5-thioxo-1,2,4-triazolyl)borato (Tr^Ph,Me) was synthetized as natrium salt and the complexes [Zn(Tr^Ph,Me)₂] · 7.5H₂O · 1.5CH₃CN (2a), [Zn(Tr^Ph,Me)₂] · 8DMF (2b), [Co(Tr^Ph,Me)₂] · 8DMF (3a), [Ni(Tr^Ph,Me)₂] · H₂O · 6DMSO (4a), [Bi(Tr^Ph,Me)₂]NO₃ (5), have been isolated and structurally characterized by X-ray diffraction. In the zinc derivatives the ligand adopts different denticity and coordination modes, η² and [S₂] for 2a and η³ and [N₃] for 2b, depending on the crystallization solvent, giving rise to tetrahedral and octahedral geometry, respectively. In the octahedral cobalt and nickel complexes the ligand is η³ and [N₃] coordinated whereas in the bismuth complex the η³ and [S₃] coordination is exhibited.     

Three ion-pair complexes containing bis(maleonitriledithiolate)copper(II) anion and substituted 4-dimethylaminopyridinium: Syntheses, crystal structures, and magnetic properties

Three new ion-pair complexes, [4RBzDMAP]₂[Cu(mnt)₂] (mnt²⁻ = maleonitriledithiolate; [4RBzDMAP]⁺ = 1-(4′-R-benzyl)-4-dimethylaminopyridinium, R = F(1), Cl(2) and Br(3)) were synthesized and characterized by elemental analyses, IR, UV, single crystal X-ray diffraction and magnetic measurements. The [Cu(mnt)₂]²⁻ anions and the cations stack alternately and form a 1D column via C-H···S, C-H···π or C-H···Cu interactions for 1 and 2. While the cations stack into a column though π···π or C-H···π interactions between pyridine and phenyl rings for 1 and 3. The change of the molecular topology of the counteraction when the 4-substituted group in the benzyl ring have been changed from F or Cl to Br atom, results in the difference in the crystal system, space group and the stacking mode of the cations and anions of 1, 2 and 3. Some weak hydrogen bonds between the adjacent columns further generate a 3D network structure. It is interesting that 1 and 2 exhibits antiferromagnetic coupling with θ = −2.372 K and θ = −14.732 K, while 3 shows weak ferromagnetic coupling feature with θ = 0.381 K.     

The role of non-classical supramolecular interactions in the structures of 2-amino-4,6-dimethylpyridinium tetrahalocuprate (II) salts

The compounds [2-amino-4,6-dimethylpyridinium]₂CuCl₄ (1) and [2-amino-4,6-dimethylpyridinium]₂CuBr₄ (2) were prepared from acidic ethanolic media containing CuX₂ and [2-amino-4,6-dimethylpyridine] in the molar ratio 1:1. The compounds were characterized by IR and single-crystal X-ray diffraction and found to be isomorphous in the space group with V = 991.2(10) Å³ for (1) and 1059.26(12) Å³ for (2) . There is no significant difference in the non-classical N-H?X hydrogen bonding between (1) and (2). The anions show essentially the same extent of distortion from tetrahedral geometry with max./min. values for the X-Cu-X bond angles of 139.72(6)°/96.78(6)° for (1) and 139.43(4)°/96.64(3)° for (2). Each [CuX₄]²⁻ anion is hydrogen bonded nonsymmetrically to four cations. In this manner, ladder chains are formed that run along the b-axis, with planar cations falling parallel to the (2, 0, 1) plane. Weaker π-π interactions exist between cations from different chains with centroid to centroid distance of 4.07 Å in (1) and a long 4.594 Å in (2). The X-π electrostatic interactions are surprisingly stronger in (2) than in (1) with a Br to centroid of pyridinic ring distance of 3.890 Å compared with 3.996 Å for the chloride analogue.     

Hepatic chemoprotective enzyme responses to 2-substituted selenazolidine-4(R)-carboxylic acids

In epidemiology and human supplementation studies, as well as many animal models, selenium has shown antitumorigenic activity. The mechanism of action, however, has not been satisfactorily resolved. Selenium supplementation affects many enzymes in addition to those where selenocysteine is an essential component. Such enzymes include cytoprotective detoxifying enzymes, and the regulation of these enzymes by a set of 2-substituted selenazolidine-4(R)-carboxylic acids (SCAs) has been investigated. Following seven consecutive daily doses of these prodrugs of L-selenocysteine, changes in hepatic enzyme activities and/or mRNA levels of glutathione transferase (GST), microsomal epoxide hydrolase (mEH), NAD(P)H-quinone oxidoreductase (NQO), UDP-glucuronosyltransferase (UGT), glutathione peroxidase (GPx), and thioredoxin reductase (TR) have been observed. Among the enzymes examined, UGTs and GPx were found to be the least affected. Among the compounds, 2-oxoSCA produced the most changes and 2-phenylSCA produced the least, none. For no two compounds was the pattern of changes identical, and for a single compound, few changes were reproduced in common by the two routes of administration investigated. In general, more changes were elicited following intraperitoneal (i.p.) administration than with the intragastric (i.g.) route. This dominance was typified by 2-butylSCA and 2-cyclohexylSCA where enzyme activity elevations (TR and mEH with both, NQO with 2-butylSCA) were seen only with the i.p. route. With 2-oxoSCA, however, GST, TR, and NQO activities were found to be elevated independent of route. Only with GST (both routes) and TR (i.p. route), elevations in mRNAs accompanied the 2-oxoSCA elicited elevations of activities at the time of sacrifice. For some enzymes, most notably mEH with compounds administered i.p., elevations in mRNAs were not manifest as increased enzyme activity. Thus, although constituting a closely related series of compounds, each 2-substituted SCA produced its own unique pattern of changes, and for most members, changes were predominant following i.p. administration.