ABSTRACT
The heme acquisition systemâ A protein secreted by Pseudomonas aeruginosa (HasA(p)) can capture several synthetic metal complexes other than heme. The crystal structures of HasA(p) harboring synthetic metal complexes revealed only small perturbation of the overall HasA(p) structure. An inhibitory effect upon heme acquisition by HasA(p) bearing synthetic metal complexes was examined by monitoring the growth of Pseudomonas aeruginosa PAO1. HasA(p) bound to iron-phthalocyanine inhibits heme acquisition in the presence of heme-bound HasA(p) as an iron source.
Subject(s)
Bacterial Proteins/metabolism , Carrier Proteins/metabolism , Coordination Complexes/metabolism , Heme/metabolism , Iron/chemistry , Pseudomonas aeruginosa/metabolism , Bacterial Proteins/chemistry , Binding Sites , Carrier Proteins/chemistry , Coordination Complexes/chemistry , Crystallography, X-Ray , Heme/chemistry , Indoles/chemistry , Indoles/metabolism , Iron/metabolism , Isoindoles , Mesoporphyrins/chemistry , Mesoporphyrins/metabolism , Molecular Dynamics Simulation , Protein Binding , Protein Structure, Tertiary , Spectrometry, Mass, Electrospray IonizationABSTRACT
Given their high neuroprotective potential, ligands that block GluN2B-containing N-methyl-D-aspartate (NMDA) receptors by interacting with the ifenprodil binding site located on the GluN2B subunit are of great interest for the treatment of various neuronal disorders. In this study, a novel class of GluN2B-selective NMDA receptor antagonists with the benzo[7]annulene scaffold was prepared and pharmacologically evaluated. The key intermediate, N-(2-methoxy-5-oxo-6,7,8,9-tetrahydro-5H-benzo[7]annulen-7-yl)acetamide (11), was obtained by cyclization of 3-acetamido-5-(3-methoxyphenyl)pentanoic acid (10 b). The final reaction steps comprise hydrolysis of the amide, reduction of the ketone, and reductive alkylation, leading to cis- and trans-configured 7-(ω-phenylalkylamino)benzo[7]annulen-5-ols. High GluN2B affinity was observed with cis-configured γ-amino alcohols substituted with a 3-phenylpropyl moiety at the amino group. Removal of the benzylic hydroxy moiety led to the most potent GluN2B antagonists of this series: 2-methoxy-N-(3-phenylpropyl)-6,7,8,9-tetrahydro-5H-benzo[7]annulen-7-amine (20 a, Ki =10 nM) and 2-methoxy-N-methyl-N-(3-phenylpropyl)-6,7,8,9-tetrahydro-5H-benzo[7]annulen-7-amine (23 a, Ki =7.9 nM). The selectivity over related receptors (phencyclidine binding site of the NMDA receptor, σ1 and σ2 receptors) was recorded. In a functional assay measuring the cytoprotective activity of the benzo[7]annulenamines, all tested compounds showed potent NMDA receptor antagonistic activity. Cytotoxicity induced via GluN2A subunit-containing NMDA receptors was not inhibited by the new ligands.
Subject(s)
Benzazepines/pharmacology , Benzocycloheptenes/pharmacology , Receptors, N-Methyl-D-Aspartate/antagonists & inhibitors , Animals , Benzazepines/chemical synthesis , Benzazepines/chemistry , Benzocycloheptenes/chemical synthesis , Benzocycloheptenes/chemistry , Cells, Cultured , Crystallography, X-Ray , Dose-Response Relationship, Drug , Mice , Models, Molecular , Molecular Structure , Structure-Activity RelationshipABSTRACT
Aromatic C-H bond hydroxylation of 1-methoxynaphthalene was efficiently catalyzed by the substrate misrecognition system of the hydrogen peroxide dependent cytochrome P450BSbeta (CYP152A1), which usually catalyzes hydroxylation of long-alkyl-chain fatty acids. Very importantly, the hydroxylation of 1-methoxynaphthalene can be monitored by a color change since the formation of 4-methoxy-1-naphthol was immediately followed by its further oxidation to yield Russig's blue. Russig's blue formation allows us to estimate the peroxygenation activity of enzymes without the use of high performance liquid chromatography, gas chromatography, and nuclear magnetic resonance measurements.
Subject(s)
Colorimetry/methods , Cytochrome P-450 Enzyme System/metabolism , Hydrocarbons, Aromatic/chemistry , Peroxidases/metabolism , Catalytic Domain , Cytochrome P-450 Enzyme System/chemistry , Hydrocarbons, Aromatic/metabolism , Hydroxylation , Models, Molecular , Molecular Structure , Naphthalenes/chemistry , Naphthalenes/metabolism , Peroxidases/chemistry , Protein Structure, TertiaryABSTRACT
Genetic transformation of many filamentous fungi is carried out by a protocol that utilizes polyethylene glycol (PEG) and calcium ion (Ca2+). This method has remained practically unchanged for more than 20 years, but the roles these molecules play are not definitively understood. To gain a better understanding, we have compared PEG transformation to a protocol using polyethylenimine (PEI) that is the basis for non-viral transfection in mammals and which has a well established molecular model for assisting DNA uptake. Protoplasts of Aspergillus nidulans could be transformed in the presence of Ca2+ with a relatively high ratio of PEI to DNA molecules. By comparing PEI and PEG in terms of interaction with DNA, fungal protoplasts, and response to different transformation conditions, we propose that the role of PEG is most likely to function after transforming DNA is incorporated into protoplasts, rather than the accepted view that it functions outside of the cell. Confirmation that protoplast fusion was not involved in DNA uptake is consistent with this hypothesis.
