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The lipid A biosynthesis pathway is essential in Pseudomonas aeruginosa. LpxA and LpxD are the first and third enzymes in this pathway respectively, and are regarded as promising antibiotic targets. The unique structural similarities between these two enzymes make them suitable targets for dual-binding inhibitors, a characteristic that would decrease the likelihood of mutational resistance and increase cell-based activity. We report the discovery of multiple small molecule ligands that bind to P. aeruginosa LpxA and LpxD, including dual-binding ligands. Binding poses were determined for select compounds by X-ray crystallography. The new structures reveal a previously uncharacterized magnesium ion residing at the core of the LpxD trimer. In addition, ligand binding in the LpxD active site resulted in conformational changes in the distal C-terminal helix-bundle, which forms extensive contacts with acyl carrier protein (ACP) during catalysis. These ligand-dependent conformational changes suggest a potential allosteric influence of reaction intermediates on ACP binding, and vice versa. Taken together, the novel small molecule ligands and their crystal structures provide new chemical scaffolds for ligand discovery targeting lipid A biosynthesis, while revealing structural features of interest for future investigation of LpxD function. [ABSTRACT FROM AUTHOR]

We analyzed the effect of a natural osmolyte, trimethylamine N-oxide (TMAO), on structural properties and conformational stabilities of several proteins under macromolecular crowding conditions by a set of biophysical techniques. We also used the solvent interaction analysis method to look at the peculiarities of the TMAO-protein interactions under crowded conditions. To this end, we analyzed the partitioning of these proteins in TMAO-free and TMAO-containing aqueous two-phase systems (ATPSs). These ATPSs had the same polymer composition of 6.0 wt.% PEG-8000 and 12.0 wt.% dextran-75, and same ionic composition of 0.01 M K/NaPB, pH 7.4. These analyses revealed that there is no direct interaction of TMAO with proteins, suggesting that the TMAO effects on the protein structure in crowded solutions occur via the effects of this osmolyte on solvent properties of aqueous media. The effects of TMAO on protein structure in the presence of polymers were rather complex and protein-specific. Curiously, our study revealed that in highly concentrated polymer solutions, TMAO does not always act to promote further protein folding. [ABSTRACT FROM AUTHOR]

Background: Major depressive disorder afflicts up to 10% of adolescents. However, nearly 50% of those afflicted are considered nonresponsive to available treatments. Ketamine, a noncompetitive N-methyl-D-aspartate receptor antagonist has shown potential as a rapid-acting and long-lasting treatment for major depressive disorder in adults. Thus, the effectiveness and functional consequences of ketamine exposure during adolescence were explored. Methods: Adolescent male rats (postnatal day [PD] 35) received two ketamine (0, 5, 10, or 20 mg/kg) injections, 4 hours apart, after exposure to day 1 of the forced swim test (FST). The next day, rats were reexposed to the FST to assess ketamine-induced antidepressant-like responses. Separate groups were exposed to chronic unpredictable stress to confirm findings from the FST. After these initial experiments, adolescent naive rats were exposed to either 1 or 15 consecutive days (PD35–49) of ketamine (20 mg/kg) twice daily. Ketamine’s influence on behavioral reactivity to rewarding (i.e., sucrose preference) and aversive (i.e., elevated plus-maze, FST) circumstances was then assessed 2 months after treatment. To control for age-dependent effects, adult rats (PD75–89) were exposed to identical experimental conditions. Results: Ketamine (20 mg/kg) reversed the chronic unpredictable stress–induced depression-like behaviors in the FST. Repeated ketamine exposure resulted in anxiolytic- and antidepressant-like responses 2 months after drug exposure. None of the ketamine doses used were capable of inducing drug-seeking behaviors as measured by place preference conditioning. Conclusions: Repeated ketamine exposure induces enduring resilient-like responses regardless of age of exposure. These findings point to ketamine, and its repeated exposure, as a potentially useful antidepressant during adolescence. [Copyright &y& Elsevier]