The hallmarks of pyridine nucleotide-dependent dehydrogenase reactions are the stereo- and regiospecific hydride transfer between the nicotinamide coenzyme and the corresponding substrate. When the hydride is delivered from NAD(P)H to reduce the keto-substrate, the site of attack is always at the carbonyl carbon. However, the apparent regioselectivity of the hydride transfer is reversed when difluoromethylene is used as a carbonyl mimic in the NADH-dependent enzyme, TDP-l-rhamnose synthase, which catalyzes the conversion of TDP-6-deoxy-l-lyxo-4-hexulose to TDP-l-rhamnose. The observed reversed regioselectivity can be explained by two mechanisms. One involves the formation of a carbene intermediate followed by a rearrangement involving 1,2-H shift. This mechanistic proposal is theoretically sound and would represent a rare example implicating the intermediacy of a carbene species in an enzyme reaction. However, our results are also consistent with a second mechanism in which the hydride addition to the difluoromethylene moiety occurs at the difluorinated end, opposite from the site predicted on the basis of the reduction of a normal keto functional group. Such a regioselectivity is well precedented in chemical models because nucleophilic addition to fluoroalkenes prefers a route in which the number of fluorines beta to the electron-rich carbon in the transition state is maximized. In this mechanism, the difluoromethylene group may be regarded as a carbonyl mimic with reversed polarity in enzyme catalysis. While further experiments are needed to discriminate between these mechanistic possibilities, the results reported here suggest that the apparent regioselectivity of hydride transfer in a pyridine nucleotide-dependent enzyme can be changed by altering the electrochemical properties of the reaction center.

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Background and ObjectiveWhen eye diseases are treated by topical administration, the success of treatment lies in the effective drug concentration in the target tissue. This is why the drug’s pharmacokinetic, in the different substructures of the eye, needs to be explored more accurately during drug development. The aim of the present analysis was to describe by rabbit model, the distribution of a drug after ocular instillation in the selected eye tissues and fluids.MethodsBy a top-down population approach, we developed and validated a population pharmacokinetics (PopPK) model, using tissue concentrations (tear, naso-lacrymal duct, cornea and aqueous humor) of a new src tyrosine kinase inhibitor (FV-60165) in each anterior segment’s tissue and fluid of the rabbit eye. Inter-individual variability was estimated and the impact of the formulation (solution or nanosuspension) was evaluated.ResultsThe model structure selected for the eye is a 4-compartment model with the formulation as a significant covariate on the first-order rate constant between tears and the naso-lacrymal duct. The model showed a good predictive performance and may be used to estimate the concentration–time profiles after single or repeated administration, in each substructure of the eye for each animal included in the analysis.ConclusionsThis analysis allowed describing the distribution of a drug in the different selected tissues and fluids in the rabbit’s eyes after instillation of the prodrug as a solution or nanosuspension.

PURPOSE. Prostaglandin F2a analogues are the first-line medication for the treatment of ocular hypertension (OHT), and prostanoid EP2 receptor agonists are under clinical development for this indication. The goal of this study was to investigate the effects of F prostanoid (FP) and EP2 receptor activation on the myofibroblast transition of primary trabecular meshwork (TM) cells, which could be a causal mechanism of TM dysfunction in glaucoma.METHODS. Human primary TM cells were treated with either latanoprost or butaprost and TGFb2. Trabecular meshwork contraction was measured in a three-dimensional (3D) TM cellpopulated collagen gel (CPCG) model. Expression of a-smooth muscle actin (a-SMA) and phosphorylation of myosin light chain (MLC) were determined by Western blot. Assembly of actin stress fibers and collagen deposition were evaluated by immunocytochemistry. Involvement of p38, extracellular signal-regulated kinase (ERK), and Rho-associated kinase (ROCK) pathways as well as matrix metalloproteinase activation was tested with specific inhibitors.RESULTS. In one source of validated adult TM cells, latanoprost induced cell contraction as observed by CPCG surface reduction and increased actin polymerization, a-SMA expression, and MLC phosphorylation, whereas butaprost inhibited TGF-b2-induced CPCG contraction, actin polymerization, and MLC phosphorylation. Both agonists inhibited TGF-b2-dependent collagen deposition. The latanoprost effects were mediated by p38 pathway.CONCLUSIONS. Latanoprost decreased TM collagen accumulation but promoted a contractile phenotype in a source of adult TM cells that could modulate the conventional outflow pathway. In contrast, butaprost attenuated both TM contraction and collagen deposition induced by TGF-b2, thereby inhibiting myofibroblast transition of TM cells. These results open new perspectives for the management of OHT.

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ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

Glucosamine-6-phosphate synthase (GlcNP-synthase) catalyzes the formation of glucosamine 6-phosphate from fructose 6-phosphate using the y-amide functionality of glutamine as the nitrogen source. In the absence of glutamine, GlcNP-synthase was recently found to catalyze the formation of glucose 6-phosphate corresponding to a phosphoglucoisomerase-like activity. Here we report active-site directed, irreversible inhibition of Escherichia coli GlcNP-synthase (k,,,,, = 0.60 -C 0.05 min-I , K,,, = 1.40 +-0.20 mM) by anhydro-l,2-hexitol 6-phosphates previously known as irreversible inhibitors of phosphoglucoisomerase. Enzyme inactivation with the tritiated affinity label, followed by tryptic digestion and purification of the radioactive fragments, allowed identification of three peptides. Two of them, accounting for 54% of the recovered radioactivity, are believed to result from the nucleophilic attack of sidechain carboxylates of GIu255 and Glu258 and thiol of Cys300 of the fructose-6-phosphate-binding site on the epoxide functionality of the inhibitor. The major peptide corresponds to derivatization of the N-terminal cysteine from the glutamine-binding site by the inhibitor. These results provide evidence for the close proximity of glutamine and fructose-6-phosphate-binding sites recently suggested by Bearne

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