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FRET experiments enable studies of the chemical and physical properties of individual molecules, which has long been a dream of chemists. However, these modern experimental techniques are still limited by the lack of information about the dynamic behavior of the fluorescent labels as well as by the use of dipole-dipole approximation even at short donor-to-acceptor distances. Our results help to suggest that these assumptions need to be carefully considered when designing experiments. We show that at short donor-acceptor separation, dipole-dipole approximation breaks down and Forster theory fails and cannot be used to obtain correct distances. We also explicitly demonstrate that dyes' linkers allow for a lot of flexibility in the fluorescent label orientation and position resulting in distances much shorter than assumed earlier.

We present the results of molecular modeling of dye-labeled, double-stranded DNA. The structural information obtained from the simulations are used as input to an analysis of energy transfer in this system. The simulations reveal the nature of the interaction between a pair of fluorophores and DNA. The donor, tetramethylrhodamine, TMR, attached to the 5′-end of DNA with a six-carbon tether, interacts primarily with DNA's minor groove, but occasionally stacks against the DNA base pairs. The acceptor, Cy5, attached to the opposite strand at positions n (n = 7, 12, 14, 16, 19, 24, 27), binds in the major groove in two distinct locations on the upper and lower part of the groove. We analyzed in detail the dye-to-dye distances, dipole orientation factors and fluorescence resonance energy transfer (FRET) rates. Tests of the validity of the Forster model were conducted using the transition density cube (TDC) method, which provides the exact Coulombic interaction within a certain model chemistry. Our studies show that the use of long tethers does not guarantee rotational freedom of the dyes, as intended in the experiments. Instead, the tethers allow Cy5 to bind in two different geometries, which causes a large uncertainty in the dye-to-dye distances. Our results also show significant fluctuation in the orientation factor, κ2, which, together with uncertainty in dye-to-dye distances, cause considerable uncertainty in interpreting FRET measurements. We suggest that molecular modeling, combined with the TDC method, provides a useful tool in designing and interpreting FRET experiments.

Utilization of cyanide as a nitrogen source by Pseudomonas fluorescens NCIMB 11764 occurs via oxidative conversion to carbon dioxide and ammonia, with the latter compound satisfying the nitrogen requirement. Substrate attack is initiated by cyanide oxygenase (CNO), which has been shown previously to have properties of a pterin-dependent hydroxylase. CNO was purified 71-fold and catalyzed the quantitative conversion of cyanide supplied at micromolar concentrations (10 to 50 μM) to formate and ammonia. The specific activity of the partially purified enzyme was approximately 500 mU/mg of protein. The pterin requirement for activity could be satisfied by supplying either the fully (tetrahydro) or partially (dihydro) reduced forms of various pterin compounds at catalytic concentrations (0.5 μM). These compounds included, for example, biopterin, monapterin, and neopterin, all of which were also identified in cell extracts. Substrate conversion was accompanied by the consumption of 1 and 2 molar equivalents of molecular oxygen and NADH, respectively. When coupled with formate dehydrogenase, the complete enzymatic system for cyanide oxidation to carbon dioxide and ammonia was reconstituted and displayed an overall reaction stoichiometry of 1:1:1 for cyanide, O 2 , and NADH consumed. Cyanide was also attacked by CNO at a higher concentration (1 mM), but in this case formamide accumulated as the major reaction product (formamide/formate ratio, 0.6:0.3) and was not further degraded. A complex reaction mechanism involving the production of isocyanate as a potential CNO monooxygenation product is proposed. Subsequent reduction of isocyanate to formamide, whose hydrolysis occurs as a CNO-bound intermediate, is further envisioned. To our knowledge, this is the first report of enzymatic conversion of cyanide to formate and ammonia by a pterin-dependent oxygenative mechanism.

Summary: Central nervous system (CNS) chemical protection depends upon discrete control of small-molecule access by the blood-brain barrier (BBB). Curiously, some drugs cause CNS side-effects despite negligible transit past the BBB. To investigate this phenomenon, we asked whether the highly BBB-enriched drug efflux transporter MDR1 has dual functions in controlling drug and endogenous molecule CNS homeostasis. If this is true, then brain-impermeable drugs could induce behavioral changes by affecting brain levels of endogenous molecules. Using computational, genetic, and pharmacologic approaches across diverse organisms, we demonstrate that BBB-localized efflux transporters are critical for regulating brain levels of endogenous steroids and steroid-regulated behaviors (sleep in Drosophila and anxiety in mice). Furthermore, we show that MDR1-interacting drugs are associated with anxiety-related behaviors in humans. We propose a general mechanism for common behavioral side effects of prescription drugs: pharmacologically challenging BBB efflux transporters disrupts brain levels of endogenous substrates and implicates the BBB in behavioral regulation. : Hindle et al. shed light on the curious finding that some drugs cause behavioral side-effects despite negligible access into the brain. These authors propose a unifying hypothesis that links blood-brain barrier drug transporter function and brain access of circulating steroids to common CNS adverse drug responses. Keywords: drug side effect mechanisms, central nervous system, blood brain barrier, behavior, toxicology, drug transporters, endobiotics, steroid hormones

The only small-molecule compound demonstrated to substantially extend survival in prion-infected mice is a biaryl hydrazone termed “Compd B” (4-pyridinecarboxaldehyde,2-[4-(5-oxazolyl)phenyl]hydrazone). However, the hydrazone moiety of Compd B results in toxic metabolites, making it a poor candidate for further drug development. We developed a pharmacophore model based on diverse antiprion compounds identified by high-throughput screening; based on this model, we generated biaryl amide analogs of Compd B. Medicinal chemistry optimization led to multiple compounds with increased potency, increased brain concentrations, and greater metabolic stability, indicating that they could be promising candidates for antiprion therapy. Replacing the pyridyl ring of Compd B with a phenyl group containing an electron-donating substituent increased potency, while adding an aryl group to the oxazole moiety increased metabolic stability. To test the efficacy of Compd B, we applied bioluminescence imaging (BLI), which was previously shown to detect prion disease onset in live mice earlier than clinical signs. In our studies, Compd B showed good efficacy in two lines of transgenic mice infected with the mouse-adapted Rocky Mountain Laboratory (RML) strain of prions, but not in transgenic mice infected with human prions. The BLI system successfully predicted the efficacies in all cases long before extension in survival could be observed. Our studies suggest that this BLI system has good potential to be applied in future antiprion drug efficacy studies.

Recently, we described the aminothiazole lead (4-biphenyl-4-ylthiazol-2-yl)-(6-methylpyridin-2-yl)-amine (1), which exhibits many desirable properties, including excellent stability in liver microsomes, oral bioavailability of ∼40%, and high exposure in the brains of mice. Despite its good pharmacokinetic properties, compound 1 exhibited only modest potency in mouse neuroblastoma cells overexpressing the disease-causing prion protein PrPSc. Accordingly, we sought to identify analogues of 1 with improved antiprion potency in ScN2a-cl3 cells while retaining similar or superior properties. Herein we report the discovery of improved lead compounds such as (6-methylpyridin-2-yl)-[4-(4-pyridin-3-yl-phenyl)thiazol-2-yl]amine and cyclopropanecarboxylic acid (4-biphenylthiazol-2-yl)amide, which exhibit brain exposure/EC50ratios at least tenfold greater than that of compound 1. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

In order to reach their pharmacologic targets, successful central nervous system (CNS) drug candidates have to cross a complex protective barrier separating brain from the blood. Being able to predict a priori which molecules can successfully penetrate this barrier could be of significant value in CNS drug discovery. Herein we report a new computational approach that combines two mechanism-based models, for passive permeation and for active efflux by P-glycoprotein, to provide insight into the multiparameter optimization problem of designing small molecules able to access the CNS. Our results indicate that this approach is capable of distinguishing compounds with high/low efflux ratios as well as CNS+/CNS- compounds and provides advantage over estimating P-glycoprotein efflux or passive permeability alone when trying to predict these emergent properties. We also demonstrate that this method could be useful for rank-ordering chemically similar compounds and that it can provide detailed mechanistic insight into the relationship between chemical structure and efflux ratios and/or CNS penetration, offering guidance as to how compounds could be modified to improve their access into the brain.

Juvenile hormones (JHs) play key roles in regulating metamorphosis and reproduction in insects. The last two steps of JH synthesis diverge depending on the insect order. In Lepidoptera, epoxidation by a P450 monooxygenase precedes esterification by a juvenile hormone acid methyltransferase (JHAMT). In Orthoptera, Dictyoptera, Coleoptera and Diptera epoxidation follows methylation. The aim of our study was to gain insight into the structural basis of JHAMT’s substrate recognition as a means to understand the divergence of these pathways. Homology modeling was used to build the structure of Aedes aegypti JHAMT. The substrate binding site was identified, as well as the residues that interact with the methyl donor (S-adenosylmethionine) and the carboxylic acid of the substrate methyl acceptors, farnesoic acid (FA) and juvenile hormone acid (JHA). To gain further insight we generated the structures of Anopheles gambiae, Bombyx mori, Drosophila melanogaster and Tribolium castaneum JHAMTs. The modeling results were compared with previous experimental studies using recombinant proteins, whole insects, corpora allata or tissue extracts. The computational study helps explain the selectivity toward the (10R)-JHA isomer and the reduced activity for palmitic and lauric acids. The analysis of our results supports the hypothesis that all insect JHAMTs are able to recognize both FA and JHA as substrates. Therefore, the order of the methylation/epoxidation reactions may be primarily imposed by the epoxidase’s substrate specificity. In Lepidoptera, epoxidase might have higher affinity than JHAMT for FA, so epoxidation precedes methylation, while in most other insects there is no epoxidation of FA, but esterification of FA to form MF, followed by epoxidation to JH III.

A juvenile hormone acid methyltransferase (JHAMT) was isolated as an abundant EST in a library of the corpora allata of the adult female mosquito Aedes aegypti. Its full length cDNA encodes a 278-aa protein that has 43% amino acid identity with BmJHAMT, a juvenile hormone acid methyltransferase previously cloned from Bombyx mori. Heterologous expression produced a recombinant protein that metabolizes farnesoic acid (FA) into methyl farnesoate, as well as juvenile hormone acid into juvenile hormone III (JH III) with exquisite stereo specificity. Real time PCR experiments showed that JHAMT mRNA levels are not an unequivocal indicator of JH III synthesis rates; the A. aegypti JHAMT gene, silent in female pupae, was transcriptionally activated just 4-6 h before adult eclosion. Radiochemical methyltransferase assays using active and inactive corpora allata glands (CA) dissected from sugar and blood-fed females respectively, clearly indicated that significant levels of JHAMT enzymatic activity are present when the CA shows very low spontaneous rates of JH III synthesis. Having the last enzymes of the JH synthetic pathway readily available all the time might be critical for the adult female mosquito to sustain rapid dynamic changes in JH III synthesis in response to nutritional changes or peripheral influences, such as mating or feeding. These results suggest that this gene has different roles in the regulation of JH synthesis in pupal and adult female mosquitoes, and support the hypothesis that the rate-limiting steps in JH III synthesis in adult female mosquitoes are located before entrance of FA into the synthetic pathway. Fil: Mayoral, Jaime G.. Florida International University; Estados Unidos Fil: Nouzova, Marcela. Florida International University; Estados Unidos Fil: Yoshiyama, Michiyo. National Institute of Agrobiological Sciences; Japón Fil: Shinoda, Tetsuro. National Institute of Agrobiological Sciences; Japón Fil: Hernandez Martinez, Salvador. Instituto Nacional de Salud Publica; México Fil: Dolghih, Elena. University of Florida; Estados Unidos Fil: Turjanski, Adrian. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; Argentina Fil: Roitberg, Adrián. University of Florida; Estados Unidos Fil: Priestap, Horacio. Florida International University; Estados Unidos Fil: Perez, Mario. Florida International University; Estados Unidos Fil: Mackenzie, Lucy. Florida International University; Estados Unidos Fil: Li, Yiping. Florida International University; Estados Unidos Fil: Noriega, Fernando G.. Florida International University; Estados Unidos

One of the many factors involved in determining the distribution and metabolism of a compound is the strength of its binding to human serum albumin. While experimental and QSAR approaches for determining binding to albumin exist, various factors limit their ability to provide accurate binding affinity for novel compounds. Thus, to complement the existing tools, we have developed a structure-based model of serum albumin binding. Our approach for predicting binding incorporated the inherent flexibility and promiscuity known to exist for albumin. We found that a weighted combination of the predicted logP and docking score most accurately distinguished between binders and nonbinders. This model was successfully used to predict serum albumin binding in a large test set of therapeutics that had experimental binding data.