17-␣-Ethynylestradiol (17EE) inactivated purified, reconstituted rat hepatic cytochrome P450 (P450) 2B1 and human P450 2B6 in a mechanism-based manner. Little or no inactivation was observed when P450s 2B2 or 2B4 were incubated with 17EE. The inactivation of P450s 2B1 and 2B6 was entirely dependent on both NADPH and 17EE and followed pseudo-first order kinetics. The maximal rate constants for the inactivation of P450s 2B1 and 2B6 at 30°C were 0.2 and 0.03 min Ϫ1 , respectively. For P450s 2B1 and 2B6 the apparent K I was 11 and 0.8 M, respectively. Incubation of P450 2B1 with 17EE and NADPH for 20 min resulted in a 75% loss in enzymatic activity and a concurrent 20 to 25% loss of the enzyme's ability to form a reduced CO complex. With P450 2B6, an 83% loss in enzymatic activity and a 5 to 10% loss in the CO reduced spectrum were observed. The extrapolated partition ratios for 17EE with P450 2B1 and 2B6 were 21 and 13, respectively. Simultaneous incubation of an alternate substrate together with 17EE protected both enzymes from inactivation. A 1.3:1 stoichiometry of labeling for binding of the radiolabeled 17EE to P450 2B1 and 2B6 was seen. These results indicate that 17EE inactivates P450s 2B1 and 2B6 in a mechanism-based manner, primarily by the binding of a reactive intermediate of 17EE to the apoprotein. Analysis of the 17EE metabolites showed that 2B enzymes that become inactivated differ primarily by their ability to generate two metabolites that were not produced by P450s 2B2 or 2B4.

Carbaryl (1-naphthyl methylcarbamate) and carbofuran (2,3-dihydro-2,2-dimethyl-7-benzofuranyl methylcarbamate) are among the most toxic insecticides, implicated in a variety of diseases including diabetes and cancer among others. Using an integrated pharmacoinformatics based screening approach, we have identified these insecticides to be structural mimics of the neurohormone melatonin and were able to bind to the putative melatonin binding sites in MT1 and MT2 melatonin receptors in silico. Carbaryl and carbofuran then were tested for competition with 2-[125I]-iodomelatonin (300 pM) binding to hMT1 or hMT2 receptors stably expressed in CHO cells. Carbaryl and carbofuran showed higher affinity for competition with 2-[125I]-iodomelatonin binding to the hMT2 compared to the hMT1 melatonin receptor (33 and 35-fold difference, respectively) as predicted by the molecular modeling. In the presence of GTP (100 μM), which decouples the G-protein linked receptors to modulate signaling, the apparent efficacy of carbaryl and carbofuran for 2-[125I]-iodomelatonin binding for the hMT1 melatonin receptor was not affected but significantly decreased for the hMT2 melatonin receptor compatible with receptor antagonist/inverse agonist and agonist efficacy, respectively. Altogether, our data points to a potentially new mechanism through which carbamate insecticides carbaryl and carbofuran could impact human health by altering the homeostatic balance of key regulatory processes by directly binding to melatonin receptors.

SummaryThe Candida albicans response regulator protein Ssk1p regulates oxidant adaptation through the MAPK HOG1 pathway. Deletion mutants lacking SSK1 are oxidant sensitive in vitro and are killed more than wild-type (WT) cells by human neutrophils. Furthermore, the mutants are avirulent in an invasive murine model, and unable to adhere to human esophageal cells. Transcriptional profiling has indicated that approximately 25% of all changes occur in genes encoding cell wall and stress adaptation functions. In this study, we have investigated the role of amino acid residues in the Ssk1p receiver (or regulatory) domain by constructing point mutants at positions D556 (putative site of protein phosphorylation) and D513 (putative role in divalent metal binding, phosphorylation and conformational switching). For each point mutant, their sensitivity to a variety of oxidant stress conditions was assessed and correlated with in vitro phosphorylation of each Ssk1p receiver domain, phosphorylation of the Hog1p MAP kinase, and translocation to the nucleus. We show that a D556N mutant is sensitive to 5 mM H 2O2 or t-butyl hydroperoxide, similar to a gene knock-out ssk1 mutant, even though Hog1p is phosphorylated in the D556N mutant. To resolve this apparent paradox, we also demonstrate that Hog1p translocation to the nucleus in the D556N mutant is significantly reduced compared with WT cells (CAF2-1). In a second point mutant, D513 was changed to a lysine residue (D513K). This mutant had WT levels of resistance to peroxide, but in comparison to WT cells and the D556N mutant, morphogenesis (yeast to hyphae transition) was inhibited in 10% serum or in M-199 medium at 37°C. In the D513K point mutant, constitutive phosphorylation of Hog1p was observed, suggesting that a non-conservative change (D513K) traps Ssk1p in an active conformation and therefore constitutive Hog1p phosphorylation. The inhibition of morphogenesis in D513K is related to a downregulation of the transcription factors of morphogenesis, EFG1 and CPH1. Another non-conserved point mutant (D556R) was also constructed and phenotypically was like the D513K mutant. The receiver domains of the D556N and the D513K mutants could not be appreciably phosphorylated in vitro indicating that constitutive activation of Hog1p occurs in vivo due to the inability of Ssk1p to be phosphorylated at least in the D513K mutant. We speculate that the nonconservative changes described above in Ssk1p response regulator may cause conformational changes in the Ssk1p that account for phenotype differences compared with the D556N mutant that are also Hog-independent.

The draft nuclear genome sequence of the snail-transmitted, dimorphic, parasitic, platyhelminth Schistosoma mansoni revealed eight genes encoding proteins that contain the Universal Stress Protein (USP) domain. Schistosoma mansoni is a causative agent of human schistosomiasis, a severe and debilitating Neglected Tropical Disease (NTD) of poverty, which is endemic in at least 76 countries. The availability of the genome sequences of Schistosoma species presents opportunities for bioinformatics and genomics analyses of associated gene families that could be targets for understanding schistosomiasis ecology, intervention, prevention and control. Proteins with the USP domain are known to provide bacteria, archaea, fungi, protists and plants with the ability to respond to diverse environmental stresses. In this research investigation, the functional annotations of the USP genes and predicted nucleotide and protein sequences were initially verified. Subsequently, sequence clusters and distinctive features of the sequences were determined. A total of twelve ligand binding sites were predicted based on alignment to the ATP-binding universal stress protein from Methanocaldococcus jannaschii. In addition, six USP sequences showed the presence of ATP-binding motif residues indicating that they may be regulated by ATP. Public domain gene expression data and RT-PCR assays confirmed that all the S. mansoni USP genes were transcribed in at least one of the developmental life cycle stages of the helminth. Six of these genes were up-regulated in the miracidium, a free-swimming stage that is critical for transmission to the snail intermediate host. It is possible that during the intra-snail stages, S. mansoni gene transcripts for universal stress proteins are low abundant and are induced to perform specialized functions triggered by environmental stressors such as oxidative stress due to hydrogen peroxide that is present in the snail hemocytes. This report serves to catalyze the formation of a network of researchers to understand the function and regulation of the universal stress proteins encoded in genomes of schistosomes and their snail intermediate hosts.

In-silico screening of flexible ligands against flexible ligand binding pockets (LBP) is an emerging approach in structure-based drug discovery. Here, we describe a molecular dynamics (MD) based docking approach to investigate the influence on the high-throughput in-silico screening of small molecules against flexible ligand binding pockets. In our approach, an ensemble of 51 energetically favorable structures of the LBP of human estrogen receptor alpha (hERalpha) were collected from 3 ns MD simulations. In-silico screening of 3500 endocrine disrupting compounds against these flexible ligand binding pockets resulted in thousands of ER-ligand complexes of which 582 compounds were unique. Detailed analysis of MD generated structures showed that only 17 of the LBP residues significantly contribute to the overall binding pocket flexibility. Using the flexible LBP conformations generated, we have identified 32 compounds that bind better to the flexible ligand-binding pockets compared to the crystal structure. These compounds, though chemically divergent, are structurally similar to the natural hormone. Our MD-based approach in conjunction with grid-based distributed computing could be applied routinely for in-silico screening of large databases against any given target.

The organized, tightly regulated signaling relays engaged by the cannabinoid receptors (CBs) and their ligands, G proteins and other effectors, together constitute the endocannabinoid system (ECS). This system governs many biological functions including cell proliferation, regulation of ion transport and neuronal messaging. This review will firstly examine the physiology of the ECS, briefly discussing some anomalies in the relay of the ECS signaling as these are consequently linked to maladies of global concern including neurological disorders, cardiovascular disease and cancer. While endogenous ligands are crucial for dispatching messages through the ECS, there are also commonalities in binding affinities with copious exogenous ligands, both natural and synthetic. Therefore, this review provides a comparative analysis of both types of exogenous ligands with emphasis on natural products given their putative safer efficacy and the role of Δ9-tetrahydrocannabinol (Δ9-THC) in uncovering the ECS. Efficacy is congruent to both types of compounds but noteworthy is the effect of a combination therapy to achieve efficacy without unideal side-effects. An example is Sativex that displayed promise in treating Huntington's disease (HD) in preclinical models allowing for its transition to current clinical investigation. Despite the in vitro and preclinical efficacy of Δ9-THC to treat neurodegenerative ailments, its psychotropic effects limit its clinical applicability to treating feeding disorders. We therefore propose further investigation of other compounds and their combinations such as the triterpene, α,β-amyrin that exhibited greater binding affinity to CB than CB and was more potent than Δ9-THC and the N-alkylamides that exhibited CB selective affinity; the latter can be explored towards peripherally exclusive ECS modulation. The synthetic CB antagonist, Rimonabant was pulled from commercial markets for the treatment of diabetes, however its analogue SR144528 maybe an ideal lead molecule towards this end and HU-210 and Org27569 are also promising synthetic small molecules.

Synthetic estrogens have diverse chemical structures and may either positively or negatively affect the estrogenic signaling pathways through interactions with the estrogen receptors (ERs). Modeling studies suggest that 4-(1-adamantyl)phenol (AdP) and 4,4 -(1,3-adamantanediyl)diphenol (AdDP) can bind in the ligand binding site of ER . We used fluorescence polarization (FP) to compare the binding affinities of AdP, AdDP and 2-(1-adamantyl)-4-methylphenol (AdMP) for human ER and ER with the binding affinities of the known ER ligands, diethylstilbestrol (DES) and 4-hydroxytamoxifen (4OHT). Competition binding experiments show that AdDP has greater affinity for both ERs than does AdP, while AdMP does not bind the receptor proteins. The relative binding affinities of AdDP and AdP are weaker than the affinity of DES or 4OHT for both ERs with the exception of AdDP, which binds ER with higher affinity than does 4OHT. We also found that AdDP and AdP cause differential conformational changes in ER and ER , which result in altered affinities of the ERs for fluorescein-labeled estrogen response elements (EREs) using a direct binding FP assay. The results show that ER liganded with either AdDP or AdP has greater affinity for human pS2 ERE than the ER -4OHT complex. The data suggest that synthetic molecules like adamantanes may function as biologically active ligands for human ERs. This demonstrates the importance of considering the potential of novel classes of synthetic compounds as selective ER modulators.

BackgroundProteins that selectively transport water across the membranes of cells are recognized as important in the normal functioning of the body systems of vertebrates. There are 13 known mammalian aquaporins (AQP0 to AQP12), some of which have been shown to have unexpected cellular roles beyond transmembrane water transport. The availability of non-mammalian vertebrate animal models has the potential to provide insight into the emergence of diverse function in the aquaporins. The domesticated chicken (Gallus gallus) is the premier avian model for biological research; however, only a limited number of studies have compared chicken and mammalian aquaporins. The identification of aquaporins that share functional motifs or are expressed in the same tissues in human and chicken could allow the further functional analyses of homologous aquaporins in both species. We hypothesize that integrative analyses of protein sequences and body site expression of human, mouse, rat and chicken aquaporins has the potential to yield novel biological hypotheses about the unexpected cellular roles of aquaporins beyond transmembrane water transport.ResultsA total of 76 aquaporin transcript models derived from 47 aquaporin genes were obtained for human, mouse, rat and chicken. Eleven body sites (brain, connective tissue, head, heart, liver, muscle, ovary, pancreas, small intestine, spleen and testis) were identified in which there is suggested expression of at least one mammalian and one chicken aquaporin. This study demonstrates that modern on-line analysis tools, a novel matrix integration technique, and the availability of the chicken genome for comparative genomics and expression analysis enables hypothesis generation in several important areas including: (i) alternative transcription and speciation effects on the conservation of functional motifs in vertebrate aquaporins; (ii) the emergence of basolateral targeting in mammalian species; (iii) the potential of the cysteine-rich AQP11 as a possible target in the pathophysiology of neurodegenerative disorders such as autism that involve Purkinje cells; and (iv) possible impairment of function of pancreas-expressed AQP12 during pancreatotropic necrosis in avian influenza virus infection.ConclusionThe investigation of aquaporin function in chicken and mammalian species has the potential to accelerate the discovery of novel knowledge of aquaporins in both avian and mammalian species.