This is an RSS newsfeed from BioMed Central It is intended to be used with an RSS reader. For more information about RSS newsfeeds from BioMed Central, visit http://www.biomedcentral.com/info/about/rss/ http://www.biomedcentral.com/bmcchembiol/ The latest articles from BMC Chemical Biology (ISSN 1472-6769) published by BioMed Central Background: Sortin2 is a low mass compound that interferes with vacuolar delivery of proteins in plants and yeast. The Sortin2 phenotype was tested in Arabidopsis thaliana and found to be reversible upon drug removal, demonstrating the ability of chemical genomics to induce reversible phenotypes that would be difficult to achieve using conventional genetics 1. However, standard genetic methods can be used to identify drug target pathways in a high-throughput manner. Results: In this study, we analyzed structure-function relationships of Sortin2 using structural analogues. The results show the key roles of sulphite substitution and a benzoic acid group. A Sortin 2 hypersensitivity screen for the induced secretion of a vacuolar cargo protein was done utilizing a yeast haploid deletion library. Using bioinformatics approaches, we highlighted functional information about the cellular pathways affected by drug treatment which included protein sorting and other endomembrane system-related processes. Conclusion: Chemical, genomic and genetics approaches were used to understand the mode of action of Sortin2, a bioactive chemical that affects the delivery of a vacuolar protein. Critical features of Sortin2 structure necessary for bioactivity suggest a binding pocket that may recognize two ends of Sortin2. The genome-wide screen shows that Sortin2 treatment in yeast affects primarily components within the endomembrane system. This approach allowed us to assign putative functions in protein sorting for fifteen genes of previously unknown function. http://www.biomedcentral.com/1472-6769/8/1 Lorena Norambuena, Jan Zouhar, Glenn R Hicks and Natasha V Raikhel BMC Chemical Biology 2008, 8:1 2008-01-07 doi:10.1186/1472-6769-8-1 BMC Chemical Biology 1472-6769 8 1 2008-01-07 Background: Stimulation of C3H 10T1/2 murine fibroblasts with interferon-γ(IFN) and bacterial lipopolysaccharide (LPS) generates reactive oxygen and nitrogen species leading to DNA damage, lipid oxidation, and tocopherol oxidation. The tocopherols possess unique chemical and biological properties that suggest they have important roles related to intracellular defense against radical-mediated damage. Results: Despite increased levels of reactive oxidants and decreased media tocopherol, cellular levels of γ-tocopherol, but not α-tocopherol, were observed to increase significantly when cells were treated with IFN/LPS. Inhibition of nitric oxide (NO) synthesis by a specific inhibitor of inducible NO synthase (iNOS) increased both intracellular α-tocopherol and γ-tocopherol concentrations, but did not significantly alter the reduction in media tocopherol levels caused by IFN/LPS treatment. Both exposure to exogenous NO and cellular synthesis of NO in cell culture increased media levels of 8-epi-prostaglandin F2α, a marker of oxidative lipid damage, whereas inhibition of endogenous NO synthesis reduced media 8-epi-prostaglandin F2α formation to control levels. Conclusion: Elevated intracellular levels of γ-tocopherol in response to the cellular inflammatory state may indicate that it serves a unique role in minimizing cellular damage resulting from endogenous NO synthesis. Results of the current study suggest that NO is an important mediator of damage within the cell, as well as in the oxidation of both α- and γ-tocopherols. The paradoxical increase in cellular tocopherol associated with the induction of NO synthesis may indicate either enhanced cellular transport/decreased export for tocopherols or recruitment of free tocopherol from tocopherol storage molecules. http://www.biomedcentral.com/1472-6769/7/2 Yuichiro Tanaka, Leslie A Lesoon Wood and Robert V Cooney BMC Chemical Biology 2007, 7:2 2007-07-03 doi:10.1186/1472-6769-7-2 BMC Chemical Biology 1472-6769 7 2 2007-07-03 Background: The effect of single and multiple amino acid substitutions in the green fluorescent protein (GFP) from Aequorea victoria has been extensively explored, yielding several proteins of diverse spectral properties. However, the role of amino acid deletions in this protein -as with most proteins- is still unknown, due to the technical difficulties involved in generating combinatorial in-phase amino acid deletions on a target region. Results: In this study, the region I129-L142 of superglo GFP (sgGFP), corresponding to the longest loop of the protein and located far away from the central chromophore, was subjected to a random amino acid deletion approach, employing an in-house recently developed mutagenesis method termed Codon-Based Random Deletion (COBARDE). Only two mutants out of 16384 possible variant proteins retained fluorescence: sgGFP-Δ I129 and sgGFP-Δ D130. Interestingly, both mutants were thermosensitive and at 30°C sgGFP-Δ D130 was more fluorescent than the parent protein. In contrast with deletions, substitutions of single amino acids from residues F131 to L142 were well tolerated. The substitution analysis revealed a particular importance of residues F131, G135, I137, L138, H140 and L142 for the stability of the protein. Conclusion: The behavior of GFP variants with both amino acid deletions and substitutions demonstrate that this loop is playing an important structural role in GFP folding. Some of the amino acids which tolerated any substitution but no deletion are simply acting as "spacers" to localize important residues in the protein structure. http://www.biomedcentral.com/1472-6769/7/1 Gabriela Flores-Ramírez, Manuel Rivera, Alfredo Morales-Pablos, Joel Osuna, Xavier Soberón and Paul Gaytán BMC Chemical Biology 2007, 7:1 2007-06-26 doi:10.1186/1472-6769-7-1 BMC Chemical Biology 1472-6769 7 1 2007-06-26 Background: Cis-planaramineplatinum(II) complexes like their trans isomers are often found to be active against cancer cell lines. The present study deals with the synthesis, characterization and determination of activity of new cis-planaramineplatinum(II) complexes. Results: Two cis-planaramineplatinum(II) complexes: cis-(3-hydroxypyridine)(ammine)dichloroplatinum(II) (code named AH3) and cis-(2,3-diaminopyridine)(ammine)dichloroplatinum(II) (code named AH7) have been prepared and characterised based on elemental analyses, IR, Raman, mass and 1H NMR spectral measurements. The interactions of the compounds with pBR322 plasmid DNA have been investigated and their activity against ovarian cancer cell lines: A2780, A2780cisR and A2780ZD047Rhave been determined. Like cisplatin, AH3 and AH7 are believed to form mainly monofunctional N7(G) and bifunctional intrastrand N7(G)N7(G) adducts with DNA, causing a local distortion of a DNA strand. As a result, gel mobility of the DNA changes. Both AH3 and AH7 are found to be less active than cisplatin against the three cell lines with AH3 being the more active compound of the two. The higher activity of AH3 is in line with its lower molar conductivity value corresponding to a lower degree of dissociation. Conclusion: The differences in activity of AH3, AH7 and cisplatin against the cell lines illustrate structure-activity relationship. http://www.biomedcentral.com/1472-6769/6/3 Ahmed Abdullah, Fazlul Huq, Ashraf Chowdhury, Hasan Tayyem, Philip Beale and Keith Fisher BMC Chemical Biology 2006, 6:3 2006-03-13 doi:10.1186/1472-6769-6-3 BMC Chemical Biology 1472-6769 6 3 2006-03-13 Background: A recent crystal structure of monastrol in a ternary complex with the kinesin Eg5 motor domain highlights a novel, induced-fit drug binding site at atomic resolution. Mutational obliteration of the monastrol binding site results in a monastrol-resistant, but otherwise catalytically active Eg5 motor domain. However, considering the conformational changes at this site, it is unclear what specific interactions stabilize the interaction between monastrol and the Eg5 motor domain. Results: To study the molecular complementarity of the monastrol-Eg5 interaction, we used a combination of synthetic chemistry and targeted mutations in Eg5 to measure the contribution of specific contacts to inhibition of Eg5 in vitro and in cultured cells. Structure-activity data on chemical derivatives, sequence analysis of Eg5 homologs from different species, and the effect of mutations near the drug binding site were consistent with the crystal structure. Conclusion: The mechanism of monastrol revealed by our data rationalizes its specificity for Eg5 over other kinesins and highlights a potential mechanism of drug resistance for anti-cancer therapy targeting this site in Eg5. http://www.biomedcentral.com/1472-6769/6/2 Zoltan Maliga and Timothy J Mitchison BMC Chemical Biology 2006, 6:2 2006-02-27 doi:10.1186/1472-6769-6-2 BMC Chemical Biology 1472-6769 6 2 2006-02-27 Background: Urokinase-type plasminogen activator (uPA) plays a major role in extracellular proteolytic events associated with tumor cell growth, migration and angiogenesis. Consequently, uPA is an attractive target for the development of small molecule active site inhibitors. Most of the recent drug development programs aimed at nonpeptidic inhibitors targeted at uPA have focused on arginino mimetics containing amidine or guanidine functional groups attached to aromatic or heterocyclic scaffolds. There is a general problem of limited bioavailability of these charged inhibitors. In the present study, uPA inhibitors were designed on an isocoumarin scaffold containing uncharged substituents. Results: 4-Chloro-3-alkoxyisocoumarins were synthesized in which the 3-alkoxy group contained a terminal bromine; these were compared with similar inhibitors that contained a charged terminal functional group. Additional variations included functional groups attached to the seven position of the isocoumarin scaffold. N- [3-(3-Bromopropoxy)-4-chloro-1-oxo-1H-isochromen-7-yl]benzamide was identified as an uncharged lead inhibitor of uPA, Ki = 0.034 μM. Molecular modeling of human uPA with these uncharged inhibitors suggests that the bromine occupies the same position as positively charged arginino mimetic groups. Conclusion: This study demonstrates that potent uncharged inhibitors of uPA can be developed based upon the isocoumarin scaffold. A tethered bromine in the three position and an aromatic group in the seven position are important contributors to binding. Although the aim was to develop compounds that act as mechanism-based inactivators, these inhibitors are competitive reversible inhibitors. http://www.biomedcentral.com/1472-6769/6/1 Justin J Heynekamp, Lucy A Hunsaker, Thomas A Vander Jagt, Lorraine M Deck and David L Vander Jagt BMC Chemical Biology 2006, 6:1 2006-02-08 doi:10.1186/1472-6769-6-1 BMC Chemical Biology 1472-6769 6 1 2006-02-08 Background: Natural products have numerous medicinal applications and play important roles in the biology of the organisms that accumulate them. Few methods are currently available for identifying proteins that bind to small molecules, therefore the discovery of cellular targets for natural products with pharmacological activity continues to pose a significant challenge in drug validation. Similarly, the identification of enzymes that participate in the biosynthesis or modification of natural products remains a formidable bottleneck for metabolic engineering. Flavonoids are one large group of natural products with a diverse number of functions in plants and in human health. The coupling of flavonoids to small ceramic and glass beads provides a first step in the development of high-throughput, solid-support base approaches to screen complex libraries to identify proteins that bind natural products. Results: The utilization of small glass and ceramic beads as solid supports for the coupling of small molecules was explored. Initial characterization of the beads indicated uniform and high capacity loading of amino groups. Once the beads were deemed adequate for the linking of small molecules by the coupling of NHS-fluorescein followed by microscopy, chemical hydrolysis and fluorometry, the flavonoid naringenin was modified with 1,4-dibromobutane, followed by the attachment of aminopropyltriethoxysilane. After NMR structural confirmation, the resulting 7-(4-(3-(triethoxysilyl)propylamino)butoxy) naringenin was attached to the ceramic beads. Conclusion: Our results demonstrate that ceramic and glass beads provide convenient solid supports for the efficient and facile coupling of small molecules. We succeeded in generating naringenin-coupled ceramic and glass beads. We also developed a convenient series of steps that can be applied for the solid-support coupling of other related flavonoids. The availability of solid-support coupled naringenin opens up new opportunities for the identification of flavonoid-binding proteins. http://www.biomedcentral.com/1472-6769/5/3 Yuhua Lu, Niloufer G Irani and Erich Grotewold BMC Chemical Biology 2005, 5:3 2005-10-10 doi:10.1186/1472-6769-5-3 BMC Chemical Biology 1472-6769 5 3 2005-10-10 Background: Electronic properties of amino acid side chains such as inductive and field effects have not been characterized in any detail. Quantum mechanics (QM) calculations and fundamental equations that account for substituent effects may provide insight into these important properties. PM3 analysis of electron distribution and polarizability was used to derive quantitative scales that describe steric factors, inductive effects, resonance effects, and field effects of amino acid side chains. Results: These studies revealed that: (1) different semiempirical QM methods yield similar results for the electronic effects of side chain groups, (2) polarizability, which reflects molecular deformability, represents steric factors in electronic terms, and (3) inductive effects contribute to the propensity of an amino acid for α-helices. Conclusion: The data provide initial characterization of the substituent effects of amino acid side chains and suggest that these properties affect electron density along the peptide backbone. http://www.biomedcentral.com/1472-6769/5/2 Donard S Dwyer BMC Chemical Biology 2005, 5:2 2005-08-03 doi:10.1186/1472-6769-5-2 BMC Chemical Biology 1472-6769 5 2 2005-08-03 Background: Study of ERK activation has thus far relied on biochemical assays that are limited to the use of phospho-specific antibodies and radioactivity in vitro, and analysis of whole cell populations in vivo. As with many systems, fluorescence resonance energy transfer (FRET) can be utilized to make highly sensitive detectors of molecular activity. Here we introduce FRET-based ERK Activity Sensors, which utilize variants of Enhanced Green Fluorescent Protein fused by an ERK-specific peptide linker to detect ERK2 activity. Results: ERK Activity Sensors display varying changes in FRET upon phosphorylation by active ERK2 in vitro depending on the composition of ERK-specific peptide linker sequences derived from known in vivo ERK targets, Ets1 and Elk1. Analysis of point mutations reveals specific residues involved in ERK binding and phosphorylation of ERK Activity Sensor 3. ERK2 also shows high in vitro specificity for these sensors over two other major MAP Kinases, p38 and pSAPK/JNK. Conclusion: EAS's are a convenient, non-radioactive alternative to study ERK dynamics in vitro. They can be utilized to study ERK activity in real-time. This new technology can be applied to studying ERK kinetics in vitro, analysis of ERK activity in whole cell extracts, and high-throughput screening technologies. http://www.biomedcentral.com/1472-6769/5/1 Harry M Green and José Alberola-Ila BMC Chemical Biology 2005, 5:1 2005-07-05 doi:10.1186/1472-6769-5-1 BMC Chemical Biology 1472-6769 5 1 2005-07-05 Background: The amino acid composition of a low molecular weight chromium binding peptide (LMWCr), isolated from bovine liver, is reportedly E:G:C:D::4:2:2:2, though its sequence has not been discovered. There is some controversy surrounding the exact biochemical forms and the action of Cr(III) in biological systems; the topic has been the subject of many experimental reports and continues to be investigated. Clarification of Cr-protein interactions will further understanding Cr(III) biochemistry and provide a basis for novel therapies based on metallocomplexes or small molecules. Results: A genomic search of the non-redundant database for all possible decapeptides of the reported composition yields three exact matches, EDGEECDCGE, DGEECDCGEE and CEGGCEEDDE. The first two sequences are found in ADAM 19 (A Disintegrin and Metalloproteinase domain 19) proteins in man and mouse; the last is found in a protein kinase in rice (Oryza sativa). A broader search for pentameric sequences (and assuming a disulfide dimer) corresponding to the stoichiometric ratio E:D:G:C::2:1:1:1, within the set of human proteins and the set of proteins in, or related to, the insulin signaling pathway, yields a match at an acidic region in the α-subunit of the insulin receptor (-EECGD-, residues 175–184). A synthetic peptide derived from this sequence binds chromium(III) and forms a metal-peptide complex that has properties matching those reported for isolated LMWCr and Cr(III)-containing peptide fractions. Conclusion: The search for an acidic decameric sequence indicates that LMWCr may not be a contiguous sequence. The identification of a distinct pentameric sequence in a significant insulin-signaling pathway protein suggests a possible identity for the LMWCr peptide. This identification clarifies directions for further investigation of LMWCr peptide fractions, chromium bio-coordination chemistry and a possible role in the insulin signaling pathway. Implications for models of chromium action in the insulin-signaling pathway are discussed. http://www.biomedcentral.com/1472-6769/4/2 Deendayal Dinakarpandian, Vincent Morrissette, Shveta Chaudhary, Kambiz Amini, Brian Bennett and J David Van Horn BMC Chemical Biology 2004, 4:2 2004-12-16 doi:10.1186/1472-6769-4-2 BMC Chemical Biology 1472-6769 4 2 2004-12-16