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Charting calcium-regulated apoptosis pathways using chemical biology: role of calmodulin kinase IIBackground:
Intracellular free calcium ([Ca2+]i) is a key element in apoptotic signaling and a number of calcium-dependent apoptosis pathways have been described. We here used a chemical biology strategy to elucidate the relative importance of such different pathways.
Results:
A set of 40 agents ("bioprobes") that induce apoptosis was first identified by screening of a chemical library. Using p53, AP-1, NFAT and NF-κB reporter cell lines, these bioprobes were verified to induce different patterns of signaling. Experiments using the calcium chelator BAPTA-AM showed that Ca2+ was involved in induction of apoptosis by the majority of the bioprobes and that Ca2+ was in general required several hours into the apoptosis process. Further studies showed that the calmodulin pathway was an important mediator of the apoptotic response. Inhibition of calmodulin kinase II (CaMKII) resulted in more effective inhibition of apoptosis compared to inhibition of calpain, calcineurin/PP2B or DAP kinase. We used one of the bioprobes, the plant alkaloid helenalin, to study the role of CaMKII in apoptosis. Helenalin induced CaMKII, ASK1 and Jun-N-terminal kinase (JNK) activity, and inhibition of these kinases inhibited apoptosis.
Conclusion:
Our study shows that calcium signaling is generally not an early event during the apoptosis process and suggests that a CaMKII/ASK1 signaling mechanism is important for sustained JNK activation and apoptosis by some types of stimuli.
http://www.biomedcentral.com/1472-6769/8/2
Maria Hägg Olofsson, Aleksandra Mandic Havelka, Slavica Brnjic, Maria C Shoshan and Stig LinderBMC Chemical Biology 2008, 8:2Number of accesses: 3842008-08-01doi:10.1186/1472-6769-8-2BMC Chemical Biology1472-6769822008-08-01Electronic properties of amino acid side chains: quantum mechanics calculation of substituent effectsBackground:
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 DwyerBMC Chemical Biology 2005, 5:2Number of accesses: 2912005-08-03doi:10.1186/1472-6769-5-2BMC Chemical Biology1472-6769522005-08-03Chemogenetic fingerprinting by analysis of cellular growth dynamicsBackground:
A fundamental goal in chemical biology is the elucidation of on- and off-target effects of drugs and biocides. To this aim chemogenetic screens that quantify drug induced changes in cellular fitness, typically taken as changes in composite growth, is commonly applied.
Results:
Using the model organism Saccharomyces cerevisiae we here report that resolving cellular growth dynamics into its individual components, growth lag, growth rate and growth efficiency, increases the predictive power of chemogenetic screens. Both in terms of drug-drug and gene-drug interactions did the individual growth variables capture distinct and only partially overlapping aspects of cell physiology. In fact, the impact on cellular growth dynamics represented functionally distinct chemical fingerprints.DiscussionOur findings suggest that the resolution and quantification of all facets of growth increases the informational and interpretational output of chemogenetic screening. Hence, by facilitating a physiologically more complete analysis of gene-drug and drug-drug interactions the here reported results may simplify the assignment of mode-of-action to orphan bioactive compounds.
http://www.biomedcentral.com/1472-6769/8/3
Jonas Warringer, Dragi Anevski, Beidong Liu and Anders BlombergBMC Chemical Biology 2008, 8:3Number of accesses: 2152008-08-22doi:10.1186/1472-6769-8-3BMC Chemical Biology1472-6769832008-08-22Covalent attachment of the plant natural product naringenin to small glass and ceramic beadsBackground:
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 GrotewoldBMC Chemical Biology 2005, 5:3Number of accesses: 1562005-10-10doi:10.1186/1472-6769-5-3BMC Chemical Biology1472-6769532005-10-10Chemical cleavage reactions of DNA on solid support: application in mutation detectionBackground:
The conventional solution-phase Chemical Cleavage of Mismatch (CCM) method is time-consuming, as the protocol requires purification of DNA after each reaction step. This paper describes a new version of CCM to overcome this problem by immobilizing DNA on silica solid supports.
Results:
DNA test samples were loaded on to silica beads and the DNA bound to the solid supports underwent chemical modification reactions with KMnO4 (potassium permanganate) and hydroxylamine in 3M TEAC (tetraethylammonium chloride) solution. The resulting modified DNA was then simultaneously cleaved by piperidine and removed from the solid supports to afford DNA fragments without the requirement of DNA purification between reaction steps.
Conclusions:
The new solid-phase version of CCM is a fast, cost-effective and sensitive method for detection of mismatches and mutations.
http://www.biomedcentral.com/1472-6769/3/1
Chinh T Bui, Andreana Lambrinakos, Jeffrey J Babon and Richard GH CottonBMC Chemical Biology 2003, 3:1Number of accesses: 1162003-05-13doi:10.1186/1472-6769-3-1BMC Chemical Biology1472-6769312003-05-13The effect of amino acid deletions and substitutions in the longest loop of GFPBackground:
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 GaytánBMC Chemical Biology 2007, 7:1Number of accesses: 1122007-06-26doi:10.1186/1472-6769-7-1BMC Chemical Biology1472-6769712007-06-26Uncharged isocoumarin-based inhibitors of urokinase-type plasminogen activatorBackground:
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 JagtBMC Chemical Biology 2006, 6:1Number of accesses: 922006-02-08doi:10.1186/1472-6769-6-1BMC Chemical Biology1472-6769612006-02-08Small-molecule and mutational analysis of allosteric Eg5 inhibition by monastrolBackground:
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 MitchisonBMC Chemical Biology 2006, 6:2Number of accesses: 922006-02-27doi:10.1186/1472-6769-6-2BMC Chemical Biology1472-6769622006-02-27Identification of cellular pathways affected by Sortin2, a synthetic compound that affects protein targeting to the vacuole in Saccharomyces cerevisiaeBackground:
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 RaikhelBMC Chemical Biology 2008, 8:1Number of accesses: 922008-01-07doi:10.1186/1472-6769-8-1BMC Chemical Biology1472-6769812008-01-07Studies on the synthesis, characterization, binding with DNA and activities of two cis-planaramineplatinum(II) complexes of the form: cis-PtL(NH3)Cl2 where L = 3-hydroxypyridine and 2,3-diaminopyridineBackground:
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 FisherBMC Chemical Biology 2006, 6:3Number of accesses: 882006-03-13doi:10.1186/1472-6769-6-3BMC Chemical Biology1472-6769632006-03-13