Down-regulating IL-6/GP130 targets improved the anti-tumor effects of 5-fluorouracil in colon cancer

Recent studies have confirmed that IL-6/GP130 targets are closely associated with tumor growth, metastasis and drug resistance. 5-Fluorouracil (5-FU) is the most common chemotherapeutic agent for colon cancer but is limited due to chemoresistance and high cytotoxicity. Bazedoxifene (BZA), a third-generation selective estrogen receptor modulator, was discovered by multiple ligand simultaneous docking and drug repositioning approaches to have a novel function as an IL-6/GP130 target inhibitor. Thus, we speculated that in colon cancer, the anti-tumor efficacy of 5-FU might be increased in combination with IL-6/GP130 inhibitors. CCK8 assay and colony formation assay were used to detect the cell proliferation and colony formation. We measured the IC₅₀ value of 5-FU alone and in combination with BZA by cell viability inhibition. Cell migration and invasion ability were tested by scratch migration assays and transwell invasion assays. Flow cytometric analysis for cell apoptosis and cell cycle. Quantitative real-time PCR was used to detect Bad, Bcl-2 and Ki-67 mRNA expression and western blotting (WB) assay analyzed protein expression of Bad/Bcl-2 signaling pathway. Further mechanism study, WB analysis detected the key proteins level in IL-6/GP130 targets and JAK/STAT3, Ras/Raf/MEK/ERK, and PI3K/AKT/mTOR signaling pathway. A colon cancer xenograft model was used to further confirm the efficacy of 5-FU and BZA in vivo. The GP130, P-STAT3, P-AKT, and P-ERK expression levels were detected by immunohistochemistry in the xenograft tumor. BZA markedly potentiates the anti-tumor function of 5-FU in vitro and in vivo. Conversely, 5-FU activation is reduced following exogenous IL-6 treatment in cells. Further mechanistic studies determined that BZA treatment enhanced 5-FU anti-tumor activation by inhibiting the IL-6/GP130 signaling pathway and the phosphorylation status of the downstream effectors AKT, ERK and STAT3. In contrast, IL-6 can attenuate 5-FU function via activating IL-6R/GP130 signaling and the P-AKT, P-ERK and P-STAT3 levels. This study firstly verifies that targeting IL-6/GP130 signaling can increase the anti-tumor function of 5-FU; in addition, this strategy can sensitize cancer cell drug sensitivity, implying that blocking IL-6/GP130 targets can reverse chemoresistance. Therefore, combining 5-FU and IL-6/GP130 target inhibitors may be a promising approach for cancer treatment.     

Polyvalency: a promising strategy for drug design

The simultaneous binding of multiple ligands on one entity to multiple receptors on another can result in an affinity that is significantly greater than that for the binding of a single ligand to a single receptor. This concept of "polyvalency" can be used to design molecules that are potent inhibitors of toxins and pathogens. We describe the design of potent polyvalent inhibitors that neutralize anthrax toxin in vivo as well as our attempts to elucidate the relationship between inhibitor structure and activity. We also highlight promising future avenues for research in polyvalent drug design.     

Direct selection of recombinant plasmids with chlorate

A pair of small plasmid vectors have been constructed, which complement chlorate-resistant chlB mutants of Escherichia coli to chlorate reduction under anaerobic conditions. This potentially lethal function can be inactivated by the insertion of DNA fragments into a multiple cloning site. After transformation of competent mutant cells with a ligation mixture, simultaneous selection for the plasmid-coded drug resistance and for the inactivation of the complementing gene directly yields colonies, which harbor recombinant plasmids. It is demonstrated that this cloning system can be used with an efficiency beyond 90%. Vector sequences are provided.     

Methods and Results for Semi-automated Cloning Using Integrated Robotics

The Joint Center for Structural Genomics (JCSG) has emphasized automation and parallel processing approaches. Here, we describe automated methods used across the cloning process with results from JCSG projects. The protocols for PCR, restriction digests and ligations, as well as for gel electrophoresis and microtiter plate assays have all been automated. The system has the capacity to routinely process 384 clones a week. This throughput can adequately supply our expression and purification pipeline with expression-ready clones, including novel targets and truncations. The utility of our system is demonstrated by our results from three diverse projects. In summary, 94% of the PCR amplicons generated to date have been successfully cloned and verified by sequencing (83% of the total attempted targets). Our results demonstrate the capabilities of this robotic platform to provide an avenue to high-throughput cloning which requires little manpower and is rapid and cost-effective while providing insights for method optimization.     

The use of biodiversity as source of new chemical entities against defined molecular targets for treatment of malaria, tuberculosis, and T-cell mediated diseases--a review

The modern approach to the development of new chemical entities against complex diseases, especially the neglected endemic diseases such as tuberculosis and malaria, is based on the use of defined molecular targets. Among the advantages, this approach allows (i) the search and identification of lead compounds with defined molecular mechanisms against a defined target (e.g. enzymes from defined pathways), (ii) the analysis of a great number of compounds with a favorable cost/benefit ratio, (iii) the development even in the initial stages of compounds with selective toxicity (the fundamental principle of chemotherapy), (iv) the evaluation of plant extracts as well as of pure substances. The current use of such technology, unfortunately, is concentrated in developed countries, especially in the big pharma. This fact contributes in a significant way to hamper the development of innovative new compounds to treat neglected diseases. The large biodiversity within the territory of Brazil puts the country in a strategic position to develop the rational and sustained exploration of new metabolites of therapeutic value. The extension of the country covers a wide range of climates, soil types, and altitudes, providing a unique set of selective pressures for the adaptation of plant life in these scenarios. Chemical diversity is also driven by these forces, in an attempt to best fit the plant communities to the particular abiotic stresses, fauna, and microbes that co-exist with them. Certain areas of vegetation (Amazonian Forest, Atlantic Forest, Araucaria Forest, Cerrado-Brazilian Savanna, and Caatinga) are rich in species and types of environments to be used to search for natural compounds active against tuberculosis, malaria, and chronic-degenerative diseases. The present review describes some strategies to search for natural compounds, whose choice can be based on ethnobotanical and chemotaxonomical studies, and screen for their ability to bind to immobilized drug targets and to inhibit their activities. Molecular cloning, gene knockout, protein expression and purification, N-terminal sequencing, and mass spectrometry are the methods of choice to provide homogeneous drug targets for immobilization by optimized chemical reactions. Plant extract preparations, fractionation of promising plant extracts, propagation protocols and definition of in planta studies to maximize product yield of plant species producing active compounds have to be performed to provide a continuing supply of bioactive materials. Chemical characterization of natural compounds, determination of mode of action by kinetics and other spectroscopic methods (MS, X-ray, NMR), as well as in vitro and in vivo biological assays, chemical derivatization, and structure-activity relationships have to be carried out to provide a thorough knowledge on which to base the search for natural compounds or their derivatives with biological activity.     

Inhibition of the function of class IIa HDACs by blocking their interaction with MEF2

Enzymes that modify the epigenetic status of cells provide attractive targets for therapy in various diseases. The therapeutic development of epigenetic modulators, however, has been largely limited to direct targeting of catalytic active site conserved across multiple members of an enzyme family, which complicates mechanistic studies and drug development. Class IIa histone deacetylases (HDACs) are a group of epigenetic enzymes that depends on interaction with Myocyte Enhancer Factor-2 (MEF2) for their recruitment to specific genomic loci. Targeting this interaction presents an alternative approach to inhibiting this class of HDACs. We have used structural and functional approaches to identify and characterize a group of small molecules that indirectly target class IIa HDACs by blocking their interaction with MEF2 on DNA.Weused X-ray crystallography and (19)F NMRto show that these compounds directly bind to MEF2. We have also shown that the small molecules blocked the recruitment of class IIa HDACs to MEF2-targeted genes to enhance the expression of those targets. These compounds can be used as tools to study MEF2 and class IIa HDACs in vivo and as leads for drug development.     

Combined localization and real-time functional studies using a GFP-tagged ABCG2 multidrug transporter

ABCG2 is a half-transporter which causes multidrug resistance when overexpressed in tumor cells. Availability of combined localization and functional assays would greatly improve cell biology and drug modulation studies for this transporter. Here we demonstrate that an N-terminally GFP-tagged version of the protein (GFP-G2) can be used to directly monitor ABCG2 expression, dimerization, localization and function in living cells. GFP-G2 is fully functional when tested for drug-stimulated ATPase activity, vesicular transport assay, subcellular localization or cell surface epitope conformational changes. By measuring both GFP and Hoechst 33342 dye fluorescence in HEK-293 cells, we provide evidence that a real-time transport assay can be reliably applied to identify ABCG2 substrates, transport modulators, as well as to monitor the cellular functions of this multidrug transporter protein. This approach also avoids the need of cloning, drug selection or other further separation or characterization of the transgene-expressing cells.     

High throughput methods for gene cloning and expression

We outline a high throughput process for the production of bacterial expression clones using automated liquid handlers. The protocol consists of a series of interlinked methods representing liquid manipulations or incubations on various stations of the automation system. The methods employ the ligation-independent cloning approach that enables the simultaneous production of plasmids for different expression systems. The current cloning protocol spans 3 days with a linear throughput of 400 targets per production run. This automated approach enables the production of large numbers of bacterial expression clones and ultimately purified proteins. Although they were developed for structural genomics, these molecular protocols can also be applied in high throughput strategies such as those used for site-specific mutagenesis or protein interaction studies.     

Engineered minichromosomes in plants

Genetic engineering for complex or combined traits requires the simultaneous expression of multiple genes, and has been considered as the bottleneck for the next generation of genetic engineering in plants. Minichromosome technology provides one solution to the stable expression and maintenance of multiple transgenes in one genome. For example, minichromosomes can be used as a platform for efficient stacking of multiple genes for insect, bacterial and fungal resistances together with herbicide tolerance and crop quality traits. All the transgenes would reside on an independent minichromosome, not linked to any endogenous genes; thus linkage drag can be avoided. Engineered minichromosomes can be easily constructed by a telomere-mediated chromosomal truncation strategy. This approach does not rely on the cloning of centromere sequences, which are species-specific, and bypasses the any complications of epigenetic components for centromere specification. Thus, this technique can be easily extended to all plant species. The engineered minichromosome technology can also be used in combination with site-specific recombination systems to facilitate the stacking of multiple transgenes.     

The use of 3-D cultures for high-throughput screening: the multicellular spheroid model

Over the past few years, establishment and adaptation of cell-based assays for drug development and testing has become an important topic in high-throughput screening (HTS). Most new assays are designed to rapidly detect specific cellular effects reflecting action at various targets. However, although more complex than cell-free biochemical test systems, HTS assays using monolayer or suspension cultures still reflect a highly artificial cellular environment and may thus have limited predictive value for the clinical efficacy of a compound. Today's strategies for drug discovery and development, be they hypothesis free or mechanism based, require facile, HTS-amenable test systems that mimic the human tissue environment with increasing accuracy in order to optimize preclinical and preanimal selection of the most active molecules from a large pool of potential effectors, for example, against solid tumors. Indeed, it is recognized that 3-dimensional cell culture systems better reflect the in vivo behavior of most cell types. However, these 3-D test systems have not yet been incorporated into mainstream drug development operations. This article addresses the relevance and potential of 3-D in vitro systems for drug development, with a focus on screening for novel antitumor drugs. Examples of 3-D cell models used in cancer research are given, and the advantages and limitations of these systems of intermediate complexity are discussed in comparison with both 2-D culture and in vivo models. The most commonly used 3-D cell culture systems, multicellular spheroids, are emphasized due to their advantages and potential for rapid development as HTS systems. Thus, multicellular tumor spheroids are an ideal basis for the next step in creating HTS assays, which are predictive of in vivo antitumor efficacy.     

Using multiplexed regulation of luciferase activity and GFP translocation to screen for FOXO modulators

Background  

Independent luciferase reporter assays and fluorescent translocation assays have been successfully used in drug discovery for several molecular targets. We developed U2transLUC, an assay system in which luciferase and fluorescent read-outs can be multiplexed to provide a powerful cell-based high content screening method.     

Birth of mice after nuclear transfer by electrofusion using tail tip cells

Mice have been successfully cloned from cumulus cells, fibroblast cells, embryonic stem cells, and immature Sertoli cells only after direct injection of their nuclei into enucleated oocytes. This technical feature of mouse nuclear transfer differentiates it from that used in domestic species, where electrofusion is routinely used for nuclear transfer. To examine whether nuclear transfer by electrofusion can be applied to somatic cell cloning in the mouse, we electrofused tail tip fibroblast cells with enucleated oocytes, and then assessed the subsequent in vitro and in vivo development of the reconstructed embryos. The rate of successful nuclear transfer (fusion and nuclear formation) was 68.8% (753/1094) and the rate of development into morulae/blastocysts was 40.8% (260/637). After embryo transfer, seven (six males and one female; 2.5% per transfer) normal fetuses were obtained at 17.5-21.5 dpc. These rates of development in vitro and in vivo are not significantly different from those after cloning by injection (44.7% to morulae/blastocysts and 4.8% to term). These results indicate that nuclear transfer by electrofusion is practical for mouse somatic cell cloning and provide an alternative method when injection of donor nuclei into recipient oocytes is technically difficult.     

Neuroprotective strategies targeting apoptotic and necrotic cell death for stroke

It has been a major challenge to develop effective therapeutics for stroke, a leading cause of death and serious debilitation. Intensive research in the past 15 years have implicated many regulators and the related mechanisms by which neuronal cell death is regulated. It is now clear that even a brief ischemic stroke may trigger complex cellular events that lead to both apoptotic and necrotic neuronal cell death in a progressive manner. Although efforts at developing specific chemical inhibitors for validated targets have been successful for in vitro enzymatic assays, the development of some of such inhibitors into human therapy has been often hindered by their in vivo bioavailability profile. Considerations for the ability to chemically target a cellular mechanism in manner compatible with disease targets in vivo might be emphasized early in the development process by putting a priority on identifying key targets that can be effectively targeted chemically. Thorough interrogation of cellular pathways by saturation chemical genetics may provide a novel strategy to identify multiple key molecular entities that can be targeted chemically in order to select a target suitable for the treatment of intended human diseases such as stroke.     

Open innovation for phenotypic drug discovery: The PD2 assay panel

Phenotypic lead generation strategies seek to identify compounds that modulate complex, physiologically relevant systems, an approach that is complementary to traditional, target-directed strategies. Unlike gene-specific assays, phenotypic assays interrogate multiple molecular targets and signaling pathways in a target "agnostic" fashion, which may reveal novel functions for well-studied proteins and discover new pathways of therapeutic value. Significantly, existing compound libraries may not have sufficient chemical diversity to fully leverage a phenotypic strategy. To address this issue, Eli Lilly and Company launched the Phenotypic Drug Discovery Initiative (PD(2)), a model of open innovation whereby external research groups can submit compounds for testing in a panel of Lilly phenotypic assays. This communication describes the statistical validation, operations, and initial screening results from the first PD(2) assay panel. Analysis of PD(2) submissions indicates that chemical diversity from open source collaborations complements internal sources. Screening results for the first 4691 compounds submitted to PD(2) have confirmed hit rates from 1.6% to 10%, with the majority of active compounds exhibiting acceptable potency and selectivity. Phenotypic lead generation strategies, in conjunction with novel chemical diversity obtained via open-source initiatives such as PD(2), may provide a means to identify compounds that modulate biology by novel mechanisms and expand the innovation potential of drug discovery.     

Biology in a gray box: targeting the emergent properties of protein complexes: 2011 Yale Chemical Biology Symposium

At the 2011 Yale Chemical Biology Symposium, Jason Gestwicki presented a novel yet intuitive approach to drug screening. This method, which he termed "gray box" screening, targets protein complexes that have been reconstituted in vitro. Therefore, the gray box screen can achieve greater phenotypic complexity than biochemical assays but avoids the need for target identification that follows cell-based assays. Dr. Gestwicki's research group was able to use the gray box screen to identify myricetin as an inhibitor of the DnaK-DnaJ chaperone complex. This review will discuss Dr. Gestwicki's approach to identifying DnaK-DnaJ inhibitors as well as where the gray box screen fits among traditional techniques in drug discovery.     

An exogenous chloroplast genome for complex sequence manipulation in algae

We demonstrate a system for cloning and modifying the chloroplast genome from the green alga, Chlamydomonas reinhardtii. Through extensive use of sequence stabilization strategies, the ex vivo genome is assembled in yeast from a collection of overlapping fragments. The assembled genome is then moved into bacteria for large-scale preparations and transformed into C. reinhardtii cells. This system also allows for the generation of simultaneous, systematic and complex genetic modifications at multiple loci in vivo. We use this system to substitute genes encoding core subunits of the photosynthetic apparatus with orthologs from a related alga, Scenedesmus obliquus. Once transformed into algae, the substituted genome recombines with the endogenous genome, resulting in a hybrid plastome comprising modifications in disparate loci. The in vivo function of the genomes described herein demonstrates that simultaneous engineering of multiple sites within the chloroplast genome is now possible. This work represents the first steps toward a novel approach for creating genetic diversity in any or all regions of a chloroplast genome.     

Tools to evaluate estrogenic potency of dietary phytoestrogens:A consensus paper from the EU Thematic Network "Phytohealth" (QLKI-2002-2453)

Phytoestrogens are naturally occurring plantderived polyphenols with estrogenic potency. They are ubiquitous in diet and therefore, generally consumed. Among Europeans, the diet is rich in multiple putative phytoestrogens including flavonoids, tannins, stilbenoids, and lignans. These compounds have been suggested to provide beneficial effects on multiple menopause-related conditions as well as on development of hormone-dependent cancers, which has increased the interest in products and foods with high phytoestrogen content. However, phytoestrogens may as well have adverse estrogenicity related effects similar to any estrogen. Therefore, the assessment of estrogenic potency of dietary compounds is of critical importance. Due to the complex nature of estrogenicity, no single comprehensive test approach is available. Instead, several in vitro and in vivo assays are applied to evaluate estrogenic potency. In vitro estrogen receptor (ER) binding assays provide information on the ability of the compound to I) interact with ERs, II) bind to estrogen responsive element on promoter of the target gene as ligand-ER complex, and III) interact between the co-activator and ERs in ligand-dependent manner. In addition, transactivation assays in cells screen for ligand-induced ERmediated gene activation. Biochemical in vitro analysis can be used to test for possible effects on protein activities and E-screen assays to measure (anti)proliferative response in estrogen responsive cells. However, for assessment of estrogenicity in organs and tissues, in vivo approaches are essential. In females, the uterotrophic assay is applicable for testing ERa agonistic and antagonistic dietary compounds in immature or adult ovariectomized animals. In addition, mammary gland targeted estrogenicity can be detected as stimulated ductal elongation and altered formation of terminal end buds in immature or peripubertal animals. In males, Hershberger assay in peri-pubertal castrated rats can be used to detect (anti)androgenic/ (anti)estrogenic responses in accessory sex glands and other hormone regulated tissues. In addition to these short-term assays, sub-acute and chronic reproductive toxicity assays as well as two-generation studies can be applied for phytoestrogens to confirm their safety in long-term use. For reliable assessment of estrogenicity of dietary phytoestrogens in vivo, special emphasis should be focused on selection of the basal diet, route and doses of administration, and possible metabolic differences between the species used and humans. In conclusion, further development and standardization of the estrogenicity test methods are needed for better interpretation of both the potential benefits and risks of increasing consumption of phytoestrogens from diets and supplements.     

Multiplex-terminal restriction fragment length polymorphism

A novel method called "multiplex-terminal restriction fragment length polymorphism (M-TRFLP)" has been recently developed which can be used for simultaneous analysis of the community composition of two or more microbial taxa (up to four). This method can also be used for microbial diagnostic purposes. For M-TRFLP analysis, primers specific to different target genes are used for multiplex-PCR, with one primer for each target being labeled with a unique fluorescent dye at its 5' end. Restriction digestion of the amplified products followed by fragment size analysis on a DNA sequencer produces profiles for targeted genes, which can be distinguished from each other by the color of the terminal fragments imparted by the unique fluorescent dye used for primer labeling. In contrast to current protocols, M-TRFLP allows multiple communities or multiple targets (genes) data to be obtained in just one reaction and therefore saves time, cost and labor. This protocol can be completed in 5-8 h.