Identification of a novel retinoid by small molecule screening with zebrafish embryos

Small molecules have played an important role in delineating molecular pathways involved in embryonic development and disease pathology. The need for novel small molecule modulators of biological processes has driven a number of targeted screens on large diverse libraries. However, due to the specific focus of such screens, the majority of the bioactive potential of these libraries remains unharnessed. In order to identify a higher proportion of compounds with interesting biological activities, we screened a diverse synthetic library for compounds that perturb the development of any of the multiple organs in zebrafish embryos. We identified small molecules that affect the development of a variety of structures such as heart, vasculature, brain, and body-axis. We utilized the previously known role of retinoic acid in anterior-posterior (A-P) patterning to identify the target of DTAB, a compound that caused A-P axis shortening in the zebrafish embryo. We show that DTAB is a retinoid with selective activity towards retinoic acid receptors gamma and beta. Thus, conducting zebrafish developmental screens using small molecules will not only enable the identification of compounds with diverse biological activities in a large chemical library but may also facilitate the identification of the target pathways of these biologically active molecules.     

Using small molecules to study big questions in cellular microbiology

High-throughput screening of small molecules is used extensively in pharmaceutical settings for the purpose of drug discovery. In the case of antimicrobials, this involves the identification of small molecules that are significantly more toxic to the microbe than to the host. Only a small percentage of the small molecules identified in these screens have been studied in sufficient detail to explain the molecular basis of their antimicrobial effect. Rarer still are small molecule screens undertaken with the explicit goal of learning more about the biology of a particular microbe or the mechanism of its interaction with its host. Recent technological advances in small molecule synthesis and high-throughput screening have made such mechanism-directed small molecule approaches a powerful and accessible experimental option. In this article, we provide an overview of the methods and technical requirements and we discuss the potential of small molecule approaches to address important and often otherwise experimentally intractable problems in cellular microbiology.     

Small molecule inhibitors as probes for estrogen and androgen receptor action

Because activated estrogen (ER) and androgen (AR) receptors stimulate cell proliferation in breast and prostate cancer, inhibiting their actions represents a major therapeutic goal. Most efforts to modulate ER and AR activity have focused on inhibiting the synthesis of estrogens or androgens or on the identification of small molecules that act by competing with agonist hormones for binding in the ligand-binding pocket of the receptor. An alternative approach is to implement screens for small molecule inhibitors that target other sites in the pathway of steroid receptor action. Many of these second-site inhibitors directly target ER or AR; others have still unknown sites of action. Small molecule inhibitors that target second sites represent new leads with clinical potential; they serve as novel modulators of receptor action; and they can reveal new and as yet unidentified interactions and pathways that modulate ER and AR action.     

Genome-wide high-throughput screens in functional genomics

The availability of complete genome sequences from many organisms has yielded the ability to perform high-throughput, genome-wide screens of gene function. Within the past year, rapid advances have been made towards this goal in many major model systems, including yeast, worms, flies, and mammals. Yeast genome-wide screens have taken advantage of libraries of deletion strains, but RNA-interference has been used in other organisms to knockdown gene function. Examples of recent large-scale functional genetic screens include drug-target identification in yeast, regulators of fat accumulation in worms, growth and viability in flies, and proteasome-mediated degradation in mammalian cells. Within the next five years, such screens are likely to lead to annotation of function of most genes across multiple organisms. Integration of such data with other genomic approaches will extend our understanding of cellular networks.     

From in silico target prediction to multi-target drug design: current databases, methods and applications

Given the tremendous growth of bioactivity databases, the use of computational tools to predict protein targets of small molecules has been gaining importance in recent years. Applications span a wide range, from the 'designed polypharmacology' of compounds to mode-of-action analysis. In this review, we firstly survey databases that can be used for ligand-based target prediction and which have grown tremendously in size in the past. We furthermore outline methods for target prediction that exist, both based on the knowledge of bioactivities from the ligand side and methods that can be applied in situations when a protein structure is known. Applications of successful in silico target identification attempts are discussed in detail, which were based partly or in whole on computational target predictions in the first instance. This includes the authors' own experience using target prediction tools, in this case considering phenotypic antibacterial screens and the analysis of high-throughput screening data. Finally, we will conclude with the prospective application of databases to not only predict, retrospectively, the protein targets of a small molecule, but also how to design ligands with desired polypharmacology in a prospective manner.     

Systematic approaches to dissect biological processes in stem cells by image-based screening

High-throughput RNAi or small molecule screens have proven to be powerful methodologies for the systematic dissection of cellular processes. In model organisms and cell lines, large-scale screens have identified key components of many cellular pathways and helped to identify novel targets in disease-relevant pathways. Image-based high-content screening has become an increasingly important tool in high-throughput screening, enabling changes in phenotype characteristics, such as cell morphology and cell differentiation, to be monitored. In this review, we discuss the use of image-based screening approaches to explore the behavior of adult, embryonic, and induced pluripotent stem cells. First, we review how current pluripotency and differentiation assays can be adapted to high-throughput formats. We then describe general aspects of image-based screening of cells and present an outlook on challenges for screening stem cells.     

Synthetic molecules: helping to unravel plant signal transduction

The application of small molecules has played a crucial role in identifying novel components involved in plant signalling. Compared to classic genetic approaches, small molecule screens offer notable advantages in dissecting plant biological processes, such as technical simplicity, low start-up costs, and most importantly, bypassing the problems of lethality and redundancy. To identify small molecules that target a biological process or protein of interest, robust and well-reasoned high-throughput screening approaches are essential. In this review, we present a series of principles and valuable approaches in small molecule screening in the plant model system Arabidopsis thaliana. We also provide an overview of small molecules that led to breakthroughs in uncovering phytohormone signalling pathways, endomembrane signalling cascades, novel growth regulators, and plant defence mechanisms. Meanwhile, the strategies to deciphering the mechanisms of these small molecules on Arabidopsis are highlighted. Moreover, the opportunities and challenges of small molecule applications in translational biology are discussed.     

MIPHENO: data normalization for high throughput metabolite analysis

Background  

High throughput methodologies such as microarrays, mass spectrometry and plate-based small molecule screens are increasingly used to facilitate discoveries from gene function to drug candidate identification. These large-scale experiments are typically carried out over the course of months and years, often without the controls needed to compare directly across the dataset. Few methods are available to facilitate comparisons of high throughput metabolic data generated in batches where explicit in-group controls for normalization are lacking.     

Identification and validation of bioactive small molecule target through phenotypic screening

For effective bioactive small molecule discovery and development into new therapeutic drug, a systematic screening and target protein identification is required. Different from the conventional screening system, herein phenotypic screening in combination with multi-omics-based target identification and validation (MOTIV) is introduced. First, phenotypic screening provides visual effect of bioactive small molecules in the cell or organism level. It is important to know the effect on the cell or organism level since small molecules affect not only a single target but the entire cellular mechanism within a cell or organism. Secondly, MOTIV provides systemic approach to discover the target protein of bioactive small molecule. With the chemical genomics and proteomics approach of target identification methods, various target protein candidates are identified. Then network analysis and validations of these candidates result in identifying the biologically relevant target protein and cellular mechanism. Overall, the combination of phenotypic screening and MOTIV will provide an effective approach to discover new bioactive small molecules and their target protein and mechanism identification.     

Transposon-mediated mutagenesis of somatic cells in the mouse for cancer gene identification

To successfully treat cancer we will likely need a much more detailed understanding of the genes and pathways meaningfully altered in individual cancer cases. One method for achieving this goal is to derive cancers in model organisms using unbiased forward genetic screens that allow cancer gene candidate discovery. We have developed a method using a “cut-and-paste” DNA transposon system called Sleeping Beauty (SB) to perform forward genetic screens for cancer genes in mice. Although the approach is conceptually similar to the use of replication competent retroviruses for cancer gene identification, the SB system promises to allow such screens in tissues previously not amenable to forward genetic screens such as the gastrointestinal tract, brain, and liver. This article describes the strains useful for SB-based screens for cancer genes in mice and how they are deployed in an experiment.     

Transforming science: cancer gene identification

Methods for cancer gene discovery include identification of viral oncogenes, identification of genes associated with recurrent chromosomal aberrations, and screens for genes capable of the transformation of cells in culture. In recent years, the completed genome sequence of human and model organisms has markedly enhanced cancer gene identification. Whole genome, high-throughput screens have been facilitated by the advent of new technologies such as murine leukemia virus-based mutagenesis, Sleeping Beauty-based mutagenesis, RNA interference, exon re-sequencing, and high-resolution methods for detecting chromosomal amplifications and deletions; these, in turn, have led to the identification of novel tumor suppressors and oncogenes. The identification of genes that are altered by mutation or expression and which are directly involved in tumor initiation and maintenance will be instrumental for understanding cancer phenotypic variation and for identifying crucial therapeutic targets.     

A new era for small molecule screening: from new targets, such as JAK2 V617F, to complex cellular screens

Traditionally reserved to research and development in pharmaceutical companies, screening of small molecule libraries is rapidly becoming an approach undertaken by academic laboratories. Novel cellular assays, sensitive systems to probe function, emerging new molecular targets are just some of the reasons explaining this shift. Targets of small molecules identified in cellular screens begin to be amenable to identification by elegant genetic approaches, such as probing toxicity of candidate small molecules on libraries of genetically modified yeast strains. Several new targets, such as JAK2 V617F, an activated JAK2 (Janus Kinase 2) mutant genetically associated with the majority of human myeloproliferative neoplasms, are being actively pursued. In this Review Series, we will learn how libraries of small molecules are harnessed to identify novel molecules, that alone or in combination, have the ability to alter cell fate, cell signalling, gene expression or response to extracellular cues.     

Chemogenomics and biotechnology

A robust knowledge of the interactions between small molecules and specific proteins aids the development of new biotechnological tools and the identification of new drug targets, and can lead to specific biological insights. Such knowledge can be obtained through chemogenomic screens. In these screens, each small molecule from a chemical library is applied to each cell type from a library of cells, and the resulting phenotypes are recorded. Chemogenomic screens have recently become very common and will continue to generate large amounts of data. The interpretation of this data will occupy biologists and chemists alike for some time to come. This review discusses methods for the acquisition and interpretation of chemogenomic data, in addition to possible applications of chemogenomics in biotechnology.     

Chemical genetic interrogation of natural variation uncovers a molecule that is glycoactivated

Natural variation in human drug metabolism and target genes can cause pharmacogenetic or interindividual variation in drug sensitivity. We reasoned that natural pharmacogenetic variation in model organisms could be systematically exploited to facilitate the characterization of new small molecules. To test this, we subjected multiple Arabidopsis thaliana accessions to chemical genetic screens and discovered 12 accession-selective hit molecules. As a model for understanding this variation, we characterized natural resistance to hypostatin, a new inhibitor of cell expansion. Map-based cloning identified HYR1, a UDP glycosyltransferase (UGT), as causative for hypostatin resistance. Multiple lines of evidence demonstrate that HYR1 glucosylates hypostatin in vivo to form a bioactive glucoside. Additionally, we delineated a HYR1 substrate motif and used it to identify another molecule modulated by glucosylation. Our results demonstrate that natural variation can be exploited to inform the biology of new small molecules, and that UGT sequence variation affects xenobiotic sensitivity across biological kingdoms.     

A systems approach for tumor pharmacokinetics

Recent advances in genome inspired target discovery, small molecule screens, development of biological and nanotechnology have led to the introduction of a myriad of new differently sized agents into the clinic. The differences in small and large molecule delivery are becoming increasingly important in combination therapies as well as the use of drugs that modify the physiology of tumors such as anti-angiogenic treatment. The complexity of targeting has led to the development of mathematical models to facilitate understanding, but unfortunately, these studies are often only applicable to a particular molecule, making pharmacokinetic comparisons difficult. Here we develop and describe a framework for categorizing primary pharmacokinetics of drugs in tumors. For modeling purposes, we define drugs not by their mechanism of action but rather their rate-limiting step of delivery. Our simulations account for variations in perfusion, vascularization, interstitial transport, and non-linear local binding and metabolism. Based on a comparison of the fundamental rates determining uptake, drugs were classified into four categories depending on whether uptake is limited by blood flow, extravasation, interstitial diffusion, or local binding and metabolism. Simulations comparing small molecule versus macromolecular drugs show a sharp difference in distribution, which has implications for multi-drug therapies. The tissue-level distribution differs widely in tumors for small molecules versus macromolecular biologic drugs, and this should be considered in the design of agents and treatments. An example using antibodies in mouse xenografts illustrates the different in vivo behavior. This type of transport analysis can be used to aid in model development, experimental data analysis, and imaging and therapeutic agent design.     

Der Zebrafisch als vielseitiges Modellsystem. Vom Zierfisch zum Forschungsobjekt

Over the past years, the zebrafish rose from an aquarium fish to a widespread model of biomedical research. It is easily maintained, produces relatively large offspring, shows rapid extra maternal development, and is transparent during early development. Hence, it was initially primarily utilized for direct microscopic observation of developmental processes. However, as it is easily genetically manipulated, it was soon used to produce transgenic lines expressing DNA derived from other organisms. These lines could be used to study the importance of specific genes for biological processes. In addition, zebrafish is used for random mutagenesis screens, allowing the unbiased detection of genes relevant for diseases, for small molecule screens applied for the discovery of biologically active substances, and increasingly as a model for cancer research.     

DNA-encoded signal conversion for sensitive microgravimetric detection of small molecule-protein interaction

Identification and quantification of small organic molecules capable of binding to a protein of interest with reasonable affinity and specificity is a central problem. Via developing DNA-encoded recognizing probe, we validate a proof-of-principle for constructing of small target-to-DNA conversion that screens the small molecule-protein interaction. Successful identification of β-indole acetic acid, abscisic acid, or 2,4-dichlorophenoxyacetic acid/corresponding antibody binding implies its fascinating potential for interrogating small molecule/protein interaction.     

Cellular phenotype recognition for high-content RNA interference genome-wide screening

Genome-wide, cell-based screens using high-content screening (HCS) techniques and automated fluorescence microscopy generate thousands of high-content images that contain an enormous wealth of cell biological information. Such screens are key to the analysis of basic cell biological principles, such as control of cell cycle and cell morphology. However, these screens will ultimately only shed light on human disease mechanisms and potential cures if the analysis can keep up with the generation of data. A fundamental step toward automated analysis of high-content screening is to construct a robust platform for automatic cellular phenotype identification. The authors present a framework, consisting of microscopic image segmentation and analysis components, for automatic recognition of cellular phenotypes in the context of the Rho family of small GTPases. To implicate genes involved in Rac signaling, RNA interference (RNAi) was used to perturb gene functions, and the corresponding cellular phenotypes were analyzed for changes. The data used in the experiments are high-content, 3-channel, fluorescence microscopy images of Drosophila Kc167 cultured cells stained with markers that allow visualization of DNA, polymerized actin filaments, and the constitutively activated Rho protein Rac(V12). The performance of this approach was tested using a cellular database that contained more than 1000 samples of 3 predefined cellular phenotypes, and the generalization error was estimated using a cross-validation technique. Moreover, the authors applied this approach to analyze the whole high-content fluorescence images of Drosophila cells for further HCS-based gene function analysis.     

From phenologs to silent suppressors: Identifying potential therapeutic targets for human disease

Orthologous phenotypes, or phenologs, are seemingly unrelated phenotypes generated by mutations in a conserved set of genes. Phenologs have been widely observed and accepted by those who study model organisms, and allow one to study a set of genes in a model organism to learn more about the function of those genes in other organisms, including humans. At the cellular and molecular level, these conserved genes likely function in a very similar mode, but are doing so in different tissues or cell types and can result in different phenotypic effects. For example, the RAS‐RAF‐MEK‐MAPK pathway in animals is a highly conserved signaling pathway that animals adopted for numerous biological processes, such as vulval induction in Caenorhabditis elegans and cell proliferation in mammalian cells; but this same gene set has been co‐opted to function in a variety of cellular contexts. In this review, I give a few examples of how suppressor screens in model organisms (with a emphasis on C. elegans) can identify new genes that function in a conserved pathway in many other organisms. I also demonstrate how the identification of such genes can lead to important insights into mammalian biology. From such screens, an occasional silent suppressor that does not cause a phenotype on its own is found; such suppressors thus make for good candidates as therapeutic targets.