Chemical genetic approaches for the elucidation of signaling pathways

New chemical methods that use small molecules to perturb cellular function in ways analogous to genetics have recently been developed. These approaches include both synthetic methods for discovering small molecules capable of acting like genetic mutations, and techniques that combine the advantages of genetics and chemistry to optimize the potency and specificity of small-molecule inhibitors. Both approaches have been used to study protein function in vivo and have provided insights into complex signaling cascades.     

植物化学遗传学：一种崭新的植物遗传学研究方法

化学遗传学（chemical genetics,也称为化学基因组学,chemical genomics）研究方法是利用生物活性小分子扰动蛋白分子互作过程来研究有关的生命现象,是常规遗传学研究方法的补充和延伸。化学遗传学在植物科学中的应用——植物化学遗传学的研究在短短几年内,凭借其作为一种新的遗传学研究方法所具备的独特优势（如能够克服常规遗传学研究中的遗传冗余、突变致死难题及可提供特异强度、作用时间点上的条件性遗传扰动等）,已开始解决一些植物分子生物学中长期存在的研究难题。本文就植物化学遗传学的一般原理及其方法,以及它作为一种新的遗传学研究方法的优势及特点作一个综述．     

Bugs, drugs and chemical genomics

The serendipitous discovery of penicillin inspired intensive research into how small molecules affect basic cellular processes and their potential to treat disease. Biochemical and genetic approaches have been fundamental for clarifying small-molecule modes of action. Genomic technologies have permitted the use of chemical-genetic strategies that comprehensively study compound-target relationships in the context of a living cell, providing a systems biology view of both the cellular targets and the interdependent networks that respond to chemical stress. These studies highlight the fact that in vitro determinations of mechanism rarely translate into a complete understanding of drug behavior in the cell. Here, we review key discoveries that gave rise to the field of chemical genetics, with particular attention to chemical-genetic strategies developed for bakers' yeast, their extension to clinically relevant microbial pathogens, and the potential of these approaches to affect antimicrobial drug discovery.     

Forward chemical genetics: progress and obstacles on the path to a new pharmacopoeia

Forward chemical genetics is a new method to systematize the discovery and use of small molecules as tools for basic biological research. This approach requires three basic components: a library of compounds; an assay, in which the library is screened for a cellular or organismal phenotype; and a method to trace an active compound to its biological target. Bioactive compounds have traditionally been isolated from natural product extracts, although 'diversity-oriented synthesis' and commercial compound collections are gaining in prominence. New techniques, such as image-based screening and the cytoblot method, have increased the throughput of phenotypic assays. Strategies are also being developed to streamline target identification using molecular biological approaches.     

Xenopus: an ideal system for chemical genetics

Chemical genetics, or chemical biology, has become an increasingly powerful method for studying biological processes. The main objective of chemical genetics is the identification and use of small molecules that act directly on proteins, allowing rapid and reversible control of activity. These compounds are extremely powerful tools for researchers, particularly in biological systems that are not amenable to genetic methods. In addition, identification of small molecule interactions is an important step in the drug discovery process. Increasingly, the African frog Xenopus is being used for chemical genetic approaches. Here, we highlight the advantages of Xenopus as a first-line in vivo model for chemical screening as well as for testing reverse engineering approaches.     

Chemical biology approaches in plant stress research

In addition to their importance as essential agrochemicals and life-saving drugs, small molecules serve as powerful research tools to address questions at all levels of biological complexity from protein function to plant biotic interactions. In certain contexts, chemical tools are complementary or even preferred to genetic analysis, since not all experimental systems are amenable for genetic dissection. For example, mutants impaired in oxygen sensing cannot easily be recovered. Pharmacological and chemical genetics approaches have come to the rescue of biologists in unraveling such genetically intractable systems. In this review, I have discussed my own efforts to analyze oxygen deprivation signaling in plants to illustrate the validity of small molecular approaches in elucidating an essential pathway such as oxygen sensing. Chemical biology is also a potent approach to tease out genetically redundant biological processes. The recent breakthrough in identifying the elusive abscisic acid receptors has clearly demonstrated the power of chemical tools in dissecting redundant pathways and led to the blossoming of this area as a distinct discipline of plant biology research. I present a summary of this work and conclude the review with potential challenges in using chemical tools.     

Cellular reprogramming: a novel tool for investigating autism spectrum disorders

Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by impairment in reciprocal social interaction and communication, as well as the manifestation of stereotyped behaviors. Despite much effort, ASDs are not yet fully understood. Advanced genetics and genomics technologies have recently identified novel ASD genes, and approaches using genetically engineered murine models or postmortem human brain have facilitated understanding ASD. Reprogramming somatic cells into induced pluripotent stem cells (iPSCs) provides unprecedented opportunities in generating human disease models. Here, we present an overview of applying iPSCs in developing cellular models for understanding ASD. We also discuss future perspectives in the use of iPSCs as a source of cell therapy and as a screening platform for identifying small molecules with efficacy for alleviating ASD.     

Chemical genetics: a small molecule approach to neurobiology

Chemical genetics, or the specific modulation of cellular systems by small molecules, has complemented classical genetic analysis throughout the history of neurobiology. We outline several of its contributions to the understanding of ion channel biology, heat and cold signal transduction, sleep and diurnal rhythm regulation, effects of immunophilin ligands, and cell surface oligosaccharides with respect to neurobiology.     

Chemical genetics: exploring and controlling cellular processes with chemical probes.

The new field of chemical biology brings together chemists and biologists who are seeking to understand and mimic the natural world. One research strategy in this new field is the development of biologically active small molecules as molecular probes. This approach, which has been called 'chemical' genetics, has allowed elucidation of several pathways that have been difficult to study using traditional genetic approaches.     

Light,heat, action: neural control of fruit fly behaviour

The fruit fly Drosophila melanogaster has emerged as a popular model to investigate fundamental principles of neural circuit operation. The sophisticated genetics and small brain permit a cellular resolution understanding of innate and learned behavioural processes. Relatively recent genetic and technical advances provide the means to specifically and reproducibly manipulate the function of many fly neurons with temporal resolution. The same cellular precision can also be exploited to express genetically encoded reporters of neural activity and cell-signalling pathways. Combining these approaches in living behaving animals has great potential to generate a holistic view of behavioural control that transcends the usual molecular, cellular and systems boundaries. In this review, we discuss these approaches with particular emphasis on the pioneering studies and those involving learning and memory.     

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.     

A Manual Small Molecule Screen Approaching High-throughput Using Zebrafish Embryos

Zebrafish have become a widely used model organism to investigate the mechanisms that underlie developmental biology and to study human disease pathology due to their considerable degree of genetic conservation with humans. Chemical genetics entails testing the effect that small molecules have on a biological process and is becoming a popular translational research method to identify therapeutic compounds. Zebrafish are specifically appealing to use for chemical genetics because of their ability to produce large clutches of transparent embryos, which are externally fertilized. Furthermore, zebrafish embryos can be easily drug treated by the simple addition of a compound to the embryo media. Using whole-mount in situ hybridization (WISH), mRNA expression can be clearly visualized within zebrafish embryos. Together, using chemical genetics and WISH, the zebrafish becomes a potent whole organism context in which to determine the cellular and physiological effects of small molecules. Innovative advances have been made in technologies that utilize machine-based screening procedures, however for many labs such options are not accessible or remain cost-prohibitive. The protocol described here explains how to execute a manual high-throughput chemical genetic screen that requires basic resources and can be accomplished by a single individual or small team in an efficient period of time. Thus, this protocol provides a feasible strategy that can be implemented by research groups to perform chemical genetics in zebrafish, which can be useful for gaining fundamental insights into developmental processes, disease mechanisms, and to identify novel compounds and signaling pathways that have medically relevant applications.     

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.     

Chemical genetics: ligand-based discovery of gene function

Chemical genetics is the study of gene-product function in a cellular or organismal context using exogenous ligands. In this approach, small molecules that bind directly to proteins are used to alter protein function, enabling a kinetic analysis of the in vivo consequences of these changes. Recent advances have strongly enhanced the power of exogenous ligands such that they can resemble genetic mutations in terms of their general applicability and target specificity. The growing sophistication of this approach raises the possibility of its application to any biological process.     

Old drugs, new tricks: using genetically sensitized yeast to reveal drug targets

A study in this issue of Cell illustrates the power of applying genomic approaches with model systems to characterize the biological activity of small molecules and to identify their cellular targets, which can clarify the mode of action of human therapeutics.     

Molecules are more than markers: new directions in molecular microbial ecology

Macromolecules such as DNA, RNA, and proteins are widely used to quantify diversity in natural communities, to monitor the dispersal of organisms and their genes, and to trace phylogenetic relationships among organisms. With such widespread use of molecules as markers, it is easy to forget that they perform functions that are integral to the survival of organisms. The structural and functional properties of macromolecules have been intensively studied in genetics, biochemistry, and physiology. These fields, however, have not generally focused on the ecological significance of molecular variation, but instead have used defective variants as tools for identifying structure and function. Understanding the significance of molecular variation for organismal success or failure is a central problem in ecology, one whose solution will require the integration of diverse approaches and perspectives from ecology, molecular biology, genetics, physiology, and evolutionary biology. I review several studies of bacterial populations evolving in simple environments that have begun to integrate these approaches and perspectives in order to examine the consequences of molecular variation for ecological performance.     

Sirtuins: multifaceted drug targets

Sirtuin (Sir2) proteins being key regulators of numerous cellular processes have been, over the recent past, the subject of intense study. Sirs have been implicated in diverse physiological processes ranging from aging and cancer to neurological dysfunctions. Studies on Sir2s using tools of genetics, molecular biology, biochemistry and structural biology have provided significant insight into the diverse functions of this class of deacetylases. This apart, medicinal chemistry approaches have enabled the discovery of modulators (both activators and inhibitors) of Sir2 activity of diverse chemical structures and properties. The availability of these small molecule modulators of Sir2 activity not only has pharmacological significance but also opens up the possibility of exploiting chemical genetic approaches in understanding the role of this multi-functional enzyme in cellular processes.     

Target identification of bioactive compounds

To fully understand the regulation of cellular events, functional analysis of each protein involved in the regulatory systems is required. Among a variety of methods to uncover protein function, chemical genetics is a remarkable approach in which small molecular compounds are used as probes to elucidate protein functions within signaling pathways. However, identifying the target of small molecular bioactive compounds isolated by cell-based assays represents a crucial hurdle that must be overcome before chemical genetic studies can commence. A variety of methods and technologies for identifying target proteins have been reported. This review therefore aims to describe approaches for identifying these molecular targets.     

Chemical biology on the genome

In this article I discuss studies towards understanding the structure and function of DNA in the context of genomes from the perspective of a chemist. The first area I describe concerns the studies that led to the invention and subsequent development of a method for sequencing DNA on a genome scale at high speed and low cost, now known as Solexa/Illumina sequencing. The second theme will feature the four-stranded DNA structure known as a G-quadruplex with a focus on its fundamental properties, its presence in cellular genomic DNA and the prospects for targeting such a structure in cels with small molecules. The final topic for discussion is naturally occurring chemically modified DNA bases with an emphasis on chemistry for decoding (or sequencing) such modifications in genomic DNA. The genome is a fruitful topic to be further elucidated by the creation and application of chemical approaches.