Chemical Genetics Research Articles

Epidermal auxin biosynthesis facilitates rhizobial infection in Lotus japonicus.

Symbiotic nitrogen fixation in legumes requires nodule organogenesis to be coordinated with infection by rhizobia. The plant hormone auxin influences symbiotic infection, but the precise timing of auxin accumulation and the genetic network governing it remain unclear. We used a Lotus japonicus optimised variant of the DII-based auxin accumulation sensor and identified a rapid accumulation of auxin in the epidermis, specifically in the root hair cells. This auxin accumulation occurs in the infected root hairs during rhizobia invasion, while Nod factor application induces this response across a broader range of root hairs. Using the DR5 auxin responsive promoter, we demonstrate that activation of auxin signalling also occurs specifically in infected root hairs. Analysis of root hair transcriptome data identified induction of an auxin biosynthesis gene of the Tryptophan Amino-transferase Related (LjTar1) family following both bacteria inoculation and Nod factor treatment. Genetic analysis showed that both expression of the LjTar1 biosynthesis gene and the auxin response requires Nod factor perception, while common symbiotic pathway transcription factors are only partially required or act redundantly to initiate auxin accumulation. Using a chemical genetics approach, we confirmed that auxin biosynthesis has a functional role in promoting symbiotic infection events in the epidermis.

A chemical genetics approach identifies PTP4A3 as a regulator of colon cancer cell adhesion.

Dysregulation of the tightly controlled protein phosphorylation networks that govern cellular behavior causes cancer. The membrane-associated, intracellular protein tyrosine phosphatase PTP4A3 is overexpressed in human colorectal cancer and contributes to cell migration and invasion. To interrogate further the role of PTP4A3 in colorectal cancer cell migration and invasion, we deleted the Ptp4a3 gene from murine colorectal tumor cells. The resulting PTP4A3-/- cells exhibited impaired colony formation, spheroid formation, migration, and adherence compared with the paired PTP4A3fl/fl cells. We replicated these phenotypic changes using the new small-molecule, allosteric PTP4A3 inhibitor JMS-053. A related structure, JMS-038, which lacked phosphatase inhibition, displayed no cellular activity. Reduction in cell viability and colony formation by JMS-053 occurred in both mouse and human colorectal cell lines and required PTP4A3 expression. Ptp4a3 deletion increased the expression of extracellular matrix (ECM) and adhesion genes, including the tumor suppressor Emilin 1. JMS-053 also increased Emilin 1 gene expression. Moreover, The Cancer Genome Atlas genomic database revealed human colorectal tumors with high Ptp4a3 expression had low Emilin 1 expression. These chemical and biologic reagents reveal a previously unknown communication between the intracellular PTP4A3 phosphatase and the ECM and support efforts to pharmacologically target PTP4A3.-McQueeney, K. E., Salamoun, J. M., Ahn J. G., Pekic, P., Blanco, I. K., Struckman, H. L., Sharlow, E. R., Wipf, P., Lazo, J. S. A chemical genetics approach identifies PTP4A3 as a regulator of colon cancer cell adhesion.

Seh1 targets GATOR2 and Nup153 to mitotic chromosomes

ABSTRACTIn metazoa, the Nup107 complex (also known as the nucleoporin Y-complex) plays a major role in formation of the nuclear pore complex in interphase and is localised to kinetochores in mitosis. The Nup107 complex shares a single highly conserved subunit, Seh1 (also known as SEH1L in mammals) with the GATOR2 complex, an essential activator of mTORC1 kinase. mTORC1/GATOR2 has a central role in the coordination of cell growth and proliferation. Here, we use chemical genetics and quantitative chromosome proteomics to study the role of the Seh1 protein in mitosis. Surprisingly, Seh1 is not required for the association of the Nup107 complex with mitotic chromosomes, but it is essential for the association of both the GATOR2 complex and nucleoporin Nup153 with mitotic chromosomes. Our analysis also reveals a role for Seh1 at human centromeres, where it is required for efficient localisation of the chromosomal passenger complex (CPC). Furthermore, this analysis detects a functional interaction between the Nup107 complex and the small kinetochore protein SKAP (also known as KNSTRN).

Optimizing the Use of a Liquid Handling Robot to Conduct a High Throughput Forward Chemical Genetics Screen of Arabidopsis thaliana.

Chemical genetics is increasingly being employed to decode traits in plants that may be recalcitrant to traditional genetics due to gene redundancy or lethality. However, the probability of a synthetic small molecule being bioactive is low; therefore, thousands of molecules must be tested in order to find those of interest. Liquid handling robotics systems are designed to handle large numbers of samples, increasing the speed with which a chemical library can be screened in addition to minimizing/standardizing error. To achieve a high-throughput forward chemical genetics screen of a library of 50,000 small molecules on Arabidopsis thaliana (Arabidopsis), protocols using a bench-top multichannel liquid handling robot were developed that require minimal technician involvement. With these protocols, 3,271 small molecules were discovered that caused visible phenotypic alterations. 1,563 compounds induced short roots, 1,148 compounds altered coloration, 383 compounds caused root hair and other, non-categorized, alterations, and 177 compounds inhibited germination.

Inhibition of Enhancer of zeste homolog 2 (EZH2) induces natural killer cell-mediated eradication of hepatocellular carcinoma cells.

Natural killer (NK) cell-mediated tumor cell eradication could inhibit tumor initiation and progression. However, the factors that regulate NK cell-mediated cancer cell eradication remain unclear. We determined that hepatocellular carcinoma (HCC) cells exhibit transcriptional down-regulation of NK group 2D (NKG2D) ligands and are largely resistant to NK cell-mediated eradication. Because the down-regulation of NKG2D ligands occurred at the transcriptional level, we tested 32 chemical inhibitors of epigenetic regulators for their ability to re-express NKG2D ligands and enhance HCC cell eradication by NK cells and found that Enhancer of zeste homolog 2 (EZH2) was a transcriptional repressor of NKG2D ligands. The inhibition of EZH2 by small-molecule inhibitors or genetic means enhanced HCC cell eradication by NK cells in a NKG2D ligand-dependent manner. Collectively, these results demonstrate that EZH2 inhibition enhances HCC eradication by NK cells and that EZH2 functions, in part, as an oncogene by inhibiting immune response.

Robust Microplate-Based Methods for Culturing and in Vivo Phenotypic Screening of Chlamydomonas reinhardtii.

Chlamydomonas reinhardtii (Cr), a unicellular alga, is routinely utilized to study photosynthetic biochemistry, ciliary motility, and cellular reproduction. Its minimal culture requirements, unicellular morphology, and ease of transformation have made it a popular model system. Despite its relatively slow doubling time, compared with many bacteria, it is an ideal eukaryotic system for microplate-based studies utilizing either, or both, absorbance as well as fluorescence assays. Such microplate assays are powerful tools for researchers in the areas of toxicology, pharmacology, chemical genetics, biotechnology, and more. However, while microplate-based assays are valuable tools for screening biological systems, these methodologies can significantly alter the conditions in which the organisms are cultured and their subsequent physiology or morphology. Herein we describe a novel method for the microplate culture and in vivo phenotypic analysis of growth, viability, and photosynthetic pigments of C. reinhardtii. We evaluated the utility of our assay by screening silver nanoparticles for their effects on growth and viability. These methods are amenable to a wide assortment of studies and present a significant advancement in the methodologies available for research involving this model organism.

Detection of biotin-streptavidin interactions based on poly(thymine)-templated copper nanoparticles coupled with Exo III-aided DNA recycling amplification

The recognition and analysis of small molecule–protein interactions plays an important role in chemical genetics, clinical diagnostics, and drug development. However, various established highly sensitive fluorescence strategies still involve the use of expensive fluorescence dye-labelled nucleic acids. Herein, we constructed a highly sensitive and label-free fluorescent assay for small molecule–protein interactions on the basis of exonuclease III (Exo III)-assisted signal amplification and poly(thymine)-templated fluorescent copper nanoparticles (CuNPs) as signal probes. Biotin–streptavidin system was taken as a model. It was designed that biotinylated double-strand DNA served as the binding probe. Biotin was labelled at the 3′-end of the trigger strand. One kind of stem-loop DNA, which consisted of the thymine rich sequence and the trigger DNA recognition domain, was also designed. Once streptavidin bind to the binding probe, with the help of Exo III digestion, the trigger strand was able to reach the cycle for multiple poly(thymine) sequences generation. The produced poly(thymine) sequences can be used as a template for the formation of CuNPs. Comparing with no Exo III-aided DNA recycling amplification, significantly improved signals for the quantitative determination of streptavidin were obtained.

How Surrogate and Chemical Genetics in Model Organisms Can Suggest Therapies for Human Genetic Diseases.

Genetic diseases are both inherited and acquired. Many genetic diseases fall under the paradigm of orphan diseases, a disease found in < 1 in 2000 persons. With rapid and cost-effective genome sequencing becoming the norm, many causal mutations for genetic diseases are being rapidly determined. In this regard, model organisms are playing an important role in validating if specific mutations identified in patients drive the observed phenotype. An emerging challenge for model organism researchers is the application of genetic and chemical genetic platforms to discover drug targets and drugs/drug-like molecules for potential treatment options for patients with genetic disease. This review provides an overview of how model organisms have contributed to our understanding of genetic disease, with a focus on the roles of yeast and zebrafish in gene discovery and the identification of compounds that could potentially treat human genetic diseases.

MycoKey Round Table Discussions of Future Directions in Research on Chemical Detection Methods, Genetics and Biodiversity of Mycotoxins.

MycoKey, an EU-funded Horizon 2020 project, includes a series of “Roundtable Discussions” to gather information on trending research areas in the field of mycotoxicology. This paper includes summaries of the Roundtable Discussions on Chemical Detection and Monitoring of mycotoxins and on the role of genetics and biodiversity in mycotoxin production. Discussions were managed by using the nominal group discussion technique, which generates numerous ideas and provides a ranking for those identified as the most important. Four questions were posed for each research area, as well as two questions that were common to both discussions. Test kits, usually antibody based, were one major focus of the discussions at the Chemical Detection and Monitoring roundtable because of their many favorable features, e.g., cost, speed and ease of use. The second area of focus for this roundtable was multi-mycotoxin detection protocols and the challenges still to be met to enable these protocols to become methods of choice for regulated mycotoxins. For the genetic and biodiversity group, both the depth and the breadth of trending research areas were notable. For some areas, e.g., microbiome studies, the suggested research questions were primarily of a descriptive nature. In other areas, multiple experimental approaches, e.g., transcriptomics, proteomics, RNAi and gene deletions, are needed to understand the regulation of toxin production and mechanisms underlying successful biological controls. Answers to the research questions will provide starting points for developing acceptable prevention and remediation processes. Forging a partnership between scientists and appropriately-placed communications experts was recognized by both groups as an essential step to communicating risks, while retaining overall confidence in the safety of the food supply and the integrity of the food production chain.

CDK activity provides temporal and quantitative cues for organizing genome duplication.

In eukaryotes, the spatial and temporal organization of genome duplication gives rise to distinctive profiles of replication origin usage along the chromosomes. While it has become increasingly clear that these programs are important for cellular physiology, the mechanisms by which they are determined and modulated remain elusive. Replication initiation requires the function of cyclin-dependent kinases (CDKs), which associate with various cyclin partners to drive cell proliferation. Surprisingly, although we possess detailed knowledge of the CDK regulators and targets that are crucial for origin activation, little is known about whether CDKs play a critical role in establishing the genome-wide pattern of origin selection. We have addressed this question in the fission yeast, taking advantage of a simplified cell cycle network in which cell proliferation is driven by a single cyclin-CDK module. This system allows us to precisely control CDK activity in vivo using chemical genetics. First, in contrast to previous reports, our results clearly show that distinct cyclin-CDK pairs are not essential for regulating specific subsets of origins and for establishing a normal replication program. Importantly, we then demonstrate that the timing at which CDK activity reaches the S phase threshold is critical for the organization of replication in distinct efficiency domains, while the level of CDK activity at the onset of S phase is a dose-dependent modulator of overall origin efficiencies. Our study therefore implicates these different aspects of CDK regulation as versatile mechanisms for shaping the architecture of DNA replication across the genome.

Taking Aim at Glycolysis with CDK8 Inhibitors

Dependence on glycolysis under aerobic conditions, a frequent metabolic derangement in cancer cells, suggests a therapeutic opportunity. Now, through chemical genetics, CDK8, a kinase associated with the Mediator transcriptional coactivator complex, has emerged as an upstream inducer of glycolysis and a possible target for anticancer drug discovery.

Functional analysis after rapid degradation of condensins and 3D-EM reveals chromatin volume is uncoupled from chromosome architecture in mitosis.

ABSTRACTThe requirement for condensin in chromosome formation in somatic cells remains unclear, as imperfectly condensed chromosomes do form in cells depleted of condensin by conventional methodologies. In order to dissect the roles of condensin at different stages of vertebrate mitosis, we have established a versatile cellular system that combines auxin-mediated rapid degradation with chemical genetics to obtain near-synchronous mitotic entry of chicken DT40 cells in the presence and absence of condensin. We analyzed the outcome by live- and fixed-cell microscopy methods, including serial block face scanning electron microscopy with digital reconstruction. Following rapid depletion of condensin, chromosomal defects were much more obvious than those seen after a slow depletion of condensin. The total mitotic chromatin volume was similar to that in control cells, but a single mass of mitotic chromosomes was clustered at one side of a bent mitotic spindle. Cultures arrest at prometaphase, eventually exiting mitosis without segregating chromosomes. Experiments where the auxin concentration was titrated showed that different condensin levels are required for anaphase chromosome segregation and formation of a normal chromosome architecture.This article has an associated First Person interview with the first author of the paper.

Inverse Drug Screening of Bioelectric Signaling and Neurotransmitter Roles: Illustrated Using a Xenopus Tail Regeneration Assay.

Xenopus embryos and larvae are an ideal model system in which to study the interplay between genetics, physiology, and anatomy in the control of structure and function. An important emerging field is the study of bioelectric signaling, the exchange of ion- and neurotransmitter-mediated messages among all types of cells (not just nerve and muscle cells), in the regulation of growth and form during embryogenesis, regeneration, and cancer. To facilitate the mechanistic investigation of bioelectric events in vivo, it is necessary to identify the endogenous signaling machinery involved in any patterning process of interest. This protocol uses the tail regeneration assay in Xenopus to perform an inverse drug screen; tiers of known compounds are used to probe the involvement of increasingly specific classes of bioelectric and neurotransmitter machinery. By using a hierarchical approach, large classes of targets are ruled out in early rounds, focusing attention on progressively narrower sets of proteins. Such a screen avoids many of the limitations of a molecular-genetic targeting approach and provides a rapid and efficient way to focus on specific targets. Usually, <10 experiments are needed to determine whether bioelectrics and/or neurotransmitter signaling are involved in the process of interest. This protocol describes the strategy in the context of a semiquantitative analysis of tail regeneration but can be applied to any assay in Xenopus or other small aquatic model system (e.g., zebrafish). Given the ever-increasing toolkit of chemical genetics, such screens represent a powerful and versatile methodology for probing the physiological circuits underlying pattern regulation.

Combined chemical genetics and data-driven bioinformatics approach identifies receptor tyrosine kinase inhibitors as host-directed antimicrobials

Antibiotic resistance poses rapidly increasing global problems in combatting multidrug-resistant (MDR) infectious diseases like MDR tuberculosis, prompting for novel approaches including host-directed therapies (HDT). Intracellular pathogens like Salmonellae and Mycobacterium tuberculosis (Mtb) exploit host pathways to survive. Only very few HDT compounds targeting host pathways are currently known. In a library of pharmacologically active compounds (LOPAC)-based drug-repurposing screen, we identify multiple compounds, which target receptor tyrosine kinases (RTKs) and inhibit intracellular Mtb and Salmonellae more potently than currently known HDT compounds. By developing a data-driven in silico model based on confirmed targets from public databases, we successfully predict additional efficacious HDT compounds. These compounds target host RTK signaling and inhibit intracellular (MDR) Mtb. A complementary human kinome siRNA screen independently confirms the role of RTK signaling and kinases (BLK, ABL1, and NTRK1) in host control of Mtb. These approaches validate RTK signaling as a drugable host pathway for HDT against intracellular bacteria.

Introducing Our Authors

ADVERTISEMENT RETURN TO ISSUEIntroducing Our Auth...Introducing Our AuthorsNEXTIntroducing Our AuthorsBrian WylerCite this: ACS Chem. Biol. 2018, 13, 1, 1–3Publication Date (Web):January 19, 2018Publication History Published online19 January 2018Published inissue 19 January 2018https://doi.org/10.1021/acschembio.8b00020Copyright © 2018 American Chemical SocietyRIGHTS & PERMISSIONSArticle Views607Altmetric-Citations-LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (5 MB) Get e-AlertsSUBJECTS:Chemical biology,Genetics,Peptides and proteins,Small molecules,Therapeutics Get e-Alerts

Identification of Chemical Inducers of the Phosphate-Starvation Signaling Pathway in A. thaliana Using Chemical Genetics.

In spite of its importance for agriculture and 30years of genetic studies, the phosphate-starvation signaling pathway, that allows plants to detect, respond, and adapt to changes in the phosphate concentration of the rhizosphere, remains poorly known. Chemical genetics has been increasingly and successfully used as a complementary approach to genetics for the dissection of signaling pathways in diverse organisms. Screens can be designed to identify chemicals interfering specifically with a pathway of interest. We designed a screen that led to the discovery of the first chemical capable to induce specifically the phosphate-starvation signaling pathway in Arabidopsis thaliana. This procedure, described here, can be adapted for the discovery of inducers or repressors of other pathways.

High-Throughput Screening of Chemical Compound Libraries for Modulators of Salicylic Acid Signaling by In Situ Monitoring of Glucuronidase-Based Reporter Gene Expression.

Salicylic acid (SA) is a vital phytohormone that is intimately involved in coordination of the complex plant defense response to pathogen attack. Many aspects of SA signaling have been unraveled by classical genetic and biochemical methods using the model plant Arabidopsis thaliana, but many details remain unknown, owing to the inherent limitations of these methods. In recent years, chemical genetics has emerged as an alternative scientific strategy to complement classical genetics by virtue of identifying bioactive chemicals or probes that act selectively on their protein targets causing either activation or inhibition. Such selective tools have the potential to create conditional and reversible chemical mutant phenotypes that may be combined with genetic mutants. Here, we describe a facile chemical screening methodology for intact Arabidopsis seedlings harboring the β-glucuronidase (GUS) reporter by directly quantifying GUS activity in situ with 4-methylumbelliferyl-β-D-glucuronide (4-MUG) as substrate. The quantitative nature of this screening assay has an obvious advantage over the also convenient histochemical GUS staining method, as it allows application of statistical procedures and unbiased hit selection based on threshold values as well as distinction between compounds with strong or weak bioactivity. We show pilot screens for chemical activators or inhibitors of salicylic acid-mediated defense signaling using the Arabidopsis line expressing the SA-inducible PR1p::GUS reporter gene. Importantly, the screening methodology provided here can be adopted for any inducible GUS reporter line.

Cheminformatics Tools for Analyzing and Designing Optimized Small Molecule Collections and Libraries

Libraries of well-annotated small molecules have many uses in chemical genetics, drug discovery and drug repurposing. Many such libraries have become available, but few data-driven approaches exist to compare these libraries and design new ones. In this paper, we describe an approach to scoring and creating libraries based on available data, which is often incomplete, on binding selectivity, target coverage and induced cellular phenotypes as well as chemical structure, stage of clinical development and user preference. The approach, implemented as R software and a Web-accessible tool also assembles small sets of compounds (typically 2-8) that target a protein of interest while having the lowest possible off target overlap. Analysis of six kinase inhibitor libraries using our approach reveals dramatic differences among them, leading us to design a new LSP-OptimalKinase library that outperforms all previous collections in terms of target coverage and compact size. We also assemble a mechanism of action library that optimally covers 1852 targets of the liganded genome. Using our tools, individual research groups and companies can quickly analyze private compound collections, public libraries can be updated based on the latest data and the confounding effects of off-target activities minimized in chemical genetic studies.

Chemical Genomics Translatability from Unicellular to Multicellular Models.

Chemical genomics has proven to be a useful and successful approach to study complex systems where conventional genetics fails to render feasible results. High-throughput phenotype screenings in model organisms have identified a large collection of powerful and selective bioactive chemicals. Nevertheless, applying chemical high-throughput screening to crops still represents a big challenge for researchers. Fortunately, a circumvent approach could be taken by means of translational research. In this case, searching bioactive chemicals in a much handy model organism would be the starting point for discovering compounds with activity in relevant plants for improving a desirable trait. In this chapter, we describe strategies that have been proven to successfully translate chemical biology and genetics from unicellular yeast to Arabidopsis thaliana and finally to crops.

Optimization of a "bump-and-hole" approach to allele-selective BET bromodomain inhibition.

Allele-specific chemical genetics enables selective inhibition within families of highly-conserved proteins. The four BET (bromodomain & extra-terminal domain) proteins - BRD2, BRD3, BRD4 and BRDT bind acetylated chromatin via their bromodomains and regulate processes such as cell proliferation and inflammation. BET bromodomains are of particular interest, as they are attractive therapeutic targets but existing inhibitors are pan-selective. We previously established a bump-&-hole system for the BET bromodomains, pairing a leucine/alanine mutation with an ethyl-derived analogue of an established benzodiazepine scaffold. Here we optimize upon this system with the introduction of a more conservative and less disruptive leucine/valine mutation. Extensive structure-activity-relationships of diverse benzodiazepine analogues guided the development of potent, mutant-selective inhibitors with desirable physiochemical properties. The active enantiomer of our best compound - 9-ME-1 - shows ∼200 nM potency, >100-fold selectivity for the L/V mutant over wild-type and excellent DMPK properties. Through a variety of in vitro and cellular assays we validate the capabilities of our optimized system, and then utilize it to compare the relative importance of the first and second bromodomains to chromatin binding. These experiments confirm the primacy of the first bromodomain in all BET proteins, but also significant variation in the importance of the second bromodomain. We also show that, despite having a minor role in chromatin recognition, BRD4 BD2 is still essential for gene expression, likely through the recruitment of non-histone proteins. The disclosed inhibitor:mutant pair provides a powerful tool for future cellular and in vivo target validation studies.