Search

Background: Microbial conversion of lignocellulosic feedstocks into biofuels remains an attractive means to produce sustainable energy. It is essential to produce lignocellulosic hydrolysates in a consistent manner in order to study microbial performance in different feedstock hydrolysates. Because of the potential to introduce microbial contamination from the untreated biomass or at various points during the process, it can be difficult to control sterility during hydrolysate production. In this study, we compared hydrolysates produced from AFEX-pretreated corn stover and switchgrass using two different methods to control contamination: either by autoclaving the pretreated feedstocks prior to enzymatic hydrolysis, or by introducing antibiotics during the hydrolysis of non-autoclaved feedstocks. We then performed extensive chemical analysis, chemical genomics, and comparative fermentations to evaluate any differences between these two different methods used for producing corn stover and switchgrass hydrolysates. Results: Autoclaving the pretreated feedstocks could eliminate the contamination for a variety of feedstocks, whereas the antibiotic gentamicin was unable to control contamination consistently during hydrolysis. Compared to the addition of gentamicin, autoclaving of biomass before hydrolysis had a minimal effect on mineral concentrations, and showed no significant effect on the two major sugars (glucose and xylose) found in these hydrolysates. However, autoclaving elevated the concentration of some furanic and phenolic compounds. Chemical genomics analyses using Saccharomyces cerevisiae strains indicated a high correlation between the AFEX-pretreated hydrolysates produced using these two methods within the same feedstock, indicating minimal differences between the autoclaving and antibiotic methods. Comparative fermentations with S. cerevisiae and Zymomonas mobilis also showed that autoclaving the AFEX-pretreated feedstocks had no significant effects on microbial performance in these hydrolysates. Conclusions: Our results showed that autoclaving the pretreated feedstocks offered advantages over the addition of antibiotics for hydrolysate production. The autoclaving method produced a more consistent quality of hydrolysate, and also showed negligible effects on microbial performance. Although the levels of some of the lignocellulose degradation inhibitors were elevated by autoclaving the feedstocks prior to enzymatic hydrolysis, no significant effects on cell growth, sugar utilization, or ethanol production were seen during bacterial or yeast fermentations in hydrolysates produced using the two different methods. [ABSTRACT FROM AUTHOR]

The increasing emergence of antimicrobial multiresistant bacteria is of great concern to public health. While these bacteria are becoming an ever more prominent cause of nosocomial and community-acquired infections worldwide, the antibiotic discovery pipeline has been stalled in the last few years with very few efforts in the research and development of novel antibacterial therapies. Some of the root causes that have hampered current antibiotic drug development are the lack of understanding of the mode of action (MOA) of novel antibiotic molecules and the poor characterization of the bacterial physiological response to antibiotics that ultimately causes resistance. Here, we review how bacterial genetic tools can be applied at the genomic level with the goal of profiling resistance to antibiotics and elucidating antibiotic MOAs. Specifically, we highlight how chemical genomic detection of the MOA of novel antibiotic molecules and antibiotic profiling by next-generation sequencing are leveraging basic antibiotic research to unprecedented levels with great opportunities for knowledge translation. [ABSTRACT FROM AUTHOR]

Although most neurodegenerative diseases have been closely related to aberrant accumulation of aggregation-prone proteins in neurons, understanding their pathogenesis remains incomplete, and there is no treatment to delay the onset or slow the progression of many neurodegenerative diseases. The availability of induced pluripotent stem cells (iPSCs) in recapitulating the phenotypes of several late-onset neurodegenerative diseases marks the new era in in vitro modeling. The iPSC collection represents a unique and well-characterized resource to elucidate disease mechanisms in these diseases and provides a novel human stem cell platform for screening new candidate therapeutics. Modeling human diseases using iPSCs has created novel opportunities for both mechanistic studies as well as for the discovery of new disease therapies. In this review, we introduce iPSC-based disease modeling in neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease, Huntington's disease, and amyotrophic lateral sclerosis. In addition, we discuss the implementation of iPSCs in drug discovery associated with some new techniques. [ABSTRACT FROM AUTHOR]

De novo formation of a new cell wall partitions the cytoplasm of the dividing cell during plant cytokinesis. The development of the cell plate, a transient sheet-like structure, requires the accumulation of vesicles directed by the phragmoplast to the cell plate assembly matrix. Fusion and fission of the accumulated vesicles are accompanied by the deposition of polysaccharides and cell wall structural proteins; together, they are leading to the stabilization of the formed structure which after insertion into the parental wall lead to the maturation of the nascent cross wall. Callose is the most abundant polysaccharide during cell plate formation and during maturation is gradually replaced by cellulose. Matrix polysaccharides such as hemicellulose, and pectins presumably are present throughout all developmental stages, being delivered to the cell plate by secretory vesicles. The availability of novel chemical probes such as endosidin 7, which inhibits callose formation at the cell plate, has proved useful for dissecting the temporal accumulation of vesicles at the cell plate and establishing the critical role of callose during cytokinesis. The use of emerging approaches such as chemical genomics combined with live cell imaging; novel techniques of polysaccharide detection including tagged polysaccharide substrates, newly characterized polysaccharide antibodies and vesicle proteomics can be used to develop a comprehensive model of cell plate development. [ABSTRACT FROM AUTHOR]

Candida albicans is a prevalent fungal pathogen of humans that can cause both superficial and life-threatening disease, primarily in immunocompromised populations. Currently, antifungal drug classes available to treat fungal infections remain limited and the emergence of drug-resistant strains threatens antifungal efficacy, necessitating the discovery and development of additional therapeutics. The construction of the C. albicans double-barcoded heterozygous deletion collection (DBC) enables the rapid and systematic assessment of haploinsufficiency phenotypes in a pooled format. Specifically, this functional genomics resource can be used to identify heterozygous deletion mutants that are hypersensitive to compounds in order to define putative cellular targets and/or other modifiers of compound activity. Here, we describe protocols to characterize the mode of action of small molecules using the C. albicans DBC, including how to prepare compound-treated cultures, isolate genomic DNA, amplify strain-specific barcodes, and prepare DNA libraries for high-throughput sequencing. This technique provides a powerful approach to elucidate the compound mechanism of action in order to bolster the antifungal pipeline., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Plant adaptation, growth and development rely on the integration of many environmental and endogenous signals that collectively determine the overall plant phenotypic plasticity. Plant signaling molecules, also known as phytohormones, are fundamental to this process. These molecules act at low concentrations and regulate multiple aspects of plant fitness and development via complex signaling networks. By its nature, phytohormone research lies at the interface between chemistry and biology. Classically, the scientific community has always used synthetic phytohormones and analogs to study hormone functions and responses. However, recent advances in synthetic and combinational chemistry, have allowed a new field, plant chemical biology, to emerge and this has provided a powerful tool with which to study phytohormone function. Plant chemical biology is helping to address some of the most enduring questions in phytohormone research such as: Are there still undiscovered plant hormones? How can we identify novel signaling molecules? How can plants activate specific hormone responses in a tissue-specific manner? How can we modulate hormone responses in one developmental context without inducing detrimental effects on other processes? The chemical genomics approaches rely on the identification of small molecules modulating different biological processes and have recently identified active forms of plant hormones and molecules regulating many aspects of hormone synthesis, transport and response. We envision that the field of chemical genomics will continue to provide novel molecules able to elucidate specific aspects of hormone-mediated mechanisms. In addition, compounds blocking specific responses could uncover how complex biological responses are regulated. As we gain information about such compounds we can design small alterations to the chemical structure to further alter specificity, enhance affinity or modulate the activity of these compounds. [ABSTRACT FROM AUTHOR]

Background Pollen tubes extend rapidly when pollen grains are incubated in defined media. Tube extension requires many critical functions of plant cells including molecular signaling, cytoskeleton remodeling, secretion, and cell wall synthesis. Consequently, pollen tube growth has been established as a way to conduct primary screens of chemical libraries to identify compounds that perturb key cellular processes in plants. Results Here we report a simple, inexpensive, rapid and semi-quantitative method for measurement of pollen tube growth in microtiter plates. The method relies on Congo Red binding to pollen tubes and correlates dye fluorescence to tube length. Conclusions This method can be used in any laboratory without specialized equipment, and has the potential to enable larger screens as chemical libraries grow and to make chemical screening accessible to researchers building specialized libraries designed to probe pathways in plant biology. [ABSTRACT FROM AUTHOR]

Owing to the absence of antiparasitic vaccines and the constant threat of drug resistance, the development of novel antiparasitic chemotherapies remains of major importance for disease control. A better understanding of drug transport (uptake and efflux), drug metabolism and the identification of drug targets, and mechanisms of drug resistance would facilitate the development of more effective therapies. Here, we focus on malaria and African trypanosomiasis. We review existing drugs and drug development, emphasizing high-throughput genomic and genetic approaches, which hold great promise for elucidating antiparasitic mechanisms. We describe the approaches and technologies that have been influential for each parasite and develop new ideas for future research directions, including mode-of-action studies for drug target deconvolution. [ABSTRACT FROM AUTHOR]

Owing to the absence of antiparasitic vaccines and the constant threat of drug resistance, the development of novel antiparasitic chemotherapies remains of major importance for disease control. A better understanding of drug transport (uptake and efflux), drug metabolism and the identification of drug targets, and mechanisms of drug resistance would facilitate the development of more effective therapies. Here, we focus on malaria and African trypanosomiasis. We review existing drugs and drug development, emphasizing high-throughput genomic and genetic approaches, which hold great promise for elucidating antiparasitic mechanisms. We describe the approaches and technologies that have been influential for each parasite and develop new ideas for future research directions, including mode-of-action studies for drug target deconvolution. [ABSTRACT FROM AUTHOR]

Abstract: Conventional protein secretion is mediated by the endomembrane system. Secreted proteins are inserted into the endomembrane system through a N-terminal signal peptide and follow the endoplasmic reticulum to the Golgi trafficking pathway en route to the plasma membrane or the extracellular apoplastic space. In mammalian and yeast cells, unconventional secretion has been identified and relatively well studied. Also in plants, evidence of unconventional secretion mechanisms is accumulating. The ever-increasing number of leaderless proteins identified in proteomic studies indicates the importance of unconventional protein secretion in plants. Novel approaches, such as chemical genomics and vesicle proteomics might be able to provide new insights into unconventional protein secretion in plants. [Copyright &y& Elsevier]

The cell wall, a crucial cell compartment, is composed of a network of polysaccharides and proteins, providing structural support and protection from external stimuli. While the cell wall structure and biosynthesis have been extensively studied, very little is known about the transport of polysaccharides and other components into the developing cell wall. This review focuses on endomembrane trafficking pathways involved in cell wall deposition. Cellulose synthase complexes are assembled in the Golgi, and are transported in vesicles to the plasma membrane. Non-cellulosic polysaccharides are synthesized in the Golgi apparatus, whereas cellulose is produced by enzyme complexes at the plasma membrane. Polysaccharides and enzymes that are involved in cell wall modification and assembly are transported by distinct vesicle types to their destinations; however, the precise mechanisms involved in selection, sorting and delivery remain to be identified. The endomembrane system orchestrates the delivery of Golgi-derived and possibly endocytic vesicles carrying cell wall and cell membrane components to the newly-formed cell plate. However, the nature of these vesicles, their membrane compositions, and the timing of their delivery are largely unknown. Emerging technologies such as chemical genomics and proteomics are promising avenues to gain insight into the trafficking of cell wall components. [ABSTRACT FROM AUTHOR]

ABSTRACT Computational life sciences and informatics are inseparably intertwined and they lie at the heart of modern biology, predictive quantitative modeling and high-performance computing. Two of the applied biological disciplines that are poised to benefit from such progress are pharmacology and toxicology. This review will describe in silico chemoinformatics methods such as (quantitative) structure-activity relationship modeling and will overview how chemoinformatic technologies are considered in applied regulatory research. Given the post-genomics era and large-scale repositories of omics data that are available, this review will also address potential applications of in silico techniques in chemical genomics. Chemical genomics utilizes small molecules to explore the complex biological phenomena that may not be not amenable to straightforward genetic approach. The reader will gain the understanding that chemoinformatics stands at the interface of chemistry and biology with enabling systems for mapping, statistical modeling, pattern recognition, imaging and database tools. The great potential of these technologies to help address complex issues in the toxicological sciences is appreciated with the applied goal of the protection of public health. Published 2012. This article is a US Government work and is in the public domain in the USA. [ABSTRACT FROM AUTHOR]

Conventional chemotherapy not only kills tumor cells but also changes gene expression in treatment-damaged tissues, inducing production of multiple tumor-supporting secreted factors. This secretory pheno-type was found here to be mediated in part by a damage-inducible cell-cycle inhibitor p21 (CDKN1A). We developed small-molecule compounds that inhibit damage-induced transcription downstream of p21. These compounds were identified as selective inhibitors of a transcription-regulating kinase CDK8 and its isoform CDK19. Remarkably, p21 was found to bind to CDK8 and stimulate its kinase activity p21 and CDK8 also cooperate in the formation of internucleo-lar bodies, where both proteins accumulate. A CDK8 inhibitor suppresses damage-induced tumor-promoting paracrine activities of tumor cells and normal fibroblasts and reverses the increase in tumor engraftment and serum mitogenic activity in mice pretreated with a chemotherapeutic drug. The inhibitor also increases the efficacy of chemotherapy against xenografts formed by tumor cell/fibroblast mixtures. Microarray data analysis revealed striking correlations between CDK8 expression and poor survival in breast and ovarian cancers. CDK8 inhibition offers a promising approach to increasing the efficacy of cancer chemotherapy. [ABSTRACT FROM AUTHOR]

Since the introduction of chemical genomics to plant biology as a tool for basic research, the field has advanced significantly. There are now examples of important basic discoveries that demonstrate the power and untapped potential of this approach. Given the combination of protein and small-molecule complexity, new phenotypes can be described through the perturbation of cellular functions that can be linked to growth and developmental phenotypes. There are now clear examples of overcoming functional redundancy in plants to dissect molecular mechanisms or critical pathways such as hormone signaling and dynamic intracellular processes. Owing to ongoing advances, including more sophisticated high-content screening and rapid approaches for target identification, the field is beginning to move forward. However, there are also challenges to improve automation, imaging, and analysis and provide chemical biology resources to the broader plant biology community. [ABSTRACT FROM AUTHOR]

Abstract: 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. [Copyright &y& Elsevier]

Background: The sulfanilamide family comprises a clinically important group of antimicrobial compounds which also display bioactivity in plants. While there is evidence that sulfanilamides inhibit folate biosynthesis in both bacteria and plants, the complete network of plant responses to these compounds remains to be characterized. As such, we initiated two forward genetic screens in Arabidopsis in order to identify mutants that exhibit altered sensitivity to sulfanilamide compounds. These screens were based on the growth phenotype of seedlings germinated in the presence of the compound sulfamethoxazole (Smex). Results: We identified a mutant with reduced sensitivity to Smex, and subsequent mapping indicated that a gene encoding 5-oxoprolinase was responsible for this phenotype. A mutation causing enhanced sensitivity to Smex was mapped to a gene lacking any functional annotation. Conclusions: The genes identified through our forward genetic screens represent novel mediators of Arabidopsis responses to sulfanilamides and suggest that these responses extend beyond the perturbation of folate biosynthesis [ABSTRACT FROM AUTHOR]

BACKGROUND: The large and increasing number of chemicals released into the environment demands more efficient and cost-effective approaches for assessing environmental chemical toxicity. The U.S. Tox21 program has responded to this challenge by proposing alternative strategies for toxicity testing, among which the quantitative high-throughput screening (qHTS) paradigm has been adopted as the primary tool for generating data from screening large chemical libraries using a wide spectrum of assays. OBJECTIVES: The goal of this study was to develop methods to evaluate the data generated from these assays to guide future assay selection and prioritization for the Tox21 program. METHODS: We examined the data from the Tox21 pilot-phase collection of approximately 3,000 environmental chemicals profiled in qHTS format against a panel of 10 human nuclear receptors (AR, ERα, FXR, GR, LXRβ, PPARγ, PPARδ, RXRα, TRβ, and VDR) for reproducibility, concordance of biological activity profiles with sequence homology of the receptor ligand binding domains, and structure-activity relationships. RESULTS: We determined the assays to be appropriate in terms of biological relevance. We found better concordance for replicate compounds for the agonist-mode than for the antagonist-mode assays, likely due to interference of cytotoxicity in the latter assays. This exercise also enabled us to formulate data-driven strategies for discriminating true signals from artifacts, and to prioritize assays based on data quality. CONCLUSIONS: The results demonstrate the feasibility of qHTS to identify the potential for environmentally relevant chemicals to interact with key toxicity pathways related to human disease induction. [ABSTRACT FROM AUTHOR]

The discovery of novel bioactive molecules advances our systems-level understanding of biological processes and is crucial for innovation in drug development. For this purpose, the emerging field of chemical genomics is currently focused on accumulating large assay data sets describing compound–protein interactions (CPIs). Although new target proteins for known drugs have recently been identified through mining of CPI databases, using these resources to identify novel ligands remains unexplored. Herein, we demonstrate that machine learning of multiple CPIs can not only assess drug polypharmacology but can also efficiently identify novel bioactive scaffold-hopping compounds. Through a machine-learning technique that uses multiple CPIs, we have successfully identified novel lead compounds for two pharmaceutically important protein families, G-proteincoupled receptors and protein kinases. These novel compounds were not identified by existing computational ligand-screening methods in comparative studies. The results of this study indicate that data derived from chemical genomics can be highly useful for exploring chemical space, and this systems biology perspective could accelerate drug discovery processes. [ABSTRACT FROM AUTHOR]

utophagy is the mass degradation system that removes long-lived proteins and malfunctioning organelles within the cell. Dysfunctional autophagic processes can cause various diseases such as cancer and neurodegenerative disorders, but the underlying mechanisms responsible for such events remain undefined. Small molecules that control autophagy could be powerful tools to reveal autophagy mechanisms, and to develop treatments for autophagy-related diseases including Alzheimer's disease, Parkinson's disease and various cancer types. This review discusses the small molecules that have been identified to control autophagy and how they can be used to understand signaling pathways important for autophagy in the context of chemical genomics. [ABSTRACT FROM AUTHOR]

Abstract: Over the past decade, the development and application of chemical genomic assays using the model organism Saccharomyces cerevisiae has provided powerful methods to identify the mechanism of action of known drugs and novel small molecules in vivo. These assays identify drug target candidates, genes involved in buffering drug target pathways and also help to define the general cellular response to small molecules. In this review, we examine current yeast chemical genomic assays and summarize the potential applications of each approach. [Copyright &y& Elsevier]

Evaluating the specificity spectra of DNA binding molecules is a nontrivial challenge that hinders the ability to decipher gene regulatory networks or engineer molecules that act on genomes. Here we compare the DNA sequence specificities for different classes of proteins and engineered DNA binding molecules across the entire sequence space. These high-content data are visualized and interpreted using an interactive "specificity landscape" which simultaneously displays the affinity and specificity of a million-plus DNA sequences. Contrary to expectation, specificity landscapes reveal that synthetic DNA ligands match, and often surpass, the specificities of eukaryotic DNA binding proteins. The landscapes also identify differential specificity constraints imposed by diverse structural folds of natural and synthetic DNA binders. Importantly, the sequence context of a binding site significantly influences binding energetics, and utilizing the full contextual information permits greater accuracy in annotating regulatory elements within a given genome. Assigningsuch context-dependent binding values to every DNA sequence across the genome yields predictive genome-wide binding landscapes (genomescapes). A genomescape of a synthetic DNA binding molecule provided insight into its differential regulatory activity in cultured cells. The approach we describe will accelerate the creation of precision-tailored DNA therapeutics and uncover principles that govern sequence-specificity of DNA binding molecules. [ABSTRACT FROM AUTHOR]

Contents The success of the genomics revolution to construct a genetic architecture of a variety of model organisms has placed functional biologists under pressure to show what each individual gene does in vivo. Traditionally, this task has fallen on geneticists who systematically perturb gene function and study the consequences. With the advent of large, easily accessible, small-molecule libraries and new methods of chemical synthesis, biologists now have new ways to probe gene function. Often called chemical genetics, this approach involves the screening of compounds that perturb a process of interest. In this scenario, each perturbing chemical is analogous to a specific mutation. Here, we summarize, with specific examples, how chemical genetics is being used in combination with traditional genetics to address problems in plant biology. Because chemical genetics is rooted in genetic analysis, we focus on how chemicals used in combination with genetics can be very powerful in dissecting a process of interest. [ABSTRACT FROM AUTHOR]

Cell-based, phenotypic screening of small molecules often identifies compounds with provocative biological properties. However, determining the cellular target(s) and/or mechanism of action (MoA) of lead compounds remains an extremely challenging and time-consuming exercise. To provide insights into a compound''s cellular action and greatly reduce the time required for MoA determination, we have developed a screening platform consisting of an extensive series of reporter gene assays (RGAs). A collection of >11,000 compounds of known MoA (e.g., World Drug Index entries) were screened against the entire panel. The output provided evidence that an RGA signature could be ascribed to numerous, biologically diverse MoAs. The reference database generated suggested novel biological activity for particular compounds. For example, the profiling data led to the prediction that the cellular target of the natural product terprenin was dihydroorotate dehydrogenase (DHODH), which was confirmed experimentally. The screening methodology developed for this endeavor renders it amenable to the future examination of compounds with unknown MoA, in an automated, inexpensive, and time-efficient manner. [Copyright &y& Elsevier]

Post hoc assignment of patterns determined by all pairwise comparisons in microarray experiments with multiple treatments has been proven to be useful in assessing treatment effects. We propose the usage of transitive directed acyclic graphs (tDAG) as the representation of these patterns and show that such representation can be useful in clustering treatment effects, annotating existing clustering methods, and analyzing sample sizes. Advantages of this approach include: (1) unique and descriptive meaning of each cluster in terms of how genes respond to all pairs of treatments; (2) insensitivity of the observed patterns to the number of genes analyzed; and (3) a combinatorial perspective to address the sample size problem by observing the rate of contractible tDAG as the number of replicates increases. The advantages and overall utility of the method in elaborating drug structure activity relationships are exemplified in a controlled study with real and simulated data. [ABSTRACT FROM AUTHOR]

We analyzed global gene expression in Arabidopsis in response to various hormones and in related experiments as part of the AtGenExpress project. The experimental agents included seven basic phytohormones (auxin, cytokinin, gibberellin, brassinosteroid, abscisic acid, jasmonate and ethylene) and their inhibitors. In addition, gene expression was investigated in hormone-related mutants and during seed germination and sulfate starvation. Hormone-inducible genes were identified from the hormone response data. The effects of each hormone and the relevance of the gene lists were verified by comparing expression profiles for the hormone treatments and related experiments using Pearson’s correlation coefficient. This approach was also used to analyze the relationships among expression profiles for hormone responses and those included in the AtGenExpress stress-response data set. The expected correlations were observed, indicating that this approach is useful to monitor the hormonal status in the stress-related samples. Global interactions among hormones-inducible genes were analyzed in a pairwise fashion, and several known and novel hormone interactions were detected. Genome-wide transcriptional gene-to-gene correlations, analyzed by hierarchical cluster analysis (HCA), indicated that our data set is useful for identification of clusters of co-expressed genes, and to predict the functions of unknown genes, even if a gene’s function is not directly related to the experiments included in AtGenExpress. Our data are available online from AtGenExpressJapan; the results of genome-wide HCA are available from PRIMe. The data set presented here will be a versatile resource for future hormone studies, and constitutes a reference for genome-wide gene expression in Arabidopsis. [ABSTRACT FROM AUTHOR]

The discovery of new small molecules and their testing in rational combination poses an ongoing problem for rare diseases, in particular, for pediatric cancers such as neuroblastoma. Despite maximal cytotoxic therapy with double autologous stem cell transplantation, outcome remains poor for children with high-stage disease. Because differentiation is aberrant in this malignancy, compounds that modulate transcription, such as histone deacetylase (HDAC) inhibitors, are of particular interest. However, as single agents, HDAC inhibitors have had limited efficacy. In the present study, we use an HDAC inhibitor as an enhancer to screen a small-molecule library for compounds inducing neuroblastoma maturation. To quantify differentiation, we use an enabling gene expression-based screening strategy. The top hit identified in the screen was aIl-trans-retinoic acid. Secondary assays confirmed greater neuroblastoma differentiation with the combination of an HDAC inhibitor and a retinoid versus either alone. Furthermore, effects of combination therapy were synergistic with respect to inhibition of cellular viability and induction of apoptosis. In a xenograft model of neuroblastoma, animals treated with combination therapy had the longest survival. This work suggests that testing of an HDAC inhibitor and retinoid in combination is warranted for children with neuroblastoma and demonstrates the success of a signature-based screening approach to prioritize compound combinations for testing in rare diseases. [ABSTRACT FROM AUTHOR]

Although it is known that proteins are delivered to and recycled from the plasma membrane (PM) via endosomes, the nature of the compartments and pathways responsible for cargo and vesicle sorting and cellular signaling is poorly understood. To define and dissect specific recycling pathways, chemical effectors of proteins involved in vesicle trafficking, especially through endosomes, would be invaluable. Thus, we identified chemicals affecting essential steps in PM/endosome trafficking, using the intensely localized PM transport at the tips of germinating pollen tubes. The basic mechanisms of this localized growth are likely similar to those of non-tip growing cells in seedlings. The compound endosidin 1 (ES1) interfered selectively with endocytosis in seedlings, providing a unique tool to dissect recycling pathways. ES1 treatment induced the rapid agglomeration of the auxin translocators PIN2 and AUX1 and the brassinosteroid receptor BRI1 into distinct endomembrane compartments termed "endosidin bodies"; however, the markers PIN1, PIN7, and other PM proteins were unaffected. Endosidin bodies were defined by the syntaxin SYP61 and the V-ATPase subunit VHA-a1, two trans-Golgi network. (TGN)/endosomal proteins. Interestingly, brassinosteroid (BR)-induced gene expression was inhibited by ES1 and treated seedlings displayed a brassinolide (BL)-insensitive phenotype similar to a bri1 loss-of-function mutant. No effect was detected in auxin signaling. Thus, PIN2, AUX1, and BRI1 use interactive pathways involvingan early SYP61/VHA-a1 endosomal compartment. [ABSTRACT FROM AUTHOR]

Chemogenomics involves the combination of a compound's effect on biological targets together with modern genomics technologies. The merger of these two methodologies is creating a new way to screen for compound—target interactions, as well as map chemical and biological space in a parallel fashion. The challenge associated with mining complex databases has initiated the development of many novel in silico tools to profile and analyze data in a systematic way. The ability to analyze the combinatorial effects of chemical libraries on biological systems will aid the discovery of new therapeutic entities. Chemogenomics provides a tool for the rapid validation of novel targeted therapeutics, where a specific molecular target is modulated by a small molecule. Along with targeted therapies comes the ability to discovery pathway nodes where a single molecular target might be an essential component of more than one disease. Several disease areas will benefit directly from the chemogenomics approach, the most advanced being cancer. A genetic loss-of-function screen can be modulated in the presence of a compound to search for genes or pathways involved in the compound's activity. Several recent papers highlight how chemogenomics is changing with RNA interference-based screening and shaping the discovery of new targeted therapies. Together, chemical and RNA interference-based screens open the door for a new way to discovery disease-associated genes and novel targeted therapies. [ABSTRACT FROM AUTHOR]

Genetic analysis in budding yeast has shown that multiple G1 cyclins and cyclin-dependent kinases control cell cycle entry, polarized growth, and spindle pole duplication. The G1 cyclins Cln1 and Cln2 associate with the cyclin-dependent kinase Cdc28 to facilitate cell cycle progression and development of the cleavage apparatus. We have developed a chemical genetic approach toward the discovery of compounds that target G1 control pathways by screening for compounds that selectively kill a yeast strain lacking the G1 cyclins Cln1 and Cln2. A class of small molecules was identified that is highly toxic toward the cln1Δ cln2Δ double mutant and has relatively little effect on wild-type yeast. We call these compounds ‘clinostatins’ for their selectivity toward the cln1/2 deletion strain. Clinostatins were used in a genome-wide chemical synthetic lethality screen to identify other genes required for growth in the presence of the drug. Other deletions that were sensitive to the drug include members of the protein kinase C(PKC)-dependent MAP kinase pathway. These results suggest an approach for combining chemical synthetic lethality and chemical genomic screens to uncover novel genetic interactions that can be applied to other eukaryotic pathways of interest. [ABSTRACT FROM AUTHOR]

Determining the sequence-recognition properties of DNA-binding proteins and small molecules remains a major challenge. To address this need, we have developed a high-throughput approach that provides a comprehensive profile of the binding properties of DNA-binding molecules. The approach is based on displaying every permutation of a duplex DNA sequence (up to 10 positional variants) on a microfabricated array. The entire sequence space is interrogated simultaneously, and the affinity of a DNA-binding molecule for every sequence is obtained in a rapid, unbiased, and unsupervised manner. Using this platform, we have determined the full molecular recognition profile of an engineered small molecule and a eukaryotic transcription factor. The approach also yielded unique insights into the altered sequence-recognition landscapes as a result of cooperative assembly of DNA-binding molecules in a ternary complex. Solution studies strongly corroborated the sequence preferences identified by the array analysis. [ABSTRACT FROM AUTHOR]

The sequencing of the human genome has created both great opportunity and great risk for the pharmaceutical industry. Although the genome codes for all potential host targets for future drug development, the scale of new targets could result in decreased efficiencies owing to efforts expended on poor targets. Effective methods are required to model drug action in vivo in a mammal before costly development efforts begin and we review mouse knockouts and RNA interference as two modeling methods. [Copyright &y& Elsevier]

With the official completion of the Human Genome Project in April 2003, we have both the opportunity and the imperative to translate this unprecedented scientific accomplishment into tangible improvements in human health. Medical benefits from the genome will come in stages and can be conceptualized as occurring in three areas: improved understanding of disease causation at the molecular level, improved diagnosis and disease classification based on genetic profiles, and new therapeutics based on targets identified in the genome. These improvements will require increased physician understanding of genetic principles applied to common diseases. [ABSTRACT FROM AUTHOR]

Combinatorial library design can be carried out at either the reagent or the product level. Various reports in the literature have come to conflicting conclusions in favor of one over the other. In this paper a reagent-based screening library design strategy is presented. The method relies on analysis of scaffolds and building blocks separately to define the overall diversity in a compound file. The primary diversity selection by properties relevant for molecular recognition and by redundancy is followed by the application of filters for molecular properties known to be relevant for drug-likeness. Filter properties are rapidly estimated at the product level using a fragmental estimation approach. Initial experimental data suggest that high diversity in vast screening libraries can be achieved by carefully applied reagent level analysis. A potential role of diverse screening libraries in chemical genomics (pharmacological knockouts) is also discussed. [ABSTRACT FROM AUTHOR]

The current achievement of genome sequence projects of a dozen eukaryote organisms (including human genome) and the development of functional genomics are providing the basic knowledge required to utilize gene-specific reagents for both basic understanding of cell physiology and therapeutical development. The field of chemical genomics has the ambitious goal of designing molecules that could act selectively on every single gene or gene product in a cell and in vivo. The progress in oligonucleotide-based approaches will be the topic of this review, however, other nucleic acid- and SELEX-based approaches as well as high sequence-specific low molecular weight DNA-specific ligands will also be discussed. [ABSTRACT FROM AUTHOR]

Chemical genomics is a new research paradigm with importantapplications in drug discovery. It links genomic targets withsmall-molecule chemistries thereby allowing for efficient targetvalidation and lead compound identification. ACADIA'schemical-genomics platform consists of a large and diverse small-moleculelibrary (800,000), a reference drug library (2,000), druggablegenomic targets (>300) and a cell-based functional assaytechnology (R-SATTM; Receptor Selection and AmplificationTechnology) that allows for ultra-high throughput screening(>500,000 data points/week) as well as high throughputpharmacology and profiling over a wide range of targets. Twoexamples are presented that illustrate the success of ourchemical-genomics approach: (i) The validation of inverse agonismat serotonin 5-HT2A receptors as an antipsychotic mechanismand the subsequent discovery of potent and selectively acting 5-HT2A inverse agonists, currently in preclinical development,and (ii) the discovery of the first ectopically binding subtype-selective muscarinic m1 agonist. [ABSTRACT FROM AUTHOR]

Small molecules that can activate abscisic acid (ABA) receptors represent valuable probes to study ABA perception and signaling. Additionally, these compounds have the potential to be used in the field to counteract the negative effect of drought stress on plant productivity. The PYR/PYL ABA receptors, in their ligand-bound conformation, inactivate protein phosphatases 2C (PP2Cs), triggering physiological responses that are essential for plant adaptation to environmental stresses, including drought. Based on this ligand-induced PP2C inactivation mechanism, we have developed an in vitro assay for the identification of ABA-receptor agonists by high-throughput screening of chemical libraries. The assay allows simultaneous use of different ABA receptors, increasing the chances to find new agonists and eliminates the need for parallel screening. In this chapter, we describe detailed procedures for the identification of ABA agonists using this multiplexed assay in a medium- (96-well plates) or a high-throughput (384-well plates) setup.

Plant stress tolerance relies on intricate signaling networks that are not fully understood. Several plant hormones are involved in the adaptation to different environmental conditions. Abscisic acid (ABA) has an essential role in stress tolerance, especially in the adaptation to drought. During the last years, chemical genomics has gained attention as an alternative approach to decipher complex traits. Additionally, chemical-based strategies have been very useful to untangle genetic redundancy, which is hard to address by other approaches such as classical genetics. Here, we describe the use of an ABA-inducible luciferase (LUC) reporter line for the high-throughput identification of chemical activators of the ABA signaling pathway. In this assay, seven-day-old pMAPKKK18-LUC + seedlings are grown on 96-well plates and treated with test compounds. Next, the activity of the LUC reporter is quantified semiautomatically by image analysis. Candidate compounds able to activate the reporter are thus identified and subjected to a secondary screen by analyzing their effect on ABA-related phenotypes (e.g., inhibition of seed germination). This assay is fast, high-throughput, nondestructive, semiquantitative and can be applied to any other luciferase reporter lines, making it ideal for forward chemical genetic screenings.

Screening of a diversity-oriented compound library led to the identification of two 6,11-dioxobenzo[ f ]pyrido[1,2- a ]indoles (DBPI) that displayed low micromolar bactericidal activity against the Erdman strain of Mycobacterium tuberculosis in vitro . The activity of these hit compounds was limited to tubercle bacilli, including the nonreplicating form, and to Mycobacterium marinum . On hit expansion and investigation of the structure activity relationship, selected modifications to the dioxo moiety of the DBPI scaffold were either neutral or led to reduction or abolition of antimycobacterial activity. To find the target, DBPI-resistant mutants of M. tuberculosis Erdman were raised and characterized first microbiologically and then by whole genome sequencing. Four different mutations, all affecting highly conserved residues, were uncovered in the essential gene rv0338c ( ispQ ) that encodes a membrane-bound protein, named IspQ, with 2Fe-2S and 4Fe-4S centers and putative iron-sulfur-binding reductase activity. With the help of a structural model, two of the mutations were localized close to the 2Fe-2S domain in IspQ and another in transmembrane segment 3. The mutant genes were recessive to the wild type in complementation experiments and further confirmation of the hit-target relationship was obtained using a conditional knockdown mutant of rv0338c in M. tuberculosis H37Rv. More mechanistic insight was obtained from transcriptome analysis, following exposure of M. tuberculosis to two different DBPI; this revealed strong upregulation of the redox-sensitive SigK regulon and genes induced by oxidative and thiol-stress. The findings of this investigation pharmacologically validate a novel target in tubercle bacilli and open a new vista for tuberculosis drug discovery.

Therapeutics targeting Gram-negative bacteria have the challenge of overcoming a formidable outer membrane (OM) barrier. Here, we characterize the action of SPR741, a novel polymyxin B (PMB) analogue shown to potentiate several large-scaffold antibiotics in Gram-negative pathogens. Probing the surface topology of Escherichia coli using atomic force microscopy revealed substantial OM disorder at concentrations of SPR741 that lead to antibiotic potentiation. Conversely, very little cytoplasmic membrane depolarization was observed at these same concentrations, indicating that SPR741 acts predominately on the OM. Truncating the lipopolysaccharide (LPS) core with genetic perturbations uniquely sensitized E. coli to SPR741, suggesting that LPS core residues keep SPR741 at the OM, where it can potentiate a codrug, rather than permit its entry to the cytoplasmic membrane. Further, a promoter activity assay revealed that SPR741 challenge induced the expression of RcsAB, a stress sensor for OM perturbation. Together, these results indicate that SPR741 interacts predominately with the OM, in contrast to the dual action of PMB and colistin at both the outer and cytoplasmic membranes.