Abstract: Unlike normal tissues, cancers experience profound alterations in protein homeostasis. Powerful innate adaptive mechanisms, especially the transcriptional response regulated by Heat Shock Factor 1 (HSF1), are activated in cancers to enable survival under these stressful conditions. Natural products that further tax these stress responses can overwhelm the ability to cope and could provide leads for the development of new, broadly effective anticancer drugs. To identify compounds that drive the HSF1-dependent stress response, we evaluated over 80,000 natural and synthetic compounds as well as partially purified natural product extracts using a reporter cell line optimized for high-throughput screening. Surprisingly, many of the strongly active compounds identified were natural products representing five diverse chemical classes (limonoids, curvularins, withanolides, celastraloids, and colletofragarones). All of these compounds share the same chemical motif, an α,β-unsaturated carbonyl functionality, with strong potential for thiol-reactivity. Despite the lack of a priori mechanistic requirements in our primary phenotypic screen, this motif was found to be necessary albeit not sufficient, for both heat-shock activation and inhibition of glioma tumor cell growth. Within the withanolide class, a promising therapeutic index for the compound withaferin A was demonstrated in vivo using a stringent orthotopic human glioma xenograft model in mice. Our findings reveal that diverse organisms elaborate structurally complex thiol-reactive metabolites that act on the stress responses of heterologous organisms including humans. From a chemical biology perspective, they define a robust approach for discovering candidate compounds that target the malignant phenotype by disrupting protein homeostasis.

Abstract: The advent of powerful genomics technologies has uncovered many fundamental aspects of biology, including the mechanisms of cancer; however, it has not been appropriately matched by the development of global approaches to discover new medicines against human diseases. Here we describe a unique high-throughput screening strategy by high-throughput sequencing, referred to as HTS2, to meet this challenge. This technology enables large-scale and quantitative analysis of gene matrices associated with specific disease phenotypes, therefore allowing screening for small molecules that can specifically intervene with disease-linked gene-expression events. By initially applying this multitarget strategy to the pressing problem of hormone-refractory prostate cancer, which tends to be accelerated by the current antiandrogen therapy, we identify Peruvoside, a cardiac glycoside, which can potently inhibit both androgen-sensitive and -resistant prostate cancer cells without triggering severe cytotoxicity. We further show that, despite transcriptional reprogramming in prostate cancer cells at different disease stages, the compound can effectively block androgen receptor-dependent gene expression by inducing rapid androgen receptor degradation via the proteasome pathway. These findings establish a genomics-based phenotypic screening approach capable of quickly connecting pathways of phenotypic response to the molecular mechanism of drug action, thus offering a unique pathway-centric strategy for drug discovery.

Abstract: The histone modifying enzymes (HME) represent particularly promising targets for the development of alternatives to praziquantel, the only currently available drug to combat schistosomiasis. The inhibition of these enzymes frequently arrests the cell cycle or induces apoptosis in cancer cells, but not in normal cells and numerous HME inhibitors are under investigation as potential anticancer agents. The recent resolution of the genome sequences of Schistosoma mansoni and Schistosoma japonicum has allowed us to identify all the schistosome genes encoding histone acetyltransferases, deacetylases, methyltransferases and demethylases. We have chosen a strategy using phylogenetic screening with inhibitors of HME classes, screening of individual HME targets by both high-throughput and reasoned (in silico docking using resolved crystal structures) approaches in a project funded by the European Community, named SEtTReND (Schistosome Epigenetics: Targets, Regulation, New Drugs). The initial focus is on the class I histone deacetylase (HDAC) 8 since the comparison of the catalytic site of the schistosome and human enzymes shows crucial differences, rendering possible the development of inhibitors specific for SmHDAC8. However, phenotypic screening shows that inhibitors of all HME classes tested were able to induce apoptosis and death in parasites in vitro, indicating that other enzymes may prove to be viable targets.

Abstract: Here, we describe the discovery of a novel antimalarial agent using phenotypic screening of Plasmodium falciparum asexual blood-stage parasites. Screening a novel compound collection created using diversity-oriented synthesis (DOS) led to the initial hit. Structure–activity relationships guided the synthesis of compounds having improved potency and water solubility, yielding a subnanomolar inhibitor of parasite asexual blood-stage growth. Optimized compound 27 has an excellent off-target activity profile in erythrocyte lysis and HepG2 assays and is stable in human plasma. This compound is available via the molecular libraries probe production centers network (MLPCN) and is designated ML238.

Abstract: Neglected tropical diseases, especially those caused by helminths, constitute some of the most common infections of the world's poorest people Development of techniques for automated, high-throughput drug screening against these diseases, especially in whole-organism settings, constitutes one of the great challenges of modern drug discovery We present a method for enabling high-throughput phenotypic drug screening against diseases caused by helminths with a focus on schistosomiasis The proposed method allows for a quantitative analysis of the systemic impact of a drug molecule on the pathogen as exhibited by the complex continuum of its phenotypic responses This method consists of two key parts: first, biological image analysis is employed to automatically monitor and quantify shape-, appearance-, and motion-based phenotypes of the parasites Next, we represent these phenotypes as time-series and show how to compare, cluster, and quantitatively reason about them using techniques of time-series analysis We present results on a number of algorithmic issues pertinent to the time-series representation of phenotypes These include results on appropriate representation of phenotypic time-series, analysis of different time-series similarity measures for comparing phenotypic responses over time, and techniques for clustering such responses by similarity Finally, we show how these algorithmic techniques can be used for quantifying the complex continuum of phenotypic responses of parasites An important corollary is the ability of our method to recognize and rigorously group parasites based on the variability of their phenotypic response to different drugs The methods and results presented in this paper enable automatic and quantitative scoring of high-throughput phenotypic screens focused on helmintic diseases Furthermore, these methods allow us to analyze and stratify parasites based on their phenotypic response to drugs Together, these advancements represent a significant breakthrough for the process of drug discovery against schistosomiasis in particular and can be extended to other helmintic diseases which together afflict a large part of humankind

Abstract: Most of current strategies for antiviral therapeutics target the virus specifically and directly, but an alternative approach to drug discovery might be to enhance the immune response to a broad range of viruses. Based on clinical observation in humans and successful genetic strategies in experimental models, we reasoned that an improved interferon (IFN) signaling system might better protect against viral infection. Here we aimed to identify small molecular weight compounds that might mimic this beneficial effect and improve antiviral defense. Accordingly, we developed a cell-based high-throughput screening (HTS) assay to identify small molecules that enhance the IFN signaling pathway components. The assay is based on a phenotypic screen for increased IFN-stimulated response element (ISRE) activity in a fully automated and robust format (Z′>0.7). Application of this assay system to a library of 2240 compounds (including 2160 already approved or approvable drugs) led to the identification of 64 compounds with significant ISRE activity. From these, we chose the anthracycline antibiotic, idarubicin, for further validation and mechanism based on activity in the sub-µM range. We found that idarubicin action to increase ISRE activity was manifest by other members of this drug class and was independent of cytotoxic or topoisomerase inhibitory effects as well as endogenous IFN signaling or production. We also observed that this compound conferred a consequent increase in IFN-stimulated gene (ISG) expression and a significant antiviral effect using a similar dose-range in a cell-culture system inoculated with encephalomyocarditis virus (EMCV). The antiviral effect was also found at compound concentrations below the ones observed for cytotoxicity. Taken together, our results provide proof of concept for using activators of components of the IFN signaling pathway to improve IFN efficacy and antiviral immune defense as well as a validated HTS approach to identify small molecules that might achieve this therapeutic benefit.

Abstract: There are no drugs that halt the progression of any age-associated neurodegenerative disease. This may be due to the failure of drug developers to recognize that while there are mutations that predispose individuals to disease as they get older, the vast majority of neurodegenerative diseases arise from a confluence of multiple toxic insults. Thus, it is unlikely that the current single-target approach is going to yield useful drugs for these conditions. The identification of multi-target lead compounds is needed and their selection should be based upon a requirement for their efficacy in phenotypic screening assays that reflect the biology of the aging brain. This approach to neurodegenerative disease drug discovery is likely to produce safe and effective drugs.

Abstract: While target-based small-molecule discovery has taken centre-stage in the pharmaceutical industry, there are many cancer-promoting proteins not easily addressed with a traditional target-based screening approach. In order to address this problem, as well as to identify modulators of biological states in the absence of knowing the protein target of the state switch, alternative phenotypic screening approaches, such as gene expression-based and high-content imaging, have been developed. With this renewed interest in phenotypic screening, however, comes the challenge of identifying the binding protein target(s) of small-molecule hits. Emerging technologies have the potential to improve the process of target identification. In this review, we discuss the application of genomic (gene expression-based), genetic (short hairpin RNA and open reading frame screening), and proteomic approaches to protein target identification.

Abstract: After the discovery of antibacterial chemicals and antibiotics through the 1950s by empirical assays based on growth inhibition, methods gradually evolved to encompass more directed screens. These were initially based upon the phenomena revealed by the action of previously discovered antibiotics and served the dual purpose of exploiting bacterial-selective pathways and aiding in recognition of novelty among natural product samples (dereplication). Later, as microbial genetics began to uncover the range of essential bacterial genes and provide tools for construction of test strains, targeted whole cell screening for antibacterials took on more of the characteristics of a hunt for genetic mutations by exploiting the expected phenotypes of desired inhibitors. Although these methods initially succeeded in expanding the repertoire of useful classes of antibiotics inhibiting cell wall synthesis, no marketed novel non-cell wall targeted antibacterial classes have been found through these methods, nor have they been found via the later genomically based targeted screening that followed. Possible reasons for this low output are speculated upon.

Abstract: Pancreatic beta-cell regeneration, for example, by inducing proliferation, remains an important goal in developing effective treatments for diabetes. However, beta cells have mainly been considered quiescent. This “static” view has recently been challenged by observations of relevant physiological conditions in which metabolic stress is compensated by an increase in beta-cell mass. Understanding the molecular mechanisms underlining these process could open the possibility of developing novel small molecules to increase beta-cell mass. Several cellular cell-cycle and signaling proteins provide attractive targets for high throughput screening, and recent advances in cell culture have enabled phenotypic screening for small molecule-induced beta-cell proliferation. We present here an overview of the current trends involving small-molecule approaches to induce beta-cell regeneration by proliferation.

Abstract: Systemic life-threatening fungal infections represent a significant unmet medical need. Cell-based, phenotypic screening can be an effective means of discovering potential novel antifungal compounds, but it does not address target identification, normally required for compound optimization by medicinal chemistry. Here, we demonstrate a combination of screening, genetic, and biochemical approaches to identify and characterize novel antifungal compounds. We isolated a set of novel non-azole antifungal compounds for which no target or mechanism of action is known, using a screen for inhibition of Saccharomyces cerevisiae proliferation. Haploinsufficiency profiling of these compounds in S. cerevisiae suggests that they target Erg11p, a cytochrome P450 family member, which is the target of azoles. Consistent with this, metabolic profiling in S. cerevisiae revealed a buildup of the metabolic intermediates prior to Erg11p activity, following compound treatment. Further, human cytochrome P450 is also inhibited in in vitro assays by these compounds. We modeled the Erg11p protein based on the human CYP51 crystal structure, and in silico docking of these compounds suggests that they interact with the heme center in a manner similar to that of azoles. Consistent with these docking observations, Candida strains carrying azole-resistant alleles of ERG11 are also resistant to the compounds in this study. Thus, we have identified non-azole Erg11p inhibitors, using a systematic approach for ligand and target characterization.

Abstract: Parkinson's disease (PD) is a devastating neurodegenerative disease that affects over one million patients in the US. Yet, no disease modifying drugs exist, only those that temporarily alleviate symptoms. Because of its poorly defined and highly complex disease etiology, it is essential to embrace unbiased and innovative approaches for identifying new chemical entities that target the underlying toxicities associated with PD. Traditional target-based drug discovery paradigm can suffer from a bias toward a small number of potential targets. Phenotypic screening of both genetic and pharmacological PD models offers an alternative approach to discover compounds that target the initiating causes and effectors of cellular toxicity. The relative paucity of reported phenotypic screens illustrates the intrinsic difficulty in establishing model systems that are both biologically meaningful and adaptable to high-throughput screening. Parallel advances in PD models and in vivo screening technologies will help create opportunities for identifying new therapeutic leads with unanticipated, breakthrough mechanisms of action.

Abstract: Summary Trypanosoma brucei is the causative agent of African sleeping sickness, putting at risk up to 50 million people in sub-Saharan Africa. Current drug therapies are limited by toxicity and difficult treatment regimes and as the development of vaccines remains unlikely, the identification of better drugs to control this deadly disease is needed. Strategies for the identification of new lead compounds include phenotypic screening or target-based approaches. Implementation of the latter has been hampered by the lack of defined targets that are both essential and druggable. In this issue of Molecular Microbiology, Jones et al. (2012) report on the characterization of T. brucei pyridoxal kinase (PdxK), an enzyme required for the salvage of vitamin B6, an essential enzymatic cofactor. Genetic knock-down and small molecule inhibitor studies were used to demonstrate that PdxK is essential for parasite growth both in vitro and in a mouse model, providing both genetic and chemical validation of the target. An enzyme assay compatible with high-throughput screening (HTS) was developed and the X-ray crystal structure solved, showing the potential for species selective inhibition. These studies add a greatly needed additional target into the drug discovery pipeline for this deadly parasitic infection.

Abstract: The many virtues that made the yeast Saccharomyces cerevisiae a dominant model organism for genetics and molecular biology, are now establishing its role in chemical genetics. Its experimental tractability (i.e., rapid doubling time, simple culture conditions) and the availability of powerful tools for drug-target identification, make yeast an ideal organism for high-throughput phenotypic screening. It may be especially applicable for the discovery of chemical probes targeting highly conserved cellular processes, such as metabolism and bioenergetics, because these probes would likely inhibit the same processes in higher eukaryotes (including man). Importantly, changes in normal cellular metabolism are associated with a variety of diseased states (including neurological disorders and cancer), and exploiting these changes for therapeutic purposes has accordingly gained considerable attention. Here, we review progress and challenges associated with forward chemical genetic screening in yeast. We also discuss evidence supporting these screens as a useful strategy for discovery of new chemical probes and new druggable targets related to cellular metabolism.

Abstract: Quantitative measurement of the levels of mRNA expression via real-time reverse transcription polymerase chain reaction (RT-PCR) has long been used for analyzing expression differences in tissue or cell lines of interest. This method has been used somewhat less frequently to measure the changes in gene expression due to perturbagens such as small molecules or siRNA. The availability of new instrumentation for liquid handling and real-time PCR analysis, as well as the commercial availability of start-to-finish kits for RT-PCR, has enabled the use of this method for high-throughput small-molecule screening on a scale comparable to traditional high-throughput screening (HTS) assays. This protocol focuses on the special considerations necessary for using quantitative RT-PCR as a primary small-molecule screening assay, including the different methods available for mRNA isolation and analysis.Curr. Protoc. Chem. Biol. 4:49-63 © 2012 by John Wiley & Sons, Inc. Keywords: real-time PCR; high-throughput screening; phenotypic screening; qRT-PCR; gene expression

Abstract: Cancer stem cells (CSCs) are a subpopulation generally thought to be responsible for cancer initiation and progression. Because CSCs are often rare in the total tumor cell population and differentiate rapidly when grown in culture, it has been challenging to uncover compounds that selectively target CSCs. We previously described CSC-emulating cells derived from breast cancer cell lines that maintained a stable undifferentiated state. We optimized a phenotypic assay with these cells and screened 1,280-bioactive compounds, identifying five that preferentially inhibited CSC-like cell proliferation. Using a compound-guided target identification approach, we found high topoisomerase I (Topo I) expression levels in breast CSC-like cells and primary breast CSCs. Structurally unrelated small molecules targeting Topo I preferentially inhibited CSC-like cells. These results illustrate the substantial power of this CSC phenotypic screening platform and promote Topo I as a potential molecular therapeutic target for therapies aimed at expunging CSCs.

Abstract: Recent drug discovery efforts have utilized high throughput screening (HTS) of large chemical libraries to identify compounds that modify the activity of discrete molecular targets. The molecular target approach to drug screening is widely used in the pharmaceutical and biotechnology industries, because of the amount of knowledge now available regarding protein structure that has been obtained by computer simulation. The molecular target approach requires that the structure of target molecules, and an understanding of their physiological functions, is known. This approach to drug discovery may, however, limit the identification of novel drugs. As an alternative, the phenotypic- or pathway-screening approach to drug discovery is gaining popularity, particularly in the academic sector. This approach not only provides the opportunity to identify promising drug candidates, but also enables novel information regarding biological pathways to be unveiled. Reporter assays are a powerful tool for the phenotypic screening of compound libraries. Of the various reporter genes that can be used in such assays, those encoding secreted proteins enable the screening of hit molecules in both living cells and animals. Cell- and animal-based screens enable simultaneous evaluation of drug metabolism or toxicity with biological activity. Therefore, drug candidates identified in these screens may have increased biological efficacy and a lower risk of side effects in humans. In this article, we review the reporter bioassay systems available for phenotypic drug discovery.

Abstract: Motivation: Cell-based phenotypic screens using small molecule inhibitors is an important technology for early drug discovery if the relationship between the disease-related cellular phenotype and inhibitors' biological targets can be determined. However, chemical inhibitors are rightfully believed to be less specific than perturbation by biological agents, such as antibody and small inference RNA. Therefore, it is often a challenge in small molecule phenotypic screening to infer the causality between a particular cellular phenotype and the inactivation of the responsible protein due to the off-target effect of the inhibitors. Results: In this article, we present a Roche in-house effort of screening 746 structurally diverse compounds for their cytotoxicity in HeLa cells measured by high content imaging technology. These compounds were also systematically profiled for the targeted and off-target binding affinity to a panel of 25 pre-selected protein kinases in a cell-free system. In an effort to search for the kinases whose activities are crucial for cell survival, we found that the simple association method such as the chi-square test yields a large number of false positives because the observed cytotoxic phenotype is likely to be the result of promiscuous action of less specific inhibitors instead of true consequence of inactivation of single relevant target. We demonstrated that a stratified categorical data analysis technique such as the Cochran–Mantel–Haenszel test is an effective approach to extract the meaningful biological connection from the spurious correlation resulted from confounding covariates. This study indicates that, empowered by appropriate statistical adjustment, small molecule inhibitor perturbation remains a powerful tool to pin down the relevant biomarker for drug safety and efficacy research. Contact: xin.wei@roche.com Supplementary information: Supplementary data are available at Bioinformatics online.

Abstract: The changes in screening philosophy over a 40 year period from in vivo phenotypic screening to a reductionist mechanism-based in vitro search for a single selective compound against a single target are described. Examples are given of the shortcomings of the reductionist paradigm and the advantages of the phenotypic and multi-target screening approaches towards drug discovery and repurposing. Non-mechanism biased phenotypic screening offers the advantages of enhanced target opportunity space and is a good match for screening of ligands covering narrow chemistry space, e.g. natural products. Retrospective analysis suggests that phenotypic screening is better than mechanistic screening in finding first in class compounds, particularly for the more complex disease targets.

Abstract: Since protein kinases play a fundamental role in cell signaling, especially as components of critical growth and differentiation pathways linked to cancer, kinase inhibitors have been commercialized as both therapeutics and research tools. This has been enabled to some degree by the chemical tractability of protein kinase ATP binding pockets, the automation of activity measurements, and the correlation of in vitro, cellular, and in vivo activities. Despite the development of a number of efficacious kinase inhibitors, the strategies for rational design of these compounds have been limited by target promiscuity. In an effort to better understand the nature of structure and activity across the kinome, especially as it relates to off-target effects, we screened a well-defined collection of kinase inhibitors using “gold standard” radiometric assays for inhibitory activity toward 234 kinases, which represent all branches of the kinome tree. We screened 158 small molecules initially identified in the literature as potent and specific inhibitors of kinases important from a therapeutic target as well as a signal transduction biology perspective. We performed hierarchical clustering of these benchmark kinase inhibitors based upon their kinome activity profiles and illustrate how they relate to chemical structure similarities; this provides new insights into inhibitor specificity and potential applications for probing new targets. Additionally, we developed a selectivity score for each kinase which we believe reflects general binding site accessibility to small molecules. Knowledge of such scoring indices can play an important role in therapeutic target selection and drug discovery strategies. Using this broad set of data, we provide a framework for assessing polypharmacology. Examples will be shown to demonstrate that we not only identify likely off-target inhibitor activities but also potential new uses for known small molecules. Moreover we demonstrate how activity data can be used to help explain the effects of small molecules observed in phenotypic screening. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1242. doi:1538-7445.AM2012-1242

Abstract: Many drugs have been derived from bioactive components isolated from plants: while some have come from studies on traditionally used herbal medicines, others have come from leads found in screening campaigns. Phenotypic screening in model organisms has developed with the use of fruit flies (Drosophila melanogaster), nematode worms (Caenorhabditis elegans), and zebrafish (Danio rerio), and functional screens can also be conducted on cells in culture, either for studies on cell death and proliferation in cancer cell lines or for detection of more specific read-outs such as activation or inhibition of a particular biochemical pathway. Molecular assays based on target proteins are particularly suited for high throughput screening, but care needs to be taken when used with plant extracts to avoid non-specific interference, and the power of such assays also depends on their relevance to the disease that is the intended target: validation of the molecular target becomes a critical issue. Examples are given of assays that have been used for plant-based drug discovery in the areas of cancer, diabetes, inflammatory disease, and infectious diseases.