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: Publisher Summary This chapter summarizes some of the novel methods used to accelerate the discovery of old drugs that could potentially treat new indications, either by the established mechanism of action or by the identification of new ones. Representative case studies of the approaches to therapeutic discoveries are also highlighted. Researchers have previously identified repositioned drugs by serendipity, novel insights, or target searching. The innovative strategies directed toward drug repositioning discussed in this chapter are phenotypic, high throughput, and in silico screening of commercial, public, and pharmaceutical compound libraries, the prospective mining of drug/activity databases, the exchange of compound information in collaborative networks, and data collection from the Internet and social networks. The term “drug repositioning” has been used interchangeably with “drug repurposing” or “drug reprofiling,” which is a promising field in drug discovery that identifies new therapeutic opportunities for existing drugs.

Abstract: In this article, we discuss the merits of both target-based and phenotypic screening strategies to find starting points for drug discovery projects in neglected tropical disease including: human African trypanosomiasis, Chagas disease, leishmaniasis and malaria. Technological advances now mean that it is possible to undertake high quality screens against isolated molecular targets at considerable scale. However target selection is a minefield of potential issues and often molecules identified and developed as potent inhibitors of targets do not translate into actives against the whole parasite. The potential for rapid resistance development is also a key issue when tackling individual molecular targets. In phenotypic screening, compounds are screened against the whole organism, looking for activity without a priori knowledge of the target(s) being modulated. This approach brings the benefits of increased chances of efficacy and potentially slowed resistance development of a successful medicine but the lack of knowledge of the molecular target can make the optimisation process more challenging. Advances in screening technologies has now brought phenotypic approaches up to the scale attained by target-based approaches and we discuss opportunities for advances in this arena concluding that a robust drug discovery portfolio for such diseases should include both phenotypic and target-based approaches.

Abstract: The identification of susceptibility genes for human disease is a major goal of current biomedical research. Both sequence and structural variation have emerged as major genetic sources of phenotypic variability and growing evidence points to copy number variation as a particularly important source of susceptibility for disease. Here we propose and validate a strategy to identify genes in which changes in dosage alter susceptibility to disease-relevant phenotypes in the mouse. Our approach relies on sensitized phenotypic screening of megabase-sized chromosomal deletion and deficiency lines carrying altered copy numbers of ∼30 linked genes. This approach offers several advantages as a method to systematically identify genes involved in disease susceptibility. To examine the feasibility of such a screen, we performed sensitized phenotyping in five therapeutic areas (metabolic syndrome, immune dysfunction, atherosclerosis, cancer and behaviour) of a 0.8 Mb reciprocal chromosomal duplication and deficiency on chromosome 11 containing 27 genes. Gene dosage in the region significantly affected risk for high-fat diet-induced metabolic syndrome, antigen-induced immune hypersensitivity, ApoE-induced atherosclerosis, and home cage activity. Follow up studies on individual gene knockouts for two candidates in the region showed that copy number variation in Stat5 was responsible for the phenotypic variation in antigen-induced immune hypersensitivity and metabolic syndrome. These data demonstrate the power of sensitized phenotypic screening of segmental aneuploidy lines to identify disease susceptibility genes.

Abstract: It is clear that viral entry, replication, and spread is a complex process involving a dialog between the virus and the targeted host cell. Viruses have evolved highly specific strategies to hijack cellular factors to promote their internalization, initiate their replication, and facilitate their eventual spread. However, the identification of many of these host cell molecules has been hindered by the requirement for robust genome-scale loss-of-function assays that are capable of targeting a wide variety of host factors. The more recent use of genome-scale or genome-wide RNA interference (RNAi) screens have extended our knowledge of the complex interplay between a virus and host and have implicated a wide variety of cellular factors required for infection of a number of viruses. Here, we describe an approach to target mammalian host cell factors involved in regulating viral infections by the use of a genome-scale RNAi library screen.

Abstract: Chemical genetics, which relies on selecting small molecules for their ability to induce a biological phenotype or to interact with a particular gene product, is a new power- ful tool for lead generation in drug discovery. Accordingly, diversity-oriented synthesis (DOS) of small-molecule peptidomimetics gives access to collections of new chemotypes bearing high structural diversity. Biological evaluation using cell growth as a phenotypic screening on Saccharomyces cerevisiae deletant strains is a powerful tool to identify new chemotypes as hit compounds in the discovery of new antifungal and anticancer agents, and also in the dissection of their mode of action. Our contribution in this field focused on the screening of morpholine-based peptidomimetic collections toward yeast deletant strains, which provided the identification of new chemotypes involved in mitochondria metabolism and respiration.

Abstract: A droplet-based microfluidic approach was investigated to develop high throughput phenotypic screening assays for chemicals, toxic to oomycete plant pathogens. Random fast moving Phytophthora sojae zoospores and an antioomycete drug were encapsulated in micro-sized droplets. Phenotypic responses of zoospores to the drug at different concentrations were quantified parallely inside the droplets with a single zoospore spatial resolution and large data sets. This droplet-based device can not only provide a high throughput phenotypic drug screening technique for plant pathogenic zoospores, but also advance our understanding of interactions of plant pathogens with chemical compounds.

Abstract: A commonly used method to understand the role of complex biological systems and how they function is to disrupt them and then to observe the result of this disruption. A classic way to create such disruptions is to generate genetic mutations and then to observe the effect of these mutations on the cell or the organism. Small organic molecules can also cause disruption to the functioning of biological systems and can be employed to understand the role of the protein with which they interact. The history of biology is full of examples of complex systems whose molecular functioning can be understood thanks to the use of drugs or ligands as well as to the characterisation of the protein targets of these ligands. One such example is the role of colchicine in the discovery of tubulin, a component protein of microtubules (SHELANSKI and TAYLOR, 1967; PETERSON and MITCHISON, 2002).

Abstract: High-content screening (HCS) aims not only to isolate molecules that are active towards a biological target but also to obtain the maximum amount of information during the screening about the effect of the molecule on this target. Here the notion of ‘biological target’ covers both molecularly defined biomolecules and complex biological systems. When the targets are identified molecularly, the effects of the targeted molecules can be studied on the isolated target in vitro (structural screening in parallel) or on the target in its cellular context, in vivo. These are procedures in reverse chemical genetics (chapter 8). It is also possible to search for molecules active on metabolic or signalling pathways without any molecular characterisation of the target. Phenotypic screening with cells or whole organisms are approaches in forward chemical genetics (chapter 8).

Abstract: Tumor-endothelium interactions are critical for tumor survival and metastasis. Melanomas can rapidly metastasize early in tumor progression, but the dependence of this aggressive behavior on tumor-stromal interaction is poorly understood. To probe the mechanisms involved, we developed a heterotypic co-culture methodology, allowing simultaneous tracking of genomic and phenotypic changes in interacting tumor and endothelial cells in vitro. We found a dramatic re-arrangement of endothelial cell networks into patterns reminiscent of vascular beds, even on plastic and glass. Multiple genes were up-regulated in the process, many coding for cell surface and secreted proteins, including Neuropilin-2 (NRP2). A critical role of NRP2 in coordinated cell patterning and growth was confirmed using the co-culture system. We conclude that NRP2 represents an important mediator of melanoma-endothelial interactions. Furthermore, the described methodology represents a powerful yet simple system to elucidate heterotypic intercellular interactions mediating diverse physiological and pathological processes.