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Autophagy facilitates the degradation of cellular content via the lysosome and is involved in cellular homeostasis and stress response pathways. As such, malfunction of autophagy is linked to a variety of diseases ranging from organ-specific illnesses like cardiomyopathy to systemic illnesses such as cancer or metabolic syndromes. Given the variety of autophagic functions within a cell and tissue, regulation of autophagy is complex and contains numerous positive and negative feedback loops. While our knowledge of mechanisms for cargo selectivity has significantly improved over the last decade, our understanding of signaling routes activating individual autophagy pathways remains rather sparse. In this resource study, we report on a well-characterized chemical library containing 77 GPCR-targeting ligands that was used to systematically analyze LC3B-based autophagy as well as ER-phagy flux upon compound treatment. Upon others, compounds TC-G 1004, BAY 60-6583, PSNCBAM-1, TC-G 1008, LPA2 Antagonist 1, ML-154, JTC-801 and ML-290 targeting adenosine receptor A2a (ADORA2A), adenosine receptor A2b (ADORA2B), cannabinoid receptor 1 (CNR1), G-protein coupled receptor 39 (GPR39), lysophosphatidic acid receptor 2 (LPAR2), neuropeptide S receptor 1 (NPSR1), opioid related nociceptin receptor 1 (OPRL1), and relaxin receptor 1 (RXFP1), respectively, were hit compounds for general autophagy flux. From these compounds, only JTC-801 markly increased ER-phagy flux. In addition, the global impact of these selected hit compounds were analyzed by TMT-based mass spectrometry and demonstrated the differential impact of targeting GPCRs on autophagy-associated proteins. This chemical screening exercise indicates to a significant cross-talk between GPCR signaling and regulation of autophagy pathways.

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Chemical genetic screens are a powerful tool for exploring how cancer cells' response to drugs is shaped by their mutations, yet they lack a molecular view of the contribution of individual genes to the response to exposure. Here, we present sci-Plex-Gene-by-Environment (sci-Plex-GxE), a platform for combined single-cell genetic and chemical screening at scale. We highlight the advantages of large-scale, unbiased screening by defining the contribution of each of 522 human kinases to the response of glioblastoma to different drugs designed to abrogate signaling from the receptor tyrosine kinase pathway. In total, we probed 14,121 gene-by-environment combinations across 1,052,205 single-cell transcriptomes. We identify an expression signature characteristic of compensatory adaptive signaling regulated in a MEK/MAPK-dependent manner. Further analyses aimed at preventing adaptation revealed promising combination therapies, including dual MEK and CDC7/CDK9 or nuclear factor κB (NF-κB) inhibitors, as potent means of preventing transcriptional adaptation of glioblastoma to targeted therapy.

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Degraders hold the promise to efficiently inactivate previously intractable disease-relevant targets. Unlike traditional inhibitors, degraders act substoichiometrically and rely on the hijacked proteolysis machinery, which can also act as an entry point for resistance. To fully harness the potential of targeted protein degradation, it is crucial to comprehend resistance mechanisms and formulate effective strategies to overcome them. We conducted a chemical screening to identify synthetic lethal vulnerabilities of cancer cells that exhibit widespread resistance to degraders. Comparative profiling followed by tailored optimization delivered the small molecule RBS-10, which shows preferential cytotoxicity against cells pan-resistant to degraders. Multiomics deconvolution of the mechanism of action revealed that RBS-10 acts as a prodrug bioactivated by the oxidoreductase enzyme NQO1, which is highly overexpressed in our resistance models. Collectively, our work informs on NQO1 as an actionable vulnerability to overcome resistance to degraders and as a biomarker to selectively exploit bioactivatable prodrugs in cancer.

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Extensive phylogenetic conservation of molecular pathways and neuroanatomical structures, associated with efficient methods for genetic modification, have been exploited increasingly to generate zebrafish models of human disease. A range of powerful approaches can be deployed to analyze these models with the ultimate goal of elucidating pathogenic mechanisms and accelerating efforts to find effective treatments. Unbiased neurobehavioral assays can provide readouts that parallel clinical abnormalities found in patients, although some of the most useful assays quantify responses that are not routinely evaluated clinically, and differences between zebrafish and human brains preclude expression of the full range of neurobehavioral abnormalities seen in disease. Imaging approaches that use fluorescent reporters and standardized brain atlases coupled with quantitative measurements of brain structure offer an unbiased means to link experimental manipulations to changes in neural architecture. Together, quantitative structural and functional analyses allow dissection of the cellular and physiological basis underlying neurological phenotypes. These approaches can be used as outputs in chemical modifier screens, which provide a major opportunity to exploit zebrafish models to identify small molecule modulators of pathophysiology that may be informative for understanding disease mechanisms and possible therapeutic approaches.

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High-throughput screening (HTS) is a vaunted technology in drug discovery, and drug repositioning a celebrated strategy with famous examples of successful stories; however, repositioned drugs have primarily resulted from serendipitous observations, retrospective studies, and pharmacological analyses as opposed to experimental routes. This observation points to a methodological paradox, considering that academic laboratories of the post-genomic era have benefited from unprecedented technological progress, and a facilitated access to powerful resources that, historically, were a prerogative of the pharma industry. This disconnect is exacerbated by financial, practical, and regulatory complexities affecting drug repositioning; however, the pivotal significance of stringent and rigorous data is what unconditionally sits at the crossroad of go/no-go decisions concerning the therapeutic significance, or predictive validity, of selected drugs. Here, I propose a visionary approach, to which I assigned the term labsourcing, to dramatically enhance efficiency and clinical relevance of academic drug screens and, ultimately, generate contextual and reproducible data for correct interpretations and reliable selection of drug candidates. The overall concept implies intra- and intermural aggregation of expertise (e.g., assay development, cell biology, statistics, bioinformatics) to perform multiple bioassays, under multiple conditions and readouts, using a common screening collection. Advantages of high input screens can be manifold: (i) to tackle discrepancies that may arise from the screens of libraries of variable size and content and assay types and conditions too narrow in scope; (ii) the opportunity to generate massive amounts of data applicable for multiple publications and funding requests; (iii) the educational benefits for students and post-docs collegially exposed to long-term programs; and (iv) the opportunity to democratize research and recruit small labs that could not otherwise join screening programs due to costs, timelines, and risks.

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Hematopoiesis is a complex, orderly and conserved developmental process, coordinated by multiple factors including transcription factors and signaling pathways. Dysregulation of any of these factors may cause developmental or functional defects in the blood system, leading to the pathogenesis of blood diseases. Zebrafish hematopoiesis and the underlying molecular mechanisms are highly conserved with those in mammals. The use of zebrafish to recapitulate abnormal changes in pathogenic factors can build models of related blood diseases, thus providing powerful tools for exploring the molecular mechanisms of pathogenesis and progression, visualization of tumorigenesis and high-throughput chemical screening. In this review, we summarize the zebrafish models of blood diseases and their applications. These disease models not only help to improve our understanding of the pathophysiology of the blood system and the molecular mechanisms on pathogeneses of blood diseases, but also provide new ideas for the treatment of clinically relevant hematological malignancies.

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Inactivation of the von Hippel Lindau tumor suppressor protein (pVHL) is a hallmark of clear cell Renal Cell Carcinoma (ccRCC), which is the most common form of kidney cancer in adults. In complex with Elongin B/C, pVHL functions as the substrate recognition subunit of a ubiquitin ligase, perhaps best known to target the hypoxia inducible factor (HIF) transcription factor for ubiquitin-dependent proteolysis. Beyond kidney cancer, the pseudo-hypoxic state caused due to chronic HIF activation in pVHL-deficient cells has become a biological model to study hypoxia's profound effects on tumor angiogenesis, metabolism, and epigenetics. However, a number of HIF-independent substrates of pVHL, which function in a broad range of biological pathways, have also been discovered. Independently, the development of high-throughput chemical and genetic screening strategies have enabled the identification of novel, HIF-independent, targetable dependencies in ccRCC. In this review we summarize the history of pVHL and HIF mediated oxygen sensing, discuss the current status of this field, and identify critical challenges that need to be overcome. The confluence of historical discovery, development of unbiased screening strategies, and the evolution of medicinal chemistry has allowed us to begin therapeutically targeting vulnerabilities that emerge due to pVHL loss in ccRCC. Ongoing mechanistic studies on the biological consequences of pVHL loss, therefore, are likely to become the cornerstones of modern therapeutics in renal cancer.

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Zebrafish (Danio rerio) has proven to be a versatile and reliable in vivo experimental model to study human hematopoiesis and hematological malignancies. As vertebrates, zebrafish has significant anatomical and biological similarities to humans, including the hematopoietic system. The powerful genome editing and genome-wide forward genetic screening tools have generated models that recapitulate human malignant hematopoietic pathologies in zebrafish and unravel cellular mechanisms involved in these diseases. Moreover, the use of zebrafish models in large-scale chemical screens has allowed the identification of new molecular targets and the design of alternative therapies. In this review we summarize the recent achievements in hematological research that highlight the power of the zebrafish model for discovery of new therapeutic molecules. We believe that the model is ready to give an immediate translational impact into the clinic.

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Phenotypic drug discovery offers some advantages over target-based methods, mainly because it allows drug leads to be tested in systems that more closely model distinct disease states. However, a potential disadvantage is the difficulty of linking the observed phenotype to a specific cellular target. To address this problem, we developed DePick, a computational target de-convolution tool to determine targets specifically linked to small-molecule phenotypic screens. We applied DePick to eight publicly available screens and predicted 59 drug target-phenotype associations. In addition to literature-based evidence for our predictions, we provide experimental support for seven predicted associations. Interestingly, our analysis led to the discovery of a previously unrecognized connection between the Wnt signaling pathway and an aromatase, CYP19A1. These results demonstrate that the DePick approach can not only accelerate target de-convolution but also aid in discovery of new functionally relevant biological relationships.

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The inner ear is a remarkably intricate structure able to detect sound, motion, and gravity. During development of the zebrafish embryo, the ear undergoes dynamic morphogenesis from a simple epithelial vesicle into a complex labyrinth, consisting of three semicircular canals and three otolithic sensory organs, each with an array of differentiated cell types. This microcosm of biology has led to advances in understanding molecular and cellular changes in epithelial patterning and morphogenesis, through to mechanisms of mechanosensory transduction and the origins of reflexive behavior. In this chapter, we describe different methods to study the zebrafish ear, including high-speed imaging of otic cilia, confocal microscopy, and light-sheet fluorescent microscopy. Many dyes, antibodies, and transgenic lines for labeling the ear are available, and we provide a comprehensive review of these resources. The developing ear is amenable to genetic, chemical, and physical manipulations, including injection and transplantation. Chemical modulation of developmental signaling pathways has paved the way for zebrafish to be widely used in drug discovery. We describe two chemical screens with relevance to the ear: a fluorescent-based screen for compounds that protect against ototoxicity, and an in situ-based screen for modulators of a signaling pathway involved in semicircular canal development. We also describe methods for dissection and imaging of the adult otic epithelia. We review both manual and automated methods to test the function of the inner ear and lateral line, defects in which can lead to altered locomotor behavior. Finally, we review a collection of zebrafish models that are generating new insights into human deafness and vestibular disorders.

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The treatment of psychiatric disorders presents three major challenges to the research and clinical community: defining a genotype associated with a disorder, characterizing the molecular pathology of each disorder and developing new therapies. This Review addresses how cellular and animal systems can help to meet these challenges, with an emphasis on the role of the zebrafish. Genetic changes account for a large proportion of psychiatric disorders and, as gene variants that predispose to psychiatric disease are beginning to be identified in patients, these are tractable for study in cellular and animal systems. Defining cellular and molecular criteria associated with each disorder will help to uncover causal physiological changes in patients and will lead to more objective diagnostic criteria. These criteria should also define co-morbid pathologies within the nervous system or in other organ systems. The definition of genotypes and of any associated pathophysiology is integral to the development of new therapies. Cell culture-based approaches can address these challenges by identifying cellular pathology and by high-throughput screening of gene variants and potential therapeutics. Whole-animal systems can define the broadest function of disorder-associated gene variants and the organismal impact of candidate medications. Given its evolutionary conservation with humans and its experimental tractability, the zebrafish offers several advantages to psychiatric disorder research. These include assays ranging from molecular to behavioural, and capability for chemical screening. There is optimism that the multiple approaches discussed here will link together effectively to provide new diagnostics and treatments for psychiatric patients.

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The zebrafish (Danio rerio) is one of the most promising new model organisms. The increasing popularity of this amazing small vertebrate is evident from the exponentially growing numbers of research articles, funded projects and new discoveries associated with the use of zebrafish for studying development, brain function, human diseases and screening for new drugs. Thanks to the development of novel technologies, the range of zebrafish research is constantly expanding with new tools synergistically enhancing traditional techniques. In this review we will highlight the past and present techniques which have made, and continue to make, zebrafish an attractive model organism for various fields of biology, with a specific focus on neuroscience.

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