Showing papers on "Phenotypic screening published in 2016"
TL;DR: This review highlights advances and successes in approaches to targeting RNA with diverse small molecules, and the potential for these technologies to pave the way to new types of RNA-targeted therapeutics.
216 citations
TL;DR: A method called PRISM is reported that allows pooled screening of mixtures of cancer cell lines by labeling each cell line with 24-nucleotide barcodes and revealed the expected patterns of cell killing seen in conventional (unpooled) assays.
Abstract: Hundreds of genetically characterized cell lines are available for the discovery of genotype-specific cancer vulnerabilities. However, screening large numbers of compounds against large numbers of cell lines is currently impractical, and such experiments are often difficult to control. Here we report a method called PRISM that allows pooled screening of mixtures of cancer cell lines by labeling each cell line with 24-nucleotide barcodes. PRISM revealed the expected patterns of cell killing seen in conventional (unpooled) assays. In a screen of 102 cell lines across 8,400 compounds, PRISM led to the identification of BRD-7880 as a potent and highly specific inhibitor of aurora kinases B and C. Cell line pools also efficiently formed tumors as xenografts, and PRISM recapitulated the expected pattern of erlotinib sensitivity in vivo.
213 citations
TL;DR: This short review discusses the most important technologies currently available for characterizing the direct and indirect target space of bioactive compounds following phenotypic screening and presents a comprehensive strategy employing complementary approaches to balance individual technology strengths and weaknesses.
121 citations
TL;DR: A versatile screening approach that combines an All-in-One Cas9D10A nickase vector with fluorescence-activated cell sorting enrichment followed by high-throughput genotypic and phenotypic clonal screening strategies to generate isogenic knockouts and knock-ins highly efficiently, with minimal off-target effects is described.
Abstract: The RNA-guided Cas9 nuclease is being widely employed to engineer the genomes of various cells and organisms. Despite the efficient mutagenesis induced by Cas9, off-target effects have raised concerns over the system's specificity. Recently a "double-nicking" strategy using catalytic mutant Cas9(D10A) nickase has been developed to minimise off-target effects. Here, we describe a Cas9(D10A)-based screening approach that combines an All-in-One Cas9(D10A) nickase vector with fluorescence-activated cell sorting enrichment followed by high-throughput genotypic and phenotypic clonal screening strategies to generate isogenic knockouts and knock-ins highly efficiently, with minimal off-target effects. We validated this approach by targeting genes for the DNA-damage response (DDR) proteins MDC1, 53BP1, RIF1 and P53, plus the nuclear architecture proteins Lamin A/C, in three different human cell lines. We also efficiently obtained biallelic knock-in clones, using single-stranded oligodeoxynucleotides as homologous templates, for insertion of an EcoRI recognition site at the RIF1 locus and introduction of a point mutation at the histone H2AFX locus to abolish assembly of DDR factors at sites of DNA double-strand breaks. This versatile screening approach should facilitate research aimed at defining gene functions, modelling of cancers and other diseases underpinned by genetic factors, and exploring new therapeutic opportunities.
116 citations
TL;DR: Recent advances in phenotypic readouts and MoA determination are highlighted by discussing several case studies in which both activities were required for understanding the chemical biology involved and, in some cases, advancing toward clinical development.
88 citations
TL;DR: The ability of this method to identify compounds known to have causal prophylactic activity in both human and animal models of malaria, as well as novel compounds, including some exclusively active against parasite exoerythrocytic stages are demonstrated.
Abstract: In order to identify the most attractive starting points for drugs that can be used to prevent malaria, a diverse chemical space comprising tens of thousands to millions of small molecules may need to be examined. Achieving this throughput necessitates the development of efficient ultra-high-throughput screening methods. Here, we report the development and evaluation of a luciferase-based phenotypic screen of malaria exoerythrocytic-stage parasites optimized for a 1536-well format. This assay uses the exoerythrocytic stage of the rodent malaria parasite, Plasmodium berghei, and a human hepatoma cell line. We use this assay to evaluate several biased and unbiased compound libraries, including two small sets of molecules (400 and 89 compounds, respectively) with known activity against malaria erythrocytic-stage parasites and a set of 9886 diversity-oriented synthesis (DOS)-derived compounds. Of the compounds screened, we obtain hit rates of 12–13 and 0.6% in preselected and naive libraries, respectively, an...
83 citations
TL;DR: A novel microfluidic-based picodroplet platform is demonstrated which enables high-throughput assessment and isolation of antibiotic- resistant bacteria in a label-free manner and can be used for rapid screening of rare antibiotic-resistant mutants to help identify novel compound/target pairs.
Abstract: The prevalence of clinically-relevant bacterial strains resistant to current antibiotic therapies is increasing and has been recognized as a major health threat. For example, multidrug-resistant tuberculosis and methicillin-resistant Staphylococcus aureus are of global concern. Novel methodologies are needed to identify new targets or novel compounds unaffected by pre-existing resistance mechanisms. Recently, water-in-oil picodroplets have been used as an alternative to conventional high-throughput methods, especially for phenotypic screening. Here we demonstrate a novel microfluidic-based picodroplet platform which enables high-throughput assessment and isolation of antibiotic-resistant bacteria in a label-free manner. As a proof-of-concept, the system was used to isolate fusidic acid-resistant mutants and estimate the frequency of resistance among a population of Escherichia coli (strain HS151). This approach can be used for rapid screening of rare antibiotic-resistant mutants to help identify novel compound/target pairs.
82 citations
TL;DR: A large-scale genetic screen in mice employing a phenotype-driven discovery platform to identify mutations resulting in age-related disease, both late-onset and progressive, reveals a number of novel pathways involved with age- related disease.
Abstract: Determining the genetic bases of age-related disease remains a major challenge requiring a spectrum of approaches from human and clinical genetics to the utilization of model organism studies. Here we report a large-scale genetic screen in mice employing a phenotype-driven discovery platform to identify mutations resulting in age-related disease, both late-onset and progressive. We have utilized N-ethyl-N-nitrosourea mutagenesis to generate pedigrees of mutagenized mice that were subject to recurrent screens for mutant phenotypes as the mice aged. In total, we identify 105 distinct mutant lines from 157 pedigrees analysed, out of which 27 are late-onset phenotypes across a range of physiological systems. Using whole-genome sequencing we uncover the underlying genes for 44 of these mutant phenotypes, including 12 late-onset phenotypes. These genes reveal a number of novel pathways involved with age-related disease. We illustrate our findings by the recovery and characterization of a novel mouse model of age-related hearing loss. Random mutagenesis can uncover novel genes involved in phenotypic traits. Here the authors perform a large-scale phenotypic screen on over 100 mouse strains generated by ENU mutagenesis to identify mice with age-related diseases, which they attribute to specific mutations revealed by whole-genome sequencing.
82 citations
TL;DR: The results implicate PDE3A modulators as candidate cancer therapeutic agents and demonstrate the power of predictive chemogenomics in small-molecule discovery.
Abstract: High cancer death rates indicate the need for new anticancer therapeutic agents. Approaches to discovering new cancer drugs include target-based drug discovery and phenotypic screening. Here, we identified phosphodiesterase 3A modulators as cell-selective cancer cytotoxic compounds through phenotypic compound library screening and target deconvolution by predictive chemogenomics. We found that sensitivity to 6-(4-(diethylamino)-3-nitrophenyl)-5-methyl-4,5-dihydropyridazin-3(2H)-one, or DNMDP, across 766 cancer cell lines correlates with expression of the gene PDE3A, encoding phosphodiesterase 3A. Like DNMDP, a subset of known PDE3A inhibitors kill selected cancer cells, whereas others do not. Furthermore, PDE3A depletion leads to DNMDP resistance. We demonstrated that DNMDP binding to PDE3A promotes an interaction between PDE3A and Schlafen 12 (SLFN12), suggestive of a neomorphic activity. Coexpression of SLFN12 with PDE3A correlates with DNMDP sensitivity, whereas depletion of SLFN12 results in decreased DNMDP sensitivity. Our results implicate PDE3A modulators as candidate cancer therapeutic agents and demonstrate the power of predictive chemogenomics in small-molecule discovery.
70 citations
TL;DR: This work uses molecular cell barcoding to enable direct in vivo phenotypic screening of small-molecule libraries and screened >700 covalent inhibitors directed toward hydrolases for their effect on pancreatic cancer metastatic seeding.
Abstract: Phenotype-based small-molecule screening is a powerful method to identify molecules that regulate cellular functions. However, such screens are generally performed in vitro under conditions that do not necessarily model complex physiological conditions or disease states. Here, we use molecular cell barcoding to enable direct in vivo phenotypic screening of small-molecule libraries. The multiplexed nature of this approach allows rapid in vivo analysis of hundreds to thousands of compounds. Using this platform, we screened >700 covalent inhibitors directed toward hydrolases for their effect on pancreatic cancer metastatic seeding. We identified multiple hits and confirmed the relevant target of one compound as the lipase ABHD6. Pharmacological and genetic studies confirmed the role of this enzyme as a regulator of metastatic fitness. Our results highlight the applicability of this multiplexed screening platform for investigating complex processes in vivo.
59 citations
TL;DR: A 3D cell culture chip was used for high-throughput screening of a human neural progenitor cell line and has potential use in phenotypic screening to elucidate molecular toxicology on human stem cells.
Abstract: A 3D cell culture chip was used for high-throughput screening of a human neural progenitor cell line The differential toxicity of 24 compounds was determined on undifferentiated and differentiating NPCs Five compounds led to significant differences in IC50 values between undifferentiated and differentiating cultures This platform has potential use in phenotypic screening to elucidate molecular toxicology on human stem cells
TL;DR: The reverse genetic and chemical SAR data support a continued investigation of SmPLK1 as a possible drug target and/or the prosecution of the benzimidazole thiophene chemotype as a source of novel anti-schistosomals.
Abstract: Author(s): Long, Thavy; Neitz, R Jeffrey; Beasley, Rachel; Kalyanaraman, Chakrapani; Suzuki, Brian M; Jacobson, Matthew P; Dissous, Colette; McKerrow, James H; Drewry, David H; Zuercher, William J; Singh, Rahul; Caffrey, Conor R | Abstract: BackgroundSchistosoma flatworm parasites cause schistosomiasis, a chronic and debilitating disease of poverty in developing countries. Praziquantel is employed for treatment and disease control. However, its efficacy spectrum is incomplete (less active or inactive against immature stages of the parasite) and there is a concern of drug resistance. Thus, there is a need to identify new drugs and drug targets.Methodology/principal findingsWe show that RNA interference (RNAi) of the Schistosoma mansoni ortholog of human polo-like kinase (huPLK)1 elicits a deleterious phenotypic alteration in post-infective larvae (schistosomula or somules). Phenotypic screening and analysis of schistosomula and adult S. mansoni with small molecule inhibitors of huPLK1 identified a number of potent anti-schistosomals. Among these was a GlaxoSmithKline (GSK) benzimidazole thiophene inhibitor that has completed Phase I clinical trials for treatment of solid tumor malignancies. We then obtained GSKs Published Kinase Inhibitor Sets (PKIS) 1 and 2, and phenotypically screened an expanded series of 38 benzimidazole thiophene PLK1 inhibitors. Computational analysis of controls and PLK1 inhibitor-treated populations of somules demonstrated a distinctive phenotype distribution. Using principal component analysis (PCA), the phenotypes exhibited by these populations were mapped, visualized and analyzed through projection to a low-dimensional space. The phenotype distribution was found to have a distinct shape and topology, which could be elicited using cluster analysis. A structure-activity relationship (SAR) was identified for the benzimidazole thiophenes that held for both somules and adult parasites. The most potent inhibitors produced marked phenotypic alterations at 1-2 μM within 1 h. Among these were compounds previously characterized as potent inhibitors of huPLK1 in cell assays.Conclusions/significanceThe reverse genetic and chemical SAR data support a continued investigation of SmPLK1 as a possible drug target and/or the prosecution of the benzimidazole thiophene chemotype as a source of novel anti-schistosomals.
TL;DR: Novel regulatory molecules are identified, which impact on PCa proliferation and are aberrantly expressed in clinical tumors, which reveals regulatory nodes with potential for therapy.
TL;DR: ECF506 is the first small molecule with subnanomolar IC50 for SRC that requires 3 orders of magnitude greater concentration to inhibit ABL, and exhibits excellent water solubility, an optimal DMPK profile and oral bioavailability, and inhibits SRC phosphorylation in tumor xenografts in mice.
Abstract: Novel pyrazolopyrimidines displaying high potency and selectivity toward SRC family kinases have been developed by combining ligand-based design and phenotypic screening in an iterative manner. Compounds were derived from the promiscuous kinase inhibitor PP1 to search for analogs that could potentially target a broad spectrum of kinases involved in cancer. Phenotypic screening against MCF7 mammary adenocarcinoma cells generated target-agnostic structure–activity relationships that biased subsequent designs toward breast cancer treatment rather than to a particular target. This strategy led to the discovery of two potent antiproliferative leads with phenotypically distinct anticancer mode of actions. Kinase profiling and further optimization resulted in eCF506, the first small molecule with subnanomolar IC50 for SRC that requires 3 orders of magnitude greater concentration to inhibit ABL. eCF506 exhibits excellent water solubility, an optimal DMPK profile and oral bioavailability, halts SRC-associated neur...
TL;DR: A meta-analysis of integrated large-scale, heterogeneous bioactivity data is performed to create an evidence-based, quantitative metric to systematically rank tool compounds for targets and demonstrate that high-TS tools show more reliably selective phenotypic profiles than lower-TS compounds.
31 Aug 2016
TL;DR: A high-throughput, high-content, whole-organism, phenotypic screen to identify small molecules that inhibit P. aeruginosa virulence and shows that 5-fluorouridine, in contrast to 5- fluorouracil, is a genuine antivirulence compound, with no bacteriostatic or bactericidal activity.
Abstract: Pseudomonas aeruginosa is an opportunistic pathogen that causes severe health problems. Despite intensive investigation, many aspects of microbial virulence remain poorly understood. We used a high-throughput, high-content, whole-organism, phenotypic screen to identify small molecules that inhibit P. aeruginosa virulence in Caenorhabditis elegans. Approximately half of the hits were known antimicrobials. A large number of hits were nonantimicrobial bioactive compounds, including the cancer chemotherapeutic 5-fluorouracil. We determined that 5-fluorouracil both transiently inhibits bacterial growth and reduces pyoverdine biosynthesis. Pyoverdine is a siderophore that regulates the expression of several virulence determinants and is critical for pathogenesis in mammals. We show that 5-fluorouridine, a downstream metabolite of 5-fluorouracil, is responsible for inhibiting pyoverdine biosynthesis. We also show that 5-fluorouridine, in contrast to 5-fluorouracil, is a genuine antivirulence compound, with no bacteriostatic or bactericidal activity. To our knowledge, this is the first report utilizing a whole-organism screen to identify novel compounds with antivirulent properties effective against P. aeruginosa. IMPORTANCE Despite intense research effort from scientists and the advent of the molecular age of biomedical research, many of the mechanisms that underlie pathogenesis are still understood poorly, if at all. The opportunistic human pathogen Pseudomonas aeruginosa causes a variety of soft tissue infections and is responsible for over 50,000 hospital-acquired infections per year. In addition, P. aeruginosa exhibits a striking degree of innate and acquired antimicrobial resistance, complicating treatment. It is increasingly important to understand P. aeruginosa virulence. In an effort to gain this information in an unbiased fashion, we used a high-throughput phenotypic screen to identify small molecules that disrupted bacterial pathogenesis and increased host survival using the model nematode Caenorhabditis elegans. This method led to the unexpected discovery that addition of a modified nucleotide, 5-fluorouridine, disrupted bacterial RNA metabolism and inhibited synthesis of pyoverdine, a critical toxin. Our results demonstrate that this compound specifically functions as an antivirulent.
TL;DR: Data indicate a novel mechanism for TNF-α-independent TNFR2 agonism in cancer immunotherapy, and demonstrate the utility of target-agnostic screening in highlighting important targets during drug discovery.
Abstract: // Geoffrey S. Williams 1 , Bina Mistry 1 , Sandrine Guillard 1 , Jane Coates Ulrichsen 1 , Alan M. Sandercock 1 , Jun Wang 2 , Andrea Gonzalez-Munoz 1 , Julie Parmentier 3 , Chelsea Black 4 , Jo Soden 5 , Jim Freeth 5 , Jelena Jovanovic 1 , Rebecca Leyland 1 , Rafia S. Al-Lamki 2 , Andrew J. Leishman 1 , Steven J. Rust 1 , Ross Stewart 1 , Lutz Jermutus 1 , John R. Bradley 2 , Vahe Bedian 3 , Viia Valge-Archer 1 , Ralph Minter 1 , Robert W. Wilkinson 1 1 MedImmune Ltd., Granta Park, Cambridge, CB21 6GH, UK 2 Department of Medicine, University of Cambridge, Addenbrooke’s Hospital, Cambridge, CB2 0QQ, UK 3 Oncology iMED, AstraZeneca-R&D Boston, Waltham, MA 02451, USA 4 MedImmune LLC, Gaithersburg, MD 20878, USA 5 Retrogenix Ltd, Whaley Bridge, High Peak, SK23 7LY, UK Correspondence to: Geoffrey S. Williams, email: williamsge@medimmune.com Keywords: TNFR2, regulatory T cell, cancer immunotherapy, drug discovery, phenotypic screening Received: May 27, 2016 Accepted: August 13, 2016 Published: September 10, 2016 ABSTRACT Antibodies that target cell-surface molecules on T cells can enhance anti-tumor immune responses, resulting in sustained immune-mediated control of cancer. We set out to find new cancer immunotherapy targets by phenotypic screening on human regulatory T (Treg) cells and report the discovery of novel activators of tumor necrosis factor receptor 2 (TNFR2) and a potential role for this target in immunotherapy. A diverse phage display library was screened to find antibody mimetics with preferential binding to Treg cells, the most Treg-selective of which were all, without exception, found to bind specifically to TNFR2. A subset of these TNFR2 binders were found to agonise the receptor, inducing iκ-B degradation and NF-κB pathway signalling in vitro . TNFR2 was found to be expressed by tumor-infiltrating Treg cells, and to a lesser extent Teff cells, from three lung cancer patients, and a similar pattern was also observed in mice implanted with CT26 syngeneic tumors. In such animals, TNFR2-specific agonists inhibited tumor growth, enhanced tumor infiltration by CD8 + T cells and increased CD8 + T cell IFN-γ synthesis. Together, these data indicate a novel mechanism for TNF-α-independent TNFR2 agonism in cancer immunotherapy, and demonstrate the utility of target-agnostic screening in highlighting important targets during drug discovery.
TL;DR: This review highlights recent advances and progress toward phenotypic screening methodologies over the past several years, with a focus on exoerythrocytic stage screens.
TL;DR: Phenotypic screening takes an unbiased approach to finding compounds that modify a particular cellular outcome of interest, usually in cell culture, but sometimes in whole organisms, such as zebrafish or C. elegans, which can be useful to identify new targets for a disease.
Abstract: The research community has witnessed a resurgence over the last several years in the enthusiasm for phenotypic screening as an approach for small-molecule discovery. In the course of running screen...
TL;DR: The research team has discovered several novel bioactive small molecules using in vivo cell-based phenotypic screening, and has investigated their modes of action using chemical genetics and chemical genomics.
TL;DR: A prospective view of a new era of PDD underpinned by a wealth of technology advances in the areas of in vitro model development, high-content imaging and image informatics, mechanism-of-action profiling and target deconvolution is offered.
Abstract: Phenotypic drug discovery (PDD) strategies are defined by screening and selection of hit or lead compounds based on quantifiable phenotypic endpoints without prior knowledge of the drug target. We outline the challenges associated with traditional phenotypic screening strategies and propose solutions and new opportunities to be gained by adopting modern PDD technologies. We highlight both historical and recent examples of approved drugs and new drug candidates discovered by modern phenotypic screening. Finally, we offer a prospective view of a new era of PDD underpinned by a wealth of technology advances in the areas of in vitro model development, high-content imaging and image informatics, mechanism-of-action profiling and target deconvolution.
TL;DR: A modified version of a bioluminescence reporter assay that allows nondestructive detection of compounds targeting either of two macromolecular processes in M. tuberculosis: cell wall biosynthesis or maintenance of DNA integrity and the potential to infer complex MOAs in which multiple cellular processes are disrupted is described.
Abstract: The tuberculosis (TB) drug discovery pipeline is fueled by compounds identified in whole-cell screens against the causative agent, Mycobacterium tuberculosis Phenotypic screening enables the selection of molecules that inhibit essential cellular functions in live, intact bacilli grown under a chosen in vitro condition. However, deducing the mechanism of action (MOA), which is important to avoid promiscuous targets, often requires significant biological resources in a lengthy process that risks decoupling medicinal chemistry and biology efforts. Therefore, there is a need to develop methods enabling rapid MOA assessment of putative "actives" for triage decisions. Here, we describe a modified version of a bioluminescence reporter assay that allows nondestructive detection of compounds targeting either of two macromolecular processes in M. tuberculosis: cell wall biosynthesis or maintenance of DNA integrity. Coupling the luxCDABE operon from Photorhabdus luminescens to mycobacterial promoters driving expression of the iniBAC operon (PiniB-LUX) or the DNA damage-inducible genes, recA (PrecA-LUX) or radA (PradA-LUX), provided quantitative detection in real time of compounds triggering expression of any of these promoters over an extended 10- to 12-day incubation. Testing against known anti-TB agents confirmed the specificity of each reporter in registering the MOA of the applied antibiotic in M. tuberculosis, independent of bactericidal or bacteriostatic activity. Moreover, profiles obtained for experimental compounds indicated the potential to infer complex MOAs in which multiple cellular processes are disrupted. These results demonstrate the utility of the reporters for early triage of compounds based on the provisional MOA and suggest their application to investigate polypharmacology in known and experimental anti-TB agents.
TL;DR: The suitability of cardiac progenitor cells for medium to high‐throughput screens to assess both proliferation and multilineage differentiation is demonstrated and activin‐like kinase 5 (transforming growth factor‐β type 1 receptor kinase) inhibitors are identified as novel and potent inducers of human CPC differentiation to cardiomyocytes.
Abstract: Several progenitor cell populations have been reported to exist in hearts that play a role in cardiac turnover and/or repair. Despite the presence of cardiac stem and progenitor cells within the myocardium, functional repair of the heart after injury is inadequate. Identification of the signaling pathways involved in the expansion and differentiation of cardiac progenitor cells (CPCs) will broaden insight into the fundamental mechanisms playing a role in cardiac homeostasis and disease and might provide strategies for in vivo regenerative therapies. To understand and exploit cardiac ontogeny for drug discovery efforts, we developed an in vitro human induced pluripotent stem cell-derived CPC model system using a highly enriched population of KDR(pos)/CKIT(neg)/NKX2.5(pos) CPCs. Using this model system, these CPCs were capable of generating highly enriched cultures of cardiomyocytes under directed differentiation conditions. In order to facilitate the identification of pathways and targets involved in proliferation and differentiation of resident CPCs, we developed phenotypic screening assays. Screening paradigms for therapeutic applications require a robust, scalable, and consistent methodology. In the present study, we have demonstrated the suitability of these cells for medium to high-throughput screens to assess both proliferation and multilineage differentiation. Using this CPC model system and a small directed compound set, we identified activin-like kinase 5 (transforming growth factor-β type 1 receptor kinase) inhibitors as novel and potent inducers of human CPC differentiation to cardiomyocytes. Significance: Cardiac disease is a leading cause of morbidity and mortality, with no treatment available that can result in functional repair. This study demonstrates how differentiation of induced pluripotent stem cells can be used to identify and isolate cell populations of interest that can translate to the adult human heart. Two separate examples of phenotypic screens are discussed, demonstrating the value of this biologically relevant and reproducible technology. In addition, this assay system was able to identify novel and potent inducers of differentiation and proliferation of induced pluripotent stem cell-derived cardiac progenitor cells.
TL;DR: This proof-of-concept study demonstrates the in silico deorphanization of phenotypic screening hits as a viable concept for future natural-product-inspired chemical biology and drug discovery efforts.
Abstract: The cyclodepsipeptide doliculide is a marine natural product with strong actin-polymerizing and anticancer activities. Evidence for doliculide acting as a potent and subtype-selective antagonist of prostanoid E receptor 3 (EP3) is presented. Computational target prediction suggested that this membrane receptor is a likely macromolecular target and enabled immediate in vitro validation. This proof-of-concept study demonstrates the in silico deorphanization of phenotypic screening hits as a viable concept for future natural-product-inspired chemical biology and drug discovery efforts.
TL;DR: P phenotypic screening with primary cardiomyocytes can be used to discover anti-hypertrophic lead compounds for heart failure drug discovery and facilitates chemical biological dissection of signaling networks that control pathological growth of the heart.
TL;DR: From a screen of approximately 4,100 bioactive tool compounds and approved drugs, a set of 22 compounds are identified that acutely protect human astrocytes from the consequences of hydrogen peroxide‐induced oxidative stress, demonstrating the relevancy and utility of using astroCytes differentiated from human stem cells as a disease model for drug discovery and development.
Abstract: UNLABELLED Astrocytes are the predominant cell type in the nervous system and play a significant role in maintaining neuronal health and homeostasis. Recently, astrocyte dysfunction has been implicated in the pathogenesis of many neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, Huntington's disease, and amyotrophic lateral sclerosis. Astrocytes are thus an attractive new target for drug discovery for neurological disorders. Using astrocytes differentiated from human embryonic stem cells, we have developed an assay to identify compounds that protect against oxidative stress, a condition associated with many neurodegenerative diseases. This phenotypic oxidative stress assay has been optimized for high-throughput screening in a 1,536-well plate format. From a screen of approximately 4,100 bioactive tool compounds and approved drugs, we identified a set of 22 that acutely protect human astrocytes from the consequences of hydrogen peroxide-induced oxidative stress. Nine of these compounds were also found to be protective of induced pluripotent stem cell-differentiated astrocytes in a related assay. These compounds are thought to confer protection through hormesis, activating stress-response pathways and preconditioning astrocytes to handle subsequent exposure to hydrogen peroxide. In fact, four of these compounds were found to activate the antioxidant response element/nuclear factor-E2-related factor 2 pathway, a protective pathway induced by toxic insults. Our results demonstrate the relevancy and utility of using astrocytes differentiated from human stem cells as a disease model for drug discovery and development. SIGNIFICANCE Astrocytes play a key role in neurological diseases. Drug discovery efforts that target astrocytes can identify novel therapeutics. Human astrocytes are difficult to obtain and thus are challenging to use for high-throughput screening, which requires large numbers of cells. Using human embryonic stem cell-derived astrocytes and an optimized astrocyte differentiation protocol, it was possible to screen approximately 4,100 compounds in titration to identify 22 that are cytoprotective of astrocytes. This study is the largest-scale high-throughput screen conducted using human astrocytes, with a total of 17,536 data points collected in the primary screen. The results demonstrate the relevancy and utility of using astrocytes differentiated from human stem cells as a disease model for drug discovery and development.
TL;DR: The results described herein demonstrate the power of combining ALIS with cell functional assays in a high-throughput, target-based approach to determine the most tractable drug discovery opportunities within a pathway.
Abstract: The primary objective of early drug discovery is to associate druggable target space with a desired phenotype. The inability to efficiently associate these often leads to failure early in the drug discovery process. In this proof-of-concept study, the most tractable starting points for drug discovery within the NF-κB pathway model system were identified by integrating affinity selection-mass spectrometry (AS-MS) with functional cellular assays. The AS-MS platform Automated Ligand Identification System (ALIS) was used to rapidly screen 15 NF-κB proteins in parallel against large-compound libraries. ALIS identified 382 target-selective compounds binding to 14 of the 15 proteins. Without any chemical optimization, 22 of the 382 target-selective compounds exhibited a cellular phenotype consistent with the respective target associated in ALIS. Further studies on structurally related compounds distinguished two chemical series that exhibited a preliminary structure-activity relationship and confirmed target-driven cellular activity to NF-κB1/p105 and TRAF5, respectively. These two series represent new drug discovery opportunities for chemical optimization. The results described herein demonstrate the power of combining ALIS with cell functional assays in a high-throughput, target-based approach to determine the most tractable drug discovery opportunities within a pathway.
TL;DR: High-throughput drug screening using HGPS iPS-derived cells reveals seven compounds that normalized the osteogenic differentiation process and, among these, all-trans retinoic acid and 13-cis-retinoic Acid, that also decreased progerin expression are highlighted.
Abstract: Hutchinson-Gilford progeria syndrome (HGPS) is a rare fatal genetic disorder that causes systemic accelerated aging in children. Thanks to the pluripotency and self-renewal properties of induced pluripotent stem cells (iPSC), HGPS iPSC-based modeling opens up the possibility of access to different relevant cell types for pharmacological approaches. In this study, 2800 small molecules were explored using high-throughput screening, looking for compounds that could potentially reduce the alkaline phosphatase activity of HGPS mesenchymal stem cells (MSCs) committed into osteogenic differentiation. Results revealed seven compounds that normalized the osteogenic differentiation process and, among these, all-trans retinoic acid and 13-cis-retinoic acid, that also decreased progerin expression. This study highlights the potential of high-throughput drug screening using HGPS iPS-derived cells, in order to find therapeutic compounds for HGPS and, potentially, for other aging-related disorders.
TL;DR: The various model organisms used in phenotypic screens are discussed, with particular focus on zebrafish, which has emerged as a leading model of in vivo phenotypesic screens.
Abstract: Target based chemical screens are a mainstay of modern drug discovery, but the effectiveness of this reductionist approach is being questioned in light of declines in pharmaceutical R & D efficiency. In recent years, phenotypic screens have gained increasing acceptance as a complementary/alternative approach to early drug discovery. We discuss the various model organisms used in phenotypic screens, with particular focus on zebrafish, which has emerged as a leading model of in vivo phenotypic screens. Additionally, we anticipate therapeutic opportunities, particularly in orphan disease space, in the context of rapid advances in human Mendelian genetics, electronic health record (EHR)-enabled genome-phenome associations, and genome editing.
TL;DR: This method has broad potential for high-throughput phenotype characterization of putative drug efflux systems in a range of organisms and represents the first drug resistance protein characterized from this superfamily in A. baumannii.
Abstract: OBJECTIVES Drug efflux pumps are one of the key machineries in bacterial drug resistance. Although quite a few of these transport systems have been functionally characterized in various organisms, due to large-scale genome sequencing efforts and improved prediction pipelines there are increasing numbers of putative drug efflux genes annotated. For phenotype identification of the proteins encoded by these genes, we developed a novel high-throughput phenotype screening strategy and demonstrated its utility in identifying phenotypes for putative efflux systems encoded by the human pathogen Acinetobacter baumannii. METHODS Seventeen putative drug efflux systems from A. baumannii were heterologously expressed in Escherichia coli. For rapid and economical phenotype screening we employed a combination of multiplexed Biolog Phenotype Microarrays and quantitative PCR. Using this method we screened these putative drug efflux pumps against 240 antimicrobial conditions, equating to 4080 simultaneous phenotypic tests. RESULTS Of the 17 putative drug efflux systems, phenotypes were confirmed for two pumps and novel drug resistance phenotypes were identified for three new A. baumannii drug transporters, which exemplified the power of this method as a high-throughput screening technique. One of the phenotypically characterized putative drug efflux systems was classified within the ATP-binding cassette superfamily of transport proteins and represents the first drug resistance protein characterized from this superfamily in A. baumannii. The remaining two proteins were members of the major facilitator superfamily of efflux pumps. CONCLUSIONS This method has broad potential for high-throughput phenotype characterization of putative drug efflux systems in a range of organisms.