High-content screening (HCS) technology combining automated microscopy and quantitative image analysis can address biological questions in academia and the pharmaceutical industry. Various HCS ...experimental applications have been utilized in the research field of in vitro toxicology. In this review, we describe several HCS application approaches used for studying the mechanism of compound toxicity, highlight some challenges faced in the toxicological community, and discuss the future directions of HCS in regards to new models, new reagents, data management, and informatics. Many specialized areas of toxicology including developmental toxicity, genotoxicity, developmental neurotoxicity/neurotoxicity, hepatotoxicity, cardiotoxicity, and nephrotoxicity will be examined. In addition, several newly developed cellular assay models including induced pluripotent stem cells (iPSCs), three-dimensional (3D) cell models, and tissues-on-a-chip will be discussed. New genome-editing technologies (e.g., CRISPR/Cas9), data analyzing tools for imaging, and coupling with high-content assays will be reviewed. Finally, the applications of machine learning to image processing will be explored. These new HCS approaches offer a huge step forward in dissecting biological processes, developing drugs, and making toxicology studies easier.
In vitro methods which incorporate metabolic capability into the assays allow us to assess the activity of metabolites from their parent compounds. These methods can be applied into high-throughput ...screening (HTS) platforms, thereby increasing the speed to identify compounds that become active via the metabolism process. HTS was originally used in the pharmaceutical industry and now is also used in academic settings to evaluate biological activity and/or toxicity of chemicals. Although most chemicals are metabolized in our body, many HTS assays lack the capability to determine compound activity via metabolism. To overcome this problem, several in vitro metabolic methods have been applied to an HTS format. In this review, we describe in vitro metabolism methods and their application in HTS assays, as well as discuss the future perspectives of HTS with metabolic activity. Each in vitro metabolism method has advantages and disadvantages. For instance, the S9 mix has a full set of liver metabolic enzymes, but it displays high cytotoxicity in cell-based assays. In vitro metabolism requires liver fractions or the use of other metabolically capable systems, including primary hepatocytes or recombinant enzymes. Several newly developed in vitro metabolic methods, including HepaRG cells, three-dimensional (3D) cell models, and organ-on-a-chip technology, will also be discussed. These newly developed in vitro metabolism approaches offer significant progress in dissecting biological processes, developing drugs, and making toxicology studies quicker and more efficient.
Recent outbreaks of Zika virus （ZIKV） highlight an urgent need for therapeutics. The protease complex NS2B- NS3 plays essential roles during flaviviral polyprotein processing, and thus represents an ...attractive drug target. Here, we developed a split luciferase complementation-based high-throughput screening assay to identify orthosteric inhibitors that directly target flavivirus NS2B-NS3 interactions. By screening a total of 2 816 approved and investigational drugs, we identified three potent candidates, temoporfin, niclosamide, and nitazoxanide, as flavivirus NS2B- NS3 interaction inhibitors with nanomolar potencies. Significantly, the most potent compound, temoporfin, not only inhibited ZIKV replication in human placental and neural progenitor cells, but also prevented ZIKV-induced viremia and mortality in mouse models. Structural docking suggests that temoporfin potentially binds NS3 pockets that hold critical NS2B residues, thus inhibiting flaviviral polyprotein processing in a non-competitive manner. As these drugs have already been approved for clinical use in other indications either in the USA or other countries, they represent promising and easily developed therapies for the management of infections by ZIKV and other flaviviruses.
In response to the current global health emergency posed by the Zika virus (ZIKV) outbreak and its link to microcephaly and other neurological conditions, we performed a drug repurposing screen of ...∼6,000 compounds that included approved drugs, clinical trial drug candidates and pharmacologically active compounds; we identified compounds that either inhibit ZIKV infection or suppress infection-induced caspase-3 activity in different neural cells. A pan-caspase inhibitor, emricasan, inhibited ZIKV-induced increases in caspase-3 activity and protected human cortical neural progenitors in both monolayer and three-dimensional organoid cultures. Ten structurally unrelated inhibitors of cyclin-dependent kinases inhibited ZIKV replication. Niclosamide, a category B anthelmintic drug approved by the US Food and Drug Administration, also inhibited ZIKV replication. Finally, combination treatments using one compound from each category (neuroprotective and antiviral) further increased protection of human neural progenitors and astrocytes from ZIKV-induced cell death. Our results demonstrate the efficacy of this screening strategy and identify lead compounds for anti-ZIKV drug development.
The recent global pandemic of the Coronavirus disease 2019 (COVID-19) caused by the new coronavirus SARS-CoV-2 presents an urgent need for the development of new therapeutic candidates. Many efforts ...have been devoted to screening existing drug libraries with the hope to repurpose approved drugs as potential treatments for COVID-19. However, the antiviral mechanisms of action of the drugs found active in these phenotypic screens remain largely unknown. In an effort to deconvolute the viral targets in pursuit of more effective anti-COVID-19 drug development, we mined our in-house database of approved drug screens against 994 assays and compared their activity profiles with the drug activity profile in a cytopathic effect (CPE) assay of SARS-CoV-2. We found that the autophagy and AP-1 signaling pathway activity profiles are significantly correlated with the anti-SARS-CoV-2 activity profile. In addition, a class of neurology/psychiatry drugs was found to be significantly enriched with anti-SARS-CoV-2 activity. Taken together, these results provide new insights into SARS-CoV-2 infection and potential targets for COVID-19 therapeutics, which can be further validated by in vivo animal studies and human clinical trials.
Target-specific, mechanism-oriented in vitro assays post a promising alternative to traditional animal toxicology studies. Here we report the first comprehensive analysis of the Tox21 effort, a ...large-scale in vitro toxicity screening of chemicals. We test ∼ 10,000 chemicals in triplicates at 15 concentrations against a panel of nuclear receptor and stress response pathway assays, producing more than 50 million data points. Compound clustering by structure similarity and activity profile similarity across the assays reveals structure-activity relationships that are useful for the generation of mechanistic hypotheses. We apply structural information and activity data to build predictive models for 72 in vivo toxicity end points using a cluster-based approach. Models based on in vitro assay data perform better in predicting human toxicity end points than animal toxicity, while a combination of structural and activity data results in better models than using structure or activity data alone. Our results suggest that in vitro activity profiles can be applied as signatures of compound mechanism of toxicity and used in prioritization for more in-depth toxicological testing.
A synopsis of the Tox21 initiative and a focus on the NIH Chemical Genomics Center's efforts within this program using
in vitro methods and quantitative high-throughput screening.
The US Tox21 ...collaborative program represents a paradigm shift in toxicity testing of chemical compounds from traditional
in vivo tests to less expensive and higher throughput
in vitro methods to prioritize compounds for further study, identify mechanisms of action and ultimately develop predictive models for adverse health effects in humans. The NIH Chemical Genomics Center (NCGC) is an integral component of the Tox21 collaboration owing to its quantitative high-throughput screening (qHTS) paradigm, in which titration-based screening is used to profile hundreds of thousands of compounds per week. Here, we describe the Tox21 collaboration, qHTS-based compound testing and the various Tox21 screening assays that have been validated and tested at the NCGC to date.
Chronic exposure to drinking water disinfection byproducts has been linked to adverse health risks. The monohaloacetic acids (monoHAAs) are generated as byproducts during the disinfection of drinking ...water and are cytotoxic, genotoxic, mutagenic, and teratogenic. Iodoacetic acid toxicity was mitigated by antioxidants, suggesting the involvement of oxidative stress. Other monoHAAs may share a similar mode of action. Each monoHAA generated a significant concentration–response increase in the expression of a β-lactamase reporter under the control of the antioxidant response element (ARE). The monoHAAs generated oxidative stress with a rank order of iodoacetic acid (IAA) > bromoacetic acid (BAA) ≫ chloroacetic acid (CAA); this rank order was observed with other toxicological end points. Toxicogenomic analysis was conducted with a nontransformed human intestinal epithelial cell line (FHs 74 Int). Exposure to the monoHAAs altered the transcription levels of multiple oxidative stress responsive genes, indicating that each exposure generated oxidative stress. The transcriptome profiles showed an increase in thioredoxin reductase 1 (TXNRD1) and sulfiredoxin (SRXN1), suggesting peroxiredoxin proteins had been oxidized during monoHAA exposures. Three possible sources of reactive oxygen species were identified, the hypohalous acid generating peroxidase enzymes lactoperoxidase (LPO) and myeloperoxidase (MPO), nicotinamide adenine dinucleotide phosphate (NADPH)-dependent oxidase 5 (NOX5), and PTGS2 (COX-2) mediated arachidonic acid metabolism. Each monoHAA exposure caused an increase in COX-2 mRNA levels. These data provide a functional association between monoHAA exposure and adverse health outcomes such as oxidative stress, inflammation, and cancer.
Loss of function mutations in Kelch-like ECH Associated Protein 1 (KEAP1), or gain-of-function mutations in nuclear factor erythroid 2-related factor 2 (NRF2), are common in non-small cell lung ...cancer (NSCLC) and associated with therapeutic resistance. To discover novel NRF2 inhibitors for targeted therapy, we conducted a quantitative high-throughput screen using a diverse set of ∼400 000 small molecules (Molecular Libraries Small Molecule Repository Library, MLSMR) at the National Center for Advancing Translational Sciences. We identified ML385 as a probe molecule that binds to NRF2 and inhibits its downstream target gene expression. Specifically, ML385 binds to Neh1, the Cap ‘N’ Collar Basic Leucine Zipper (CNC-bZIP) domain of NRF2, and interferes with the binding of the V-Maf Avian Musculoaponeurotic Fibrosarcoma Oncogene Homologue G (MAFG)-NRF2 protein complex to regulatory DNA binding sequences. In clonogenic assays, when used in combination with platinum-based drugs, doxorubicin or taxol, ML385 substantially enhances cytotoxicity in NSCLC cells, as compared to single agents. ML385 shows specificity and selectivity for NSCLC cells with KEAP1 mutation, leading to gain of NRF2 function. In preclinical models of NSCLC with gain of NRF2 function, ML385 in combination with carboplatin showed significant antitumor activity. We demonstrate the discovery and validation of ML385 as a novel and specific NRF2 inhibitor and conclude that targeting NRF2 may represent a promising strategy for the treatment of advanced NSCLC.
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