In vitro assay data have recently emerged as a potential alternative to traditional animal toxicity studies to aid in the prediction of adverse effects of chemicals on humans. Here we evaluate the data generated from a battery of quantitative high-throughput screening (qHTS) assays applied to a large and diverse collection of chemicals, including approved drugs, for their capacity in predicting human toxicity. Models were built with animal in vivo toxicity data, in vitro human cell-based assay data, as well as in combination with chemical structure and/or drug-target information to predict adverse effects observed for drugs in humans. Interestingly, we found that the models built with the human cell-based assay data performed close to those of the models based on animal in vivo toxicity data. Furthermore, expanding the biological space coverage of assays by including additional drug-target annotations was shown to significantly improve model performance. We identified a small set of targets, which, when added to the current suite of in vitro human cell-based assay data, result in models that greatly outperform those built with the existing animal toxicity data. Assays can be developed for this set of targets to screen compounds for construction of robust models for human toxicity prediction.

Many flaviviruses, such as Zika virus (ZIKV), Dengue virus (DENV1-4) and yellow fever virus (YFV), are significant human pathogens. Infection with ZIKV, an emerging mosquito-borne flavivirus, is associated with increased risk of microcephaly in newborns and Guillain-Barré syndrome and other complications in adults. Currently, specific therapy does not exist for any flavivirus infections. In this study, we found that erythrosin B, an FDA-approved food additive, is a potent inhibitor for flaviviruses, including ZIKV and DENV2. Erythrosin B was found to inhibit the DENV2 and ZIKV NS2B-NS3 proteases with IC50 in low micromolar range, via a non-competitive mechanism. Erythrosin B can significantly reduce titers of representative flaviviruses, DENV2, ZIKV, YFV, JEV, and WNV, with micromolar potency and with excellent cytotoxicity profile. Erythrosin B can also inhibit ZIKV replication in ZIKV-relevant human placental and neural progenitor cells. As a pregnancy category B food additive, erythrosin B may represent a promising and easily developed therapy for management of infections by ZIKV and other flaviviruses.

The new strategy for chemical toxicity testing and modeling is to use in vitro human cell-based assays in conjunction with quantitative high-throughput screening (qHTS) technology, to identify molecular mechanisms and predict in vivo responses. Stem cells are more physiologically relevant than immortalized cell lines because of their unique proliferation and differentiation potentials. We established a robust two stem cells-two lineages assay system, encompassing human mesenchymal stem cells (hMSCs) along osteogenesis and human induced pluripotent stem cells (hiPSCs) along hepatogenesis. We performed qHTS phenotypic screening of LOPAC1280 and identified 38 preliminary hits for hMSCs. This was followed by validation of a selected number of hits and determination of their IC50 values and mechanistic studies of idarubicin and cantharidin treatments using proteomics and bioinformatics. In general, hiPSCs were more sensitive than hMSCs to chemicals, and differentiated progenies were less sensitive than their progenitors. We showed that chemical toxicity depends on both stem cell types and their differentiation stages. Proteomics identified and quantified over 3000 proteins for both stem cells. Bioinformatics identified apoptosis and G2/M as the top pathways conferring idarubicin toxicity. Our Omics-based assays of stem cells provide mechanistic insights into chemical toxicity and may help prioritize chemicals for in-depth toxicological evaluations.

The estrogen-related receptor α (ERRα) is an orphan nuclear receptor (NR) that plays a role in energy homeostasis and controls mitochondrial oxidative respiration. Increased expression of ERRα in certain ovarian, breast, and colon cancers has a negative prognosis, indicating an important role for ERRα in cancer progression. An interaction between ERRα and peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) has also recently been shown to regulate an enzyme in the β-oxidation of free fatty acids, thereby suggesting that ERRα plays an important role in obesity and type 2 diabetes. Therefore, it would be prudent to identify compounds that can act as activators of ERRα. In this study, we screened ∼10,000 (8311 unique) compounds, known as the Tox21 10K collection, to identify agonists of ERRα. We performed this screen using two stably transfected HEK 293 cell lines, one with the ERRα-reporter alone and the other with both ERRα-reporter and PGC-1α expression vectors. After the primary screening, we identified more than five agonist clusters based on compound structural similarity analysis (e.g., statins). By examining the activities of the confirmed ERRα modulators in other Tox21 NR assays, eliminating those with promiscuous NR activity, and performing follow-up assays (e.g., small interfering RNA knockdown), we identified compounds that might act as endocrine disrupters through effects on ERRα signaling. To our knowledge, this study is the first comprehensive analysis in discovering potential endocrine disrupters that affect the ERRα signaling pathway.

A chemical genomics "Toxmatrix" method was developed to elucidate mechanisms of cytotoxicity using neuronal models. Quantitative high-throughput screening (qHTS) was applied to systematically screen each toxicant against a panel of 70 modulators, drugs or chemicals that act on a known target, to identify interactions that either protect or sensitize cells to each toxicant. Thirty-two toxicants were tested at 10 concentrations for cytotoxicity to SH-SY5Y human neuroblastoma cells, with results fitted to the Hill equation to determine an IC50 for each toxicant. Thirty-three toxicant:modulator interactions were identified in SH-SY5Y cells for 14 toxicants, as modulators that shifted toxicant IC50 values lower or higher. The target of each modulator that sensitizes cells or protects cells from a toxicant suggests a mode of toxicant action or cellular adaptation. In secondary screening, we tested modulator-toxicant pairs identified from the SH-SY5Y primary screening for interactions in three differentiated neuronal human cell lines: dSH-SY5Y, conditionally immortalized dopaminergic neurons (LUHMES), and neural stem cells. Twenty toxicant-modulator pairs showed pronounced interactions in one or several differentiated cell models. Additional testing confirmed that several modulators acted through their primary targets. For example, several chelators protected differentiated LUHMES neurons from four toxicants by chelation of divalent cations and buthionine sulphoximine sensitized cells to 6-hydroxydopamine and 4-(methylamino)phenol hemisulfate by blocking glutathione synthesis. Such modulators that interact with multiple neurotoxicants suggest these may be vulnerable toxicity pathways in neurons. Thus, the Toxmatrix method is a systematic high-throughput approach that can identify mechanisms of toxicity and cellular adaptation.

There are tremendous unmet needs in drug development for rare diseases. Computational drug repositioning is a promising approach and has been successfully applied to the development of treatments for diseases. However, how to utilize this knowledge and effectively conduct and implement computational drug repositioning approaches for rare disease therapies is still an open issue. Here, we focus on the means of utilizing accumulated genomic data for accelerating and facilitating drug repositioning for rare diseases. First, we summarize the current genome landscape of rare diseases. Second, we propose several promising bioinformatics approaches and pipelines for computational drug repositioning for rare diseases. Finally, we discuss recent regulatory incentives and other enablers in rare disease drug development and outline the remaining challenges.

Angiogenesis is an important hallmark of cancer, contributing to tumor formation and metastasis. In vitro angiogenesis models for analyzing tube formation serve as useful tools to study these processes. However, current in vitro co-culture models using primary cells have limitations in usefulness and consistency. Therefore, in the present study, an in vitro co-culture assay system was optimized in a 1536-well format for high-throughput screening using human telomerase reverse transcriptase (hTERT)-immortalized mesenchymal stem cells and aortic endothelial cells. The National Center for Advancing Translational Sciences (NCATS) Pharmaceutical Collection (NPC) library containing 2816 drugs was evaluated using the in vitro co-culture assay. From the screen, 35 potent inhibitors (IC50 ≤1 µM) were identified, followed by 15 weaker inhibitors (IC50 1-50 µM). Moreover, many known angiogenesis inhibitors were identified, such as topotecan, docetaxel, and bortezomib. Several potential novel angiogenesis inhibitors were also identified from this study, including thimerosal and podofilox. Among the inhibitors, some compounds were proved to be involved in the hypoxia-inducible factor-1α (HIF-1α) and the nuclear factor-kappa B (NF-κB) pathways. The co-culture model developed by using hTERT-immortalized cell lines described in this report provides a consistent and robust in vitro system for antiangiogenic drug screening.

Genotoxicity potential is a critical component of any comprehensive toxicological profile. Compounds that induce DNA or chromosomal damage often activate p53, a transcription factor essential to cell cycle regulation. Thus, within the US Tox21 Program, we screened a library of ∼10,000 (∼8,300 unique) environmental compounds and drugs for activation of the p53-signaling pathway using a quantitative high-throughput screening assay employing HCT-116 cells (p53(+/+) ) containing a stably integrated β-lactamase reporter gene under control of the p53 response element (p53RE). Cells were exposed (-S9) for 16 hr at 15 concentrations (generally 1.2 nM to 92 μM) three times, independently. Excluding compounds that failed analytical chemistry analysis or were suspected of inducing assay interference, 365 (4.7%) of 7,849 unique compounds were concluded to activate p53. As part of an in-depth characterization of our results, we first compared them with results from traditional in vitro genotoxicity assays (bacterial mutation, chromosomal aberration); ∼15% of known, direct-acting genotoxicants in our library activated the p53RE. Mining the Comparative Toxicogenomics Database revealed that these p53 actives were significantly associated with increased expression of p53 downstream genes involved in DNA damage responses. Furthermore, 53 chemical substructures associated with genotoxicity were enriched in certain classes of p53 actives, for example, anthracyclines (antineoplastics) and vinca alkaloids (tubulin disruptors). Interestingly, the tubulin disruptors manifested unusual nonmonotonic concentration response curves suggesting activity through a unique p53 regulatory mechanism. Through the analysis of our results, we aim to define a role for this assay as one component of a comprehensive toxicological characterization of large compound libraries. Environ. Mol. Mutagen. 58:494-507, 2017. © 2017 Wiley Periodicals, Inc.

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.

Recent reports have noted that a number of compounds that block the human Ether-à-go-go related gene (hERG) ion channel also induce phospholipidosis (PLD). To explore a hypothesis explaining why most PLD inducers are also hERG inhibitors, a modeling approach was undertaken with data sets comprised of 4096 compounds assayed for hERG inhibition and 5490 compounds assayed for PLD induction. To eliminate the chemical domain effect, a filtered data set of 567 compounds tested in quantitative high-throughput screening (qHTS) format for both hERG inhibition and PLD induction was constructed. Partial least squares (PLS) modeling followed by 3D-SDAR mapping of the most frequently occurring bins and projection on to the chemical structure suggested that both adverse effects are driven by similar structural features, namely two aromatic rings and an amino group forming a three-center toxicophore. Non-parametric U-tests performed on the original 3D-SDAR bins indicated that the distance between the two aromatic rings is the main factor determining the differences in activity; at distances of up to about 5.5 Å, a phospholipidotic compound would also inhibit hERG, while at longer distances, a sharp reduction of the PLD-inducing potential leaves only a well-pronounced hERG blocking effect. The hERG activity itself diminishes after the distance between the centroids of the two aromatic rings exceeds 12.5 Å. Further comparison of the two toxicophores revealed that the almost identical aromatic rings to amino group distances play no significant role in distinguishing between PLD and hERG activity. The hypothesis that the PLD toxicophore appears to be a subset of the hERG toxicophore explains why about 80% of all phospholipidotic chemicals (the remaining 20% are thought to act via a different mechanism) also inhibit the hERG ion channel. These models were further validated in large-scale qHTS assays testing 1085 chemicals for their PLD-inducing potential and 1570 compounds for hERG inhibition. After removal of the modeling and experimental inconclusive compounds, the area under the receiver-operating characteristic (ROC) curve was 0.92 for the PLD model and 0.87 for the hERG model. Due to the exceptional ability of these models to recognize safe compounds (negative predictive values of 0.99 for PLD and 0.94 for hERG were achieved), their use in regulatory settings might be particularly useful.