The pregnane X receptor (PXR; NR1I2) is an important nuclear receptor whose main function is to regulate enzymes within drug metabolism. The main drug metabolizing enzyme regulated by PXR, cytochrome P450 (CYP) 3A4, accounts for the metabolism of nearly 50% of all marketed drugs. Recently, PXR has also been identified as playing a role in energy homeostasis, immune response, and cancer. Due to its interaction with these important roles, alongside its drug-drug interaction function, it is imperative to identify compounds which can modulate PXR. In this study, we screened the Tox21 10,000 compound collection to identify hPXR agonists using a stable hPXR-Luc HepG2 cell line. A pharmacological study in the presence of a PXR antagonist was performed to confirm the activity of the chosen potential hPXR agonists in the same cells. Finally, metabolically competent cell lines - HepaRG and HepaRG-PXR-Knockout (KO) - were used to further confirm the potential PXR activators. We identified a group of structural clusters and singleton compounds which included potentially novel hPXR agonists. Of the 21 selected compounds, 11 potential PXR activators significantly induced CYP3A4 mRNA expression in HepaRG cells. All 11 of these compounds lost their induction when treating HepaRG-PXR-KO cells, confirming their PXR activation. Etomidoline presented as a potentially selective agonist of PXR. In conclusion, the current study has identified 11 compounds as potentially novel or not well-characterized PXR activators. These compounds should further be studied for their potential effects on drug metabolism and drug-drug interactions due to the immense implications of being a PXR agonist.

Many flaviviruses including the Dengue virus (DENV), Zika virus (ZIKV), West Nile virus, Yellow Fever virus, and Japanese encephalitis virus are significant human pathogens, unfortunately without any specific therapy. Here, we demonstrate that methylene blue, an FDA-approved drug, is a broad-spectrum and potent antiviral against Zika virus and Dengue virus both in vitro and in vivo. We found that methylene blue can considerably inhibit the interactions between viral protease NS3 and its NS2B co-factor, inhibit viral protease activity, inhibit viral growth, protect 3D mini-brain organoids from ZIKV infection, and reduce viremia in a mouse model. Mechanistic studies confirmed that methylene blue works in both entry and post entry steps, reduces virus production in replicon cells and inhibited production of processed NS3 protein. Overall, we have shown that methylene blue is a potent antiviral for management of flavivirus infections, particularly for Zika virus. As an FDA-approved drug, methylene blue is well-tolerated for human use. Therefore, methylene blue represents a promising and easily developed therapy for management of infections by ZIKV and other flaviviruses.

Dysregulation of the Wnt/β-catenin signaling pathway has been widely recognized as a pathogenic mechanism for colorectal cancer (CRC). Although numerous Wnt inhibitors have been developed, they commonly suffer from toxicity and unintended effects. Moreover, concerns have been raised in targeting this pathway because of its critical roles in maintaining stem cells and regenerating tissues and organs. On the basis of the anthelmintic drug pyrvinium and previous lead FX1128, we have developed a compound YW2065 (1c) which demonstrated excellent anti-CRC effects in vitro and in vivo. YW2065 achieves its inhibitory activity for Wnt signaling by stabilizing Axin-1, a scaffolding protein that regulates proteasome degradation of β-catenin. Simultaneously, YW2065 also led to the activation of the tumor suppressor AMPK, providing an additional anticancer mechanism. In addition, YW2065 showed favorable pharmacokinetic properties without obvious toxicity. The anti-CRC effect of YW2065 was highlighted by its promising efficacy in a mice xenograft model.

The transforming growth factor beta (TGFβ) superfamily of secreted signaling molecules and their cognate receptors regulate cell fate and behaviors relevant to many developmental and disease processes. Disruption of TGFβ signaling during embryonic development can, for example, affect morphogenesis and differentiation through complex pathways that may be SMAD (Small Mothers Against Decapentaplegic) dependent or SMAD independent. In the present study, the SMAD Binding Element (SBE)-beta lactamase (bla) HEK 293T cell line, which responds to the activation of the SMAD2/3/4 complex, was used in a quantitative high-throughput screening (qHTS) assay to identify potential TGFβ disruptors in the Tox21 10K compound library. From the primary screening we identified several kinase inhibitors, organometallic compounds, and dithiocarbamates (DTCs) that inhibited TGFβ1-induced SMAD signaling of reporter gene activation independent of cytotoxicity. Counterscreen of SBE antagonists on human embryonic neural stem cells demonstrated cytotoxicity, providing additional evidence to support evaluation of these compounds for developmental toxicity. We profiled the inhibitory patterns of putative SBE antagonists toward other developmental signaling pathways, including wingless-related integration site (WNT), retinoic acid α receptor (RAR), and sonic hedgehog (SHH). The profiling results from SBE-bla assay identify chemicals that disrupt TGFβ/SMAD signaling as part of an integrated qHTS approach for prioritizing putative developmental toxicants.

BACKGROUND: Thyroid hormone receptors (TRs) are critical endocrine receptors that regulate a multitude of processes in adult and developing organisms, and thyroid hormone disruption is of high concern for neurodevelopmental and reproductive toxicities in particular. To date, only a small number of chemical classes have been identified as possible TR modulators, and the receptors appear highly selective with respect to the ligand structural diversity. Thus, the question of whether TRs are an important screening target for protection of human and wildlife health remains. OBJECTIVE: Our goal was to evaluate the hypothesis that there is limited structural diversity among environmentally relevant chemicals capable of modulating TR activity via the collaborative interagency Tox21 project. METHODS: We screened the Tox21 chemical library (8,305 unique structures) in a quantitative high-throughput, cell-based reporter gene assay for TR agonist or antagonist activity. Active compounds were further characterized using additional orthogonal assays, including mammalian one-hybrid assays, coactivator recruitment assays, and a high-throughput, fluorescent imaging, nuclear receptor translocation assay. RESULTS: Known agonist reference chemicals were readily identified in the TR transactivation assay, but only a single novel, direct agonist was found, the pharmaceutical betamipron. Indirect activation of TR through activation of its heterodimer partner, the retinoid-X-receptor (RXR), was also readily detected by confirmation in an RXR agonist assay. Identifying antagonists with high confidence was a challenge with the presence of significant confounding cytotoxicity and other, non-TR-specific mechanisms common to the transactivation assays. Only three pharmaceuticals-mefenamic acid, diclazuril, and risarestat-were confirmed as antagonists. DISCUSSION: The results support limited structural diversity for direct ligand effects on TR and imply that other potential target sites in the thyroid hormone axis should be a greater priority for bioactivity screening for thyroid axis disruptors. https://doi.org/10.1289/EHP5314.

Natural products and their derivatives continue to be wellsprings of nascent therapeutic potential. However, many laboratories have limited resources for biological evaluation, leaving their previously isolated or synthesized compounds largely or completely untested. To address this issue, the Canvass library of natural products was assembled, in collaboration with academic and industry researchers, for quantitative high-throughput screening (qHTS) across a diverse set of cell-based and biochemical assays. Characterization of the library in terms of physicochemical properties, structural diversity, and similarity to compounds in publicly available libraries indicates that the Canvass library contains many structural elements in common with approved drugs. The assay data generated were analyzed using a variety of quality control metrics, and the resultant assay profiles were explored using statistical methods, such as clustering and compound promiscuity analyses. Individual compounds were then sorted by structural class and activity profiles. Differential behavior based on these classifications, as well as noteworthy activities, are outlined herein. One such highlight is the activity of (-)-2(S)-cathafoline, which was found to stabilize calcium levels in the endoplasmic reticulum. The workflow described here illustrates a pilot effort to broadly survey the biological potential of natural products by utilizing the power of automation and high-throughput screening.

The constitutive androstane receptor (CAR; NR1I3) is a nuclear receptor involved in all phases of drug metabolism and disposition. However, recently it's been implicated in energy metabolism, tumor progression, and cancer therapy as well. It is, therefore, important to identify compounds that induce hCAR activation to predict drug-drug interactions and potential therapeutic usage. In this study, we screen the Tox21 10,000 compound collection to characterize hCAR activators. A novel structural cluster of compounds was identified, which included nitazoxanide and tenonitrozole, while known structural clusters, such as flavones and prazoles, were also detected. Four compounds, neticonazole, diphenamid, phenothrin, and rimcazole, have been identified as novel hCAR activators, one of which, rimcazole, shows potential selectivity towards hCAR over its sister receptor, the pregnane X receptor (PXR). All four compounds translocated hCAR from the cytoplasm into the nucleus demonstrating the first step to CAR activation. Profiling these compounds as hCAR activators would enable an estimation of drug-drug interactions, as well as identify prospective therapeutically beneficial drugs.

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.

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.