BACKGROUND: Mitochondrial dysfunction has been implicated in the pathogenesis of a variety of disorders including cancer, diabetes, and neurodegenerative and cardiovascular diseases. Understanding whether different environmental chemicals and druglike molecules impact mitochondrial function represents an initial step in predicting exposure-related toxicity and defining a possible role for such compounds in the onset of various diseases. OBJECTIVES: We sought to identify individual chemicals and general structural features associated with changes in mitochondrial membrane potential (MMP). METHODS: We used a multiplexed [two end points in one screen; MMP and adenosine triphosphate (ATP) content] quantitative high throughput screening (qHTS) approach combined with informatics tools to screen the Tox21 library of 10,000 compounds (~ 8,300 unique chemicals) at 15 concentrations each in triplicate to identify chemicals and structural features that are associated with changes in MMP in HepG2 cells. RESULTS: Approximately 11% of the compounds (913 unique compounds) decreased MMP after 1 hr of treatment without affecting cell viability (ATP content). In addition, 309 compounds decreased MMP over a concentration range that also produced measurable cytotoxicity [half maximal inhibitory concentration (IC50) in MMP assay/IC50 in viability assay ≤ 3; p < 0.05]. More than 11% of the structural clusters that constitute the Tox21 library (76 of 651 clusters) were significantly enriched for compounds that decreased the MMP. CONCLUSIONS: Our multiplexed qHTS approach allowed us to generate a robust and reliable data set to evaluate the ability of thousands of drugs and environmental compounds to decrease MMP. The use of structure-based clustering analysis allowed us to identify molecular features that are likely responsible for the observed activity.

The farnesoid X receptor (FXR) regulates the homeostasis of bile acids, lipids, and glucose. Because endogenous chemicals bind and activate FXR, it is important to examine which xenobiotic compounds would disrupt normal receptor function. We used a cell-based human FXR β-lactamase (Bla) reporter gene assay to profile the Tox21 10K compound collection of environmental chemicals and drugs. Structure-activity relationships of FXR-active compounds revealed by this screening were then compared against the androgen receptor, estrogen receptor α, peroxisome proliferator-activated receptors δ and γ, and the vitamin D receptor. We identified several FXR-active structural classes including anthracyclines, benzimidazoles, dihydropyridines, pyrethroids, retinoic acids, and vinca alkaloids. Microtubule inhibitors potently decreased FXR reporter gene activity. Pyrethroids specifically antagonized FXR transactivation. Anthracyclines affected reporter activity in all tested assays, suggesting non-specific activity. These results provide important information to prioritize chemicals for further investigation, and suggest possible modes of action of compounds in FXR signaling.

The U.S. Tox21 program has screened a library of approximately 10,000 (10K) environmental chemicals and drugs in three independent runs for estrogen receptor alpha (ERα) agonist and antagonist activity using two types of ER reporter gene cell lines, one with an endogenous full length ERα (ER-luc; BG1 cell line) and the other with a transfected partial receptor consisting of the ligand binding domain (ER-bla; ERα β-lactamase cell line), in a quantitative high-throughput screening (qHTS) format. The ability of the two assays to correctly identify ERα agonists and antagonists was evaluated using a set of 39 reference compounds with known ERα activity. Although both assays demonstrated adequate (i.e. >80%) predictivity, the ER-luc assay was more sensitive and the ER-bla assay more specific. The qHTS assay results were compared with results from previously published ERα binding assay data and showed >80% consistency. Actives identified from both the ER-bla and ER-luc assays were analyzed for structure-activity relationships (SARs) revealing known and potentially novel ERα active structure classes. The results demonstrate the feasibility of qHTS to identify environmental chemicals with the potential to interact with the ERα signaling pathway and the two different assay formats improve the confidence in correctly identifying these chemicals.

INTRODUCTION: Breast cancer is a devastating disease that results in approximately 40,000 deaths each year in the USA. Current drug screening and chemopreventatitive methods are suboptimal, due in part to the poor specificity of compounds for cancer cells. Poly (ADP-ribose) polymerase 1 (PARP1) inhibitor (PARPi)-mediated therapy is a promising approach for familial breast cancers caused by mutations of breast cancer-associated gene-1 and -2 (BRCA1/2), yet drug resistance frequently occurs during the treatment. Moreover, PARPis exhibit very little effect on cancers that are proficient for DNA repair and clinical efficacy for PARPis as single-agent therapies has yet to be illustrated. METHODS: Using a quantitative high-throughput screening approach, we screened a library containing 2,816 drugs, most of which are approved for human or animal use by the Food and Drug Administration (FDA) or other countries, to identify compounds that sensitize breast cancer cells to PARPi. After initial screening, we performed further cellular and molecular analysis on lestaurtinib, which is an orally bioavailable multikinase inhibitor and has been used in clinical trials for myeloproliferative disorders and acute myelogenous leukemia. RESULTS: Our study indicated that lestaurtinib is highly potent against breast cancers as a mono-treatment agent. It also strongly enhanced the activity of the potent PARPi AG14361 on breast cancer cell growth both in vitro and in vivo conditions. The inhibition of cancer growth is measured by increased apoptosis and reduced cell proliferation. Consistent with this, the treatment results in activation of caspase 3/7, and accumulation of cells in the G2 phase of the cell cycle, irrespective of their BRCA1 status. Finally, we demonstrated that AG14361 inhibits NF-κB signaling, which is further enhanced by lestaurtinib treatment. CONCLUSIONS: Lestaurtinib amplifies the ability of the PARP1 inhibitor AG14361 to kill BRCA1 mutant and wild-type breast cancer cells, at least in part, by inhibiting NF-κB signaling. Each of these drugs has been approved for clinical trials for several different cancers, thus, their combination treatment should be applicable for a breast cancer trial in the future.

Domiphen bromide and didecyl dimethylammonium bromide were widely used environmental chemicals with potent activity on blockade of human ether-a-go-go related gene (HERG) channels. But the mechanism of their action is not clear. The kinetics of block of HERG channels by domiphen bromide and didecyl dimethylammonium bromide was studied in order to characterize the inhibition of HERG currents by these quaternary ammonium compounds (QACs). Domiphen bromide and didecyl dimethylammonium bromide inhibited HERG channel currents in a dose-dependent manner with IC50 values of 9nM and 5nM, respectively. Block of HERG channel by domiphen bromide and didecyl dimethylammonium bromide was voltage-dependent and use-dependent. Domiphen bromide and didecyl dimethylammonium bromide caused substantial negative shift of the activation curves, accelerated activated process, but had no effects on the deactivation and reactivation processes. The docking models implied that these two compounds bound to PAS domain of HERG channels and inhibited its function. Our data demonstrated that domiphen bromide and didecyl dimethylammonium bromide blocked the HERG channel with a preference for the activated channel state.

Anti-cancer topoisomerase I (Top1) inhibitors (camptothecin and its derivatives irinotecan and topotecan, and indenoisoquinolines) induce lethal DNA lesions by stabilizing Top1-DNA cleavage complex (Top1cc). These lesions are repaired by parallel repair pathways including the tyrosyl-DNA phosphodiesterase 1 (TDP1)-related pathway and homologous recombination. As TDP1-deficient cells in vertebrates are hypersensitive to Top1 inhibitors, small molecules inhibiting TDP1 should augment the cytotoxicity of Top1 inhibitors. We developed a cell-based high-throughput screening assay for the discovery of inhibitors for human TDP1 using a TDP1-deficient chicken DT40 cell line (TDP1-/-) complemented with human TDP1 (hTDP1). Any compounds showing a synergistic effect with the Top1 inhibitor camptothecin (CPT) in hTDP1 cells should either be a TDP1-related pathway inhibitor or an inhibitor of alternate repair pathways for Top1cc. We screened the 400,000-compound Small Molecule Library Repository (SMLR, NIH Molecular Libraries) against hTDP1 cells in the absence or presence of CPT. After confirmation in a secondary screen using both hTDP1 and TDP1-/- cells in the absence or presence of CPT, five compounds were confirmed as potential TDP1 pathway inhibitors. All five compounds showed synergistic effect with CPT in hTDP1 cells, but not in TDP1-/- cells, indicating that the compounds inhibited a TDP1-related repair pathway. Yet, in vitro gel-based assay revealed that the five compounds did not inhibit TDP1 catalytic activity directly. We tested the compounds for their ability to inhibit poly(ADP-ribose)polymerase (PARP) because PARP inhibitors are known to potentiate the cytotoxicity of CPT by inhibiting the recruitment of TDP1 to Top1cc. Accordingly, we found that the five compounds inhibit catalytic activity of PARP by ELISA and Western blotting. We identified the most potent compound (Cpd1) that offers characteristic close to veliparib, a leading clinical PARP inhibitor. Cpd1 may represent a new scaffold for the development of PARP inhibitors.

To adapt the use of GH3.TRE-Luc reporter gene cell line for a quantitative high-throughput screening (qHTS) platform, we miniaturized the reporter gene assay to a 1536-well plate format. 1280 chemicals from the Library of Pharmacologically Active Compounds (LOPAC) and the National Toxicology Program (NTP) 1408 compound collection were analyzed to identify potential thyroid hormone receptor (TR) agonists and antagonists. Of the 2688 compounds tested, eight scored as potential TR agonists when the positive hit cut-off was defined at ≥10% efficacy, relative to maximal triiodothyronine (T3) induction, and with only one of those compounds reaching ≥20% efficacy. One common class of compounds positive in the agonist assays were retinoids such as all-trans retinoic acid, which are likely acting via the retinoid-X receptor, the heterodimer partner with the TR. Five potential TR antagonists were identified, including the antiallergy drug tranilast and the anxiolytic drug SB 205384 but also some cytotoxic compounds like 5-fluorouracil. None of the inactive compounds were structurally related to T3, nor had been reported elsewhere to be thyroid hormone disruptors, so false negatives were not detected. None of the low potency (>100µM) TR agonists resembled T3 or T4, thus these may not bind directly in the ligand-binding pocket of the receptor. For TR agonists, in the qHTS, a hit cut-off of ≥20% efficacy at 100 µM may avoid identification of positives with low or no physiological relevance. The miniaturized GH3.TRE-Luc assay offers a promising addition to the in vitro test battery for endocrine disruption, and given the low percentage of compounds testing positive, its high-throughput nature is an important advantage for future toxicological screening.

Endocrine disrupting chemicals (EDCs) interfere with the biosynthesis, metabolism, and functions of steroid hormones, including estrogens and androgens. Aromatase enzyme converts androgen to estrogen. Thus, EDCs against aromatase significantly impact estrogen- and/or androgen-dependent functions, including the development of breast cancer. The current study aimed to develop a biologically relevant cell-based high-throughput screening assay to identify EDCs that act as aromatase inhibitors (AIs), estrogen receptor (ER) agonists, and/or ER antagonists. The AroER tri-screen assay was developed by stable transfection of ER-positive, aromatase-expressing MCF-7 breast cancer cells with an estrogen responsive element (ERE) driven luciferase reporter plasmid. The AroER tri-screen can identify: estrogenic EDCs, which increase luciferase signal without 17β-estradiol (E2); anti-estrogenic EDCs, which inhibit the E2-induced luciferase signal; and AI-like EDCs, which suppress a testosterone-induced luciferase signal. The assay was first optimized in a 96-well plate format and then miniaturized into a 1536-well plate format. The AroER tri-screen was demonstrated to be suitable for high-throughput screening in the 1536-well plate format, with a 6.9-fold signal-to-background ratio, a 5.4% coefficient of variation, and a screening window coefficient (Z-factor) of 0.78. The assay suggested that bisphenol A (BPA) functions mainly as an ER agonist. Results from screening the 446 drugs in the National Institutes of Health Clinical Collection revealed 106 compounds that modulated ER and/or aromatase activities. Among these, two AIs (bifonazole and oxiconazole) and one ER agonist (paroxetine) were confirmed through alternative aromatase and ER activity assays. These findings indicate that AroER tri-screen is a useful high-throughput screening system for identifying ER ligands and aromatase-inhibiting chemicals.

Chordoma is a rare primary bone malignancy that arises in the skull base, spine and sacrum and originates from remnants of the notochord. These tumors are typically resistant to conventional chemotherapy, and to date there are no FDA-approved agents to treat chordoma. The lack of in vivo models of chordoma has impeded the development of new therapies for this tumor. Primary tumor from a sacral chordoma was xenografted into NOD/SCID/IL-2R γ-null mice. The xenograft is serially transplantable and was characterized by both gene expression analysis and whole genome SNP genotyping. The NIH Chemical Genomics Center performed high-throughput screening of 2,816 compounds using two established chordoma cell lines, U-CH1 and U-CH2B. The screen yielded several compounds that showed activity and two, sunitinib and bortezomib, were tested in the xenograft. Both agents slowed the growth of the xenograft tumor. Sensitivity to an inhibitor of IκB, as well as inhibition of an NF-κB gene expression signature demonstrated the importance of NF-κB signaling for chordoma growth. This serially transplantable chordoma xenograft is thus a practical model to study chordomas and perform in vivo preclinical drug testing.

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.