Quantitative Bioactivity Signatures of Dietary Supplements and Natural Products.

Dietary supplements and natural products are often marketed as safe and effective alternatives to conventional drugs, but their safety and efficacy are not well regulated. To address the lack of scientific data in these areas, we assembled a collection of Dietary Supplements and Natural Products (DSNP), as well as Traditional Chinese Medicinal (TCM) plant extracts. These collections were then profiled in a series of in vitro high-throughput screening assays, including a liver cytochrome p450 enzyme panel, CAR/PXR signaling pathways, and P-glycoprotein (P-gp) transporter assay activities. This pipeline facilitated the interrogation of natural product-drug interaction (NaPDI) through prominent metabolizing pathways. In addition, we compared the activity profiles of the DSNP/TCM substances with those of an approved drug collection (the NCATS Pharmaceutical Collection or NPC). Many of the approved drugs have well-annotated mechanisms of action (MOAs), while the MOAs for most of the DSNP and TCM samples remain unknown. Based on the premise that compounds with similar activity profiles tend to share similar targets or MOA, we clustered the library activity profiles to identify overlap with the NPC to predict the MOAs of the DSNP/TCM substances. Our results suggest that many of these substances may have significant bioactivity and potential toxicity, and they provide a starting point for further research on their clinical relevance.

Mining of high throughput screening database reveals AP-1 and autophagy pathways as potential targets for COVID-19 therapeutics.

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.

Biological activity-based modeling identifies antiviral leads against SARS-CoV-2.

Computational approaches for drug discovery, such as quantitative structure-activity relationship, rely on structural similarities of small molecules to infer biological activity but are often limited to identifying new drug candidates in the chemical spaces close to known ligands. Here we report a biological activity-based modeling (BABM) approach, in which compound activity profiles established across multiple assays are used as signatures to predict compound activity in other assays or against a new target. This approach was validated by identifying candidate antivirals for Zika and Ebola viruses based on high-throughput screening data. BABM models were then applied to predict 311 compounds with potential activity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Of the predicted compounds, 32% had antiviral activity in a cell culture live virus assay, the most potent compounds showing a half-maximal inhibitory concentration in the nanomolar range. Most of the confirmed anti-SARS-CoV-2 compounds were found to be viral entry inhibitors and/or autophagy modulators. The confirmed compounds have the potential to be further developed into anti-SARS-CoV-2 therapies.

Application of In Vitro Metabolism Activation in High-Throughput Screening.

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.

Identification of Estrogen-Related Receptor α Agonists in the Tox21 Compound Library.

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 ß-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.

Identification of Angiogenesis Inhibitors Using a Co-culture Cell Model in a High-Content and High-Throughput Screening Platform.

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.

One-Step Seeding of Neural Stem Cells with Vitronectin-Supplemented Medium for High-Throughput Screening Assays.

Human neuronal cells differentiated from induced pluripotent cells have emerged as a new model system for the study of disease pathophysiology and evaluation of drug efficacy. Differentiated neuronal cells are more similar in genetics and biological content to human brain cells than other animal disease models. However, culture of neuronal cells in assay plates requires a labor-intensive procedure of plate precoating, hampering its applications in high-throughput screening (HTS). We developed a simplified method with one-step seeding of neural stem cells in assay plates by supplementing the medium with a recombinant human vitronectin (VTN), thus avoiding plate precoating. Robust results were obtained from cell viability, calcium response, and neurite outgrowth assays using this new method. Our data demonstrate that this approach greatly simplifies high-throughput assays using neuronal cells differentiated from human stem cells for translational research.

Identification of known drugs targeting the endoplasmic reticulum stress response.

The endoplasmic reticulum (ER), a multifunctional organelle, plays a central role in cellular signaling, development, and stress response. Dysregulation of ER homeostasis has been associated with human diseases, such as cancer, inflammation, and diabetes. A broad spectrum of stressful stimuli including hypoxia as well as a variety of pharmacological agents can lead to the ER stress response. In this study, we have developed a stable ER stress reporter cell line that stably expresses a ß-lactamase reporter gene under the control of the ER stress response element (ESRE) present in the glucose-regulated protein, 78 kDa (GRP78) gene promoter. This assay has been optimized and miniaturized into a 1536-well plate format. In order to identify clinically used drugs that induce ER stress response, we screened approximately 2800 drugs from the NIH Chemical Genomics Center Pharmaceutical Collection (NPC library) using a quantitative high-throughput screening (qHTS) platform. From this study, we have identified several known ER stress inducers, such as 17-AAG (via HSP90 inhibition), as well as several novel ER stress inducers such as AMI-193 and spiperone. The confirmed drugs were further studied for their effects on the phosphorylation of eukaryotic initiation factor 2α (eIF2α), the X-box-binding protein (XBP1) splicing, and GRP78 gene expression. These results suggest that the ER stress inducers identified from the NPC library using the qHTS approach could shed new lights on the potential therapeutic targets of these drugs.

Predictive models for cytochrome p450 isozymes based on quantitative high throughput screening data.

The human cytochrome P450 (CYP450) isozymes are the most important enzymes in the body to metabolize many endogenous and exogenous substances including environmental toxins and therapeutic drugs. Any unnecessary interactions between a small molecule and CYP450 isozymes may raise a potential to disarm the integrity of the protection. Accurately predicting the potential interactions between a small molecule and CYP450 isozymes is highly desirable for assessing the metabolic stability and toxicity of the molecule. The National Institutes of Health Chemical Genomics Center (NCGC) has screened a collection of over 17,000 compounds against the five major isozymes of CYP450 (1A2, 2C9, 2C19, 2D6, and 3A4) in a quantitative high throughput screening (qHTS) format. In this study, we developed support vector classification (SVC) models for these five isozymes using a set of customized generic atom types. The CYP450 data sets were randomly split into equal-sized training and test sets. The optimized SVC models exhibited high predictive power against the test sets for all five CYP450 isozymes with accuracies of 0.93, 0.89, 0.89, 0.85, and 0.87 for 1A2, 2C9, 2C19, 2D6, and 3A4, respectively, as measured by the area under the receiver operating characteristic (ROC) curves. The important atom types and features extracted from the five models are consistent with the structural preferences for different CYP450 substrates reported in the literature. We also identified novel features with significant discerning power to separate CYP450 actives from inactives. These models can be useful in prioritizing compounds in a drug discovery pipeline or recognizing the toxic potential of environmental chemicals.

Identification of quaternary ammonium compounds as potent inhibitors of hERG potassium channels.

The human ether-a-go-go-related gene (hERG) channel, a member of a family of voltage-gated potassium (K(+)) channels, plays a critical role in the repolarization of the cardiac action potential. The reduction of hERG channel activity as a result of adverse drug effects or genetic mutations may cause QT interval prolongation and potentially leads to acquired long QT syndrome. Thus, screening for hERG channel activity is important in drug development. Cardiotoxicity associated with the inhibition of hERG channels by environmental chemicals is also a public health concern. To assess the inhibitory effects of environmental chemicals on hERG channel function, we screened the National Toxicology Program (NTP) collection of 1408 compounds by measuring thallium influx into cells through hERG channels. Seventeen compounds with hERG channel inhibition were identified with IC(50) potencies ranging from 0.26 to 22µM. Twelve of these compounds were confirmed as hERG channel blockers in an automated whole cell patch clamp experiment. In addition, we investigated the structure-activity relationship of seven compounds belonging to the quaternary ammonium compound (QAC) series on hERG channel inhibition. Among four active QAC compounds, tetra-n-octylammonium bromide was the most potent with an IC(50) value of 260nM in the thallium influx assay and 80nM in the patch clamp assay. The potency of this class of hERG channel inhibitors appears to depend on the number and length of their aliphatic side-chains surrounding the charged nitrogen. Profiling environmental compound libraries for hERG channel inhibition provides information useful in prioritizing these compounds for cardiotoxicity assessment in vivo.

The pilot phase of the NIH Chemical Genomics Center.

The NIH Chemical Genomics Center (NCGC) was the inaugural center of the Molecular Libraries and Screening Center Network (MLSCN). Along with the nine other research centers of the MLSCN, the NCGC was established with a primary goal of bringing industrial technology and experience to empower the scientific community with small molecule compounds for use in their research. We intend this review to serve as 1) an introduction to the NCGC standard operating procedures, 2) an overview of several of the lessons learned during the pilot phase and 3) a review of several of the innovative discoveries reported during the pilot phase of the MLSCN.

Cardiac glycosides inhibit p53 synthesis by a mechanism relieved by Src or MAPK inhibition.

p53 is regulated at multiple levels. We report here that p53, in multiple lines of human cancer cells, is down-regulated by cardiac glycoside drugs digoxin and ouabain, potent inhibitors of Na(+)/K(+)-ATPase. These drugs reduced the basal levels of p53 protein at nanomolar concentrations in a dose-, time-, and cancer cell line-dependent manner, but independent of p53 status of wild-type or mutant. The drugs also reduced the levels of p53 induced by its activators as well as p53 transfected into human cancer cells, regardless of its status. Interestingly, the drugs had no effect on endogenous p53 in two immortalized human cell lines. Mechanistically, p53 reduction occurred not at the mRNA levels but at the protein levels, as a result of reduced protein synthesis rather than enhanced degradation. The cellular sensitivity to drug-induced p53 reduction was not associated with the levels of alphasubunits of Na(+)/K(+)-ATPase in different cell lines. Although lowering extracellular K(+) did not reduce p53 as did ouabain and digoxin, it did potentiate both digoxin- and ouabain-induced p53 reduction in sensitive lines. Finally, p53 reduction seems to be triggered by activation of Src/mitogen-activated protein kinase (MAPK) signaling pathways upon drug binding to the Na(+)/K(+)-ATPase and can be completely blocked by the inhibitors of Src or MAP/ERK kinase. This is the first report that cardiac glycoside drugs, by initiating the Src/MAPK signaling pathways, reduce the p53 levels via inhibition of p53 protein synthesis. The drugs may be useful in the treatment of human cancers with a gain-of-function p53 mutation.

A new homogeneous high-throughput screening assay for profiling compound activity on the human ether-a-go-go-related gene channel.

Long QT syndrome, either inherited or acquired from drug treatments, can result in ventricular arrhythmia (torsade de pointes) and sudden death. Human ether-a-go-go-related gene (hERG) channel inhibition by drugs is now recognized as a common reason for the acquired form of long QT syndrome. It has been reported that more than 100 known drugs inhibit the activity of the hERG channel. Since 1997, several drugs have been withdrawn from the market due to the long QT syndrome caused by hERG inhibition. Food and Drug Administration regulations now require safety data on hERG channels for investigative new drug (IND) applications. The assessment of compound activity on the hERG channel has now become an important part of the safety evaluation in the process of drug discovery. During the past decade, several in vitro assay methods have been developed and significant resources have been used to characterize hERG channel activities. However, evaluation of compound activities on hERG have not been performed for large compound collections due to technical difficulty, lack of throughput, and/or lack of biological relevance to function. Here we report a modified form of the FluxOR thallium flux assay, capable of measuring hERG activity in a homogeneous 1536-well plate format. To validate the assay, we screened a 7-point dilution series of the LOPAC 1280 library collection and reported rank order potencies of ten common hERG inhibitors. A correlation was also observed for the hERG channel activities of 10 known hERG inhibitors determined in this thallium flux assay and in the patch clamp experiment. Our findings indicate that this thallium flux assay can be used as an alternative method to profile large-volume compound libraries for compound activity on the hERG channel.

Identification of chemical compounds that induce HIF-1alpha activity.

Cellular metabolism depends on the availability of oxygen and the major regulator of oxygen homeostasis is hypoxia-inducible factor 1 (HIF-1), a highly conserved transcription factor that plays an essential role in cellular and systemic homeostatic responses to hypoxia. HIF-1 is a heterodimeric transcription factor composed of hypoxia-inducible HIF-1alpha and constitutively expressed HIF-1beta. Under hypoxic conditions, the two subunits dimerize, allowing translocation of the HIF-1 complex to the nucleus where it binds to hypoxia-response elements (HREs) and activates expression of target genes implicated in angiogenesis, cell growth, and survival. The HIF-1 pathway is essential to normal growth and development, and is involved in the pathophysiology of cancer, inflammation, and ischemia. Thus, there is considerable interest in identifying compounds that modulate the HIF-1 signaling pathway. To assess the ability of environmental chemicals to stimulate the HIF-1 signaling pathway, we screened a National Toxicology Program collection of 1408 compounds using a cell-based beta-lactamase HRE reporter gene assay in a quantitative high-throughput screening (qHTS) format. Twelve active compounds were identified. These compounds were tested in a confirmatory assay for induction of vascular endothelial growth factor, a known hypoxia target gene, and confirmed compounds were further tested for their ability to mimic the effect of a reduced-oxygen environment on hypoxia-regulated promoter activity. Based on this testing strategy, three compounds (o-phenanthroline, iodochlorohydroxyquinoline, cobalt sulfate heptahydrate) were confirmed as hypoxia mimetics, whereas two compounds (7-diethylamino-4-methylcoumarin and 7,12-dimethylbenz(a)anthracence) were found to interact with HIF-1 in a manner different from hypoxia. These results demonstrate the effectiveness of qHTS in combination with secondary assays for identification of HIF-1alpha inducers and for distinguishing among inducers based on their pattern of activated hypoxic target genes. Identification of environmental compounds having HIF-1alpha activation activity in cell-based assays may be useful for prioritizing chemicals for further testing as hypoxia-response inducers in vivo.

Characterization of diversity in toxicity mechanism using in vitro cytotoxicity assays in quantitative high throughput screening.

Assessing the potential health risks of environmental chemical compounds is an expensive undertaking that has motivated the development of new alternatives to traditional in vivo toxicological testing. One approach is to stage the evaluation, beginning with less expensive and higher throughput in vitro testing before progressing to more definitive trials. In vitro testing can be used to generate a hypothesis about a compound's mechanism of action, which can then be used to design an appropriate in vivo experiment. Here we begin to address the question of how to design such a battery of in vitro cell-based assays by combining data from two different types of assays, cell viability and caspase activation, with the aim of elucidating the mechanism of action. Because caspase activation is a transient event during apoptosis, it is not possible to design a single end-point assay protocol that would identify all instances of compound-induced caspase activation. Nevertheless, useful information about compound mechanism of action can be obtained from these assays in combination with cell viability data. Unsupervised clustering in combination with Dunn's cluster validity index is a robust method for identifying mechanisms of action without requiring any a priori knowledge about mechanisms of toxicity. The performance of this clustering method is evaluated by comparing the clustering results against literature annotations of compound mechanisms.

High-throughput screening assays for the identification of chemical probes.

High-throughput screening (HTS) assays enable the testing of large numbers of chemical substances for activity in diverse areas of biology. The biological responses measured in HTS assays span isolated biochemical systems containing purified receptors or enzymes to signal transduction pathways and complex networks functioning in cellular environments. This Review addresses factors that need to be considered when implementing assays for HTS and is aimed particularly at investigators new to this field. We discuss assay design strategies, the major detection technologies and examples of HTS assays for common target classes, cellular pathways and simple cellular phenotypes. We conclude with special considerations for configuring sensitive, robust, informative and economically feasible HTS assays.