Technical evaluation and standardization of the human thyroid microtissue assay.

The success and sustainability of U.S. EPA efforts to reduce, refine, and replace in vivo animal testing depends on the ability to translate toxicokinetic and toxicodynamic data from in vitro and in silico new approach methods (NAMs) to human-relevant exposures and health outcomes. Organotypic culture models employing primary human cells enable consideration of human health effects and inter-individual variability but present significant challenges for test method standardization, transferability, and validation. Increasing confidence in the information provided by these in vitro NAMs requires setting appropriate performance standards and benchmarks, defined by the context of use, to consider human biology and mechanistic relevance without animal data. The human thyroid microtissue (hTMT) assay utilizes primary human thyrocytes to reproduce structural and functional features of the thyroid gland that enable testing for potential thyroid-disrupting chemicals. As a variable-donor assay platform, conventional principles for assay performance standardization need to be balanced with the ability to predict a range of human responses. The objectives of this study were to (1) define the technical parameters for optimal donor procurement, primary thyrocyte qualification, and performance in the hTMT assay, and (2) set benchmark ranges for reference chemical responses. Thyrocytes derived from a cohort of 32 demographically diverse euthyroid donors were characterized across a battery of endpoints to evaluate morphological and functional variability. Reference chemical responses were profiled to evaluate the range and chemical-specific variability of donor-dependent effects within the cohort. The data-informed minimum acceptance criteria for donor qualification and set benchmark parameters for method transfer proficiency testing and validation of assay performance.

Discovery and Preclinical Development of Novel Intraocular Pressure-Lowering Rho Kinase Inhibitor: Corticosteroid Conjugates.

Purpose: A new class of ocular steroids designed to mitigate steroid-induced intraocular pressure (IOP) elevation while maintaining anti-inflammatory activity was developed. Herein is described the discovery and preclinical characterization of ROCK'Ster compound 1. Methods: Codrugs consisting of a Rho kinase inhibitor (ROCKi) and a corticosteroid were synthesized. Compounds were initially screened in vitro for ROCKi activity and anti-inflammatory activity against the proinflammatory interleukin 23 and bacterial lipopolysaccharide (LPS) pathways. Selected compounds were then screened for solubility, chemical stability, and ex vivo corneal metabolism. Lead compound 1 was evaluated for IOP lowering in the Dutch Belted rabbit and for anti-inflammatory efficacy in both a postcataract surgery model and an allergic eye disease (AED) mouse model. Results: Several ROCK'Sters were found to be potent inhibitors of ROCK (Kis < 50 nM), have high anti-inflammatory activity in vitro (IC50s < 50 nM), display sufficient stability in topical ophthalmic formulations, and have a moderate rate of corneal metabolism. Compound 1 (0.1% and 0.25%, quater in die [QID]-4 times a day) demonstrated IOP-lowering capability without inducing hyperemia in our rabbit model. When compared with the marketed steroids, Durezol® and Pred Forte®, compound 1 (0.1%, 0.25%) demonstrated noninferiority in clinical scoring in a rabbit model of inflammation after surgery. In addition, anti-inflammatory outcomes were observed with compound 1 (0.1%) relative to Lotemax® or vehicle control in an AED mouse model. Conclusion: ROCK'Ster compound 1 is a novel compound suitable for topical ocular dosing that possesses IOP-lowering capability along with similar anti-inflammatory activity compared with marketed steroids.

A Qualitative Modeling Approach for Whole Genome Prediction Using High-Throughput Toxicogenomics Data and Pathway-Based Validation.

Efficient high-throughput transcriptomics (HTT) tools promise inexpensive, rapid assessment of possible biological consequences of human and environmental exposures to tens of thousands of chemicals in commerce. HTT systems have used relatively small sets of gene expression measurements coupled with mathematical prediction methods to estimate genome-wide gene expression and are often trained and validated using pharmaceutical compounds. It is unclear whether these training sets are suitable for general toxicity testing applications and the more diverse chemical space represented by commercial chemicals and environmental contaminants. In this work, we built predictive computational models that inferred whole genome transcriptional profiles from a smaller sample of surrogate genes. The model was trained and validated using a large scale toxicogenomics database with gene expression data from exposure to heterogeneous chemicals from a wide range of classes (the Open TG-GATEs data base). The method of predictor selection was designed to allow high fidelity gene prediction from any pre-existing gene expression data set, regardless of animal species or data measurement platform. Predictive qualitative models were developed with this TG-GATES data that contained gene expression data of human primary hepatocytes with over 941 samples covering 158 compounds. A sequential forward search-based greedy algorithm, combining different fitting approaches and machine learning techniques, was used to find an optimal set of surrogate genes that predicted differential expression changes of the remaining genome. We then used pathway enrichment of up-regulated and down-regulated genes to assess the ability of a limited gene set to determine relevant patterns of tissue response. In addition, we compared prediction performance using the surrogate genes found from our greedy algorithm (referred to as the SV2000) with the landmark genes provided by existing technologies such as L1000 (Genometry) and S1500 (Tox21), finding better predictive performance for the SV2000. The ability of these predictive algorithms to predict pathway level responses is a positive step toward incorporating mode of action (MOA) analysis into the high throughput prioritization and testing of the large number of chemicals in need of safety evaluation.

An in vitro approach for prioritization and evaluation of chemical effects on glucocorticoid receptor mediated adipogenesis.

Rising obesity rates worldwide have socio-economic ramifications. While genetics, diet, and lack of exercise are major contributors to obesity, environmental factors may enhance susceptibility through disruption of hormone homeostasis and metabolic processes. The obesogen hypothesis contends that chemical exposure early in development may enhance adipocyte differentiation, thereby increasing the number of adipocytes and predisposing for obesity and metabolic disease. We previously developed a primary human adipose stem cell (hASC) assay to evaluate the effect of environmental chemicals on PPARG-dependent adipogenesis. Here, the assay was modified to determine the effects of chemicals on the glucocorticoid receptor (GR) pathway. In differentiation cocktail lacking the glucocorticoid agonist dexamethasone (DEX), hASCs do not differentiate into adipocytes. In the presence of GR agonists, adipocyte maturation was observed using phenotypic makers for lipid accumulation, adipokine secretion, and expression of key genes. To evaluate the role of environmental compounds on adipocyte differentiation, progenitor cells were treated with 19 prioritized compounds previously identified by ToxPi as having GR-dependent bioactivity, and multiplexed assays were used to confirm a GR-dependent mode of action. Five chemicals were found to be strong agonists. The assay was also modified to evaluate GR-antagonists, and 8/10 of the hypothesized antagonists inhibited adipogenesis. The in vitro bioactivity data was put into context with extrapolated human steady state concentrations (Css) and clinical exposure data (Cmax). These data support using a human adipose-derived stem cell differentiation assay to test the potential of chemicals to alter human GR-dependent adipogenesis.

Editor's Highlight: Screening ToxCast Prioritized Chemicals for PPARG Function in a Human Adipose-Derived Stem Cell Model of Adipogenesis.

The developmental origins of obesity hypothesis posits a multifaceted contribution of factors to the fetal origins of obesity and metabolic disease. Adipocyte hyperplasia in gestation and early childhood may result in predisposition for obesity later in life. Rodent in vitro and in vivo studies indicate that some chemicals may directly affect adipose progenitor cell differentiation, but the human relevance of these findings is unclear. The nuclear receptor peroxisome proliferator-activated receptor gamma (PPARG) is the master regulator of adipogenesis. Human adipose-derived stem cells (hASC) isolated from adipose tissue express endogenous isoforms of PPARG and represent a biologically relevant cell-type for evaluating activity of PPARG ligands. Here, a multi-endpoint approach based on a phenotypic adipogenesis assay was applied to screen a set of 60 chemical compounds identified in ToxCast Phase I as PPARG active (49) or inactive (11). Chemicals showing activity in the adipogenesis screen were further evaluated in a series of 4 orthogonal assays representing 7 different key events in PPARG-dependent adipogenesis, including gene transcription, protein expression, and adipokine secretion. An siRNA screen was also used to evaluate PPARG-dependence of the adipogenesis phenotype. A universal concentration-response design enabled inter-assay comparability and implementation of a weight-of-evidence approach for bioactivity classification. Collectively, a total of 14/49 (29%) prioritized chemicals were identified with moderate-to-strong activity for human adipogenesis. These results provide the first integrated screening approach of prioritized ToxCast chemicals in a human stem cell model of adipogenesis and provide insight into the capacity of PPARG-activating chemicals to modulate early life programming of adipose tissue.

Discovery of novel aminoquinazolin-7-yl 6,7-dihydro-indol-4-ones as potent, selective inhibitors of heat shock protein 90.

A novel class of Hsp90 inhibitors, structurally distinct from previously reported scaffolds, was developed from rational design and optimization of a compound library screen hit. These aminoquinazoline derivatives, represented by compound 15 (SNX-6833) or 1-(2-amino-4-methylquinazolin-7-yl)-3,6,6-trimethyl-6,7-dihydro-1H-indol-4(5H)-one, selectively bind to Hsp90 and inhibit its cellular activities at concentrations as low as single digit nanomolar.

Application of chemoproteomics to drug discovery: identification of a clinical candidate targeting hsp90.

A chemoproteomics-based drug discovery strategy is presented that utilizes a highly parallel screening platform, encompassing more than 1000 targets, with a focused chemical library prior to target selection. This chemoproteomics-based process enables a data-driven selection of both the biological target and chemical hit after the screen is complete. The methodology has been exemplified for the purine binding proteome (proteins utilizing ATP, NAD, FAD). Screening of an 8000 member library yielded over 1500 unique protein-ligand interactions, which included novel hits for the oncology target Hsp90. The approach, which also provides broad target selectivity information, was used to drive the identification of a potent and orally active Hsp90 inhibitor, SNX-5422, which is currently in phase 1 clinical studies.

Discovery of novel 2-aminobenzamide inhibitors of heat shock protein 90 as potent, selective and orally active antitumor agents.

A novel class of heat shock protein 90 (Hsp90) inhibitors was developed from an unbiased screen to identify protein targets for a diverse compound library. These indol-4-one and indazol-4-one derived 2-aminobenzamides showed strong binding affinity to Hsp90, and optimized analogues exhibited nanomolar antiproliferative activity across multiple cancer cell lines. Heat shock protein 70 (Hsp70) induction and specific client protein degradation in cells on treatment with the inhibitors supported Hsp90 inhibition as the mechanism of action. Computational chemistry and X-ray crystallographic analysis of selected member compounds clearly defined the protein-inhibitor interaction and assisted the design of analogues. 4-[6,6-Dimethyl-4-oxo-3-(trifluoromethyl)-4,5,6,7-tetrahydro-1H-indazol-1-yl]-2-[(trans-4-hydroxycyclohexyl)amino]benzamide (SNX-2112, 9) was identified as highly selective and potent (IC(50) Her2 = 11 nM, HT-29 = 3 nM); its prodrug amino-acetic acid 4-[2-carbamoyl-5-(6,6-dimethyl-4-oxo-3-trifluoromethyl-4,5,6,7-tetrahydro-indazol-1-yl)-phenylamino]-cyclohexyl ester methanesulfonate (SNX-5422, 10) was orally bioavailable and efficacious in a broad range of xenograft tumor models (e.g. 67% growth delay in a HT-29 model) and is now in multiple phase I clinical trials.

Novel carbazole and acyl-indole antimitotics.

In the course of our Heat Shock 90 program, certain carbazole compounds were identified which had an off-target antiproliferative activity. To understand the off-target activity, we studied one analog with strong activity. We discovered that it had an effect on tubulin polymerization kinetics and was competitive with colchicine. Additional analogs were made, and a number of potent compounds were identified.

Targeting of multiple signaling pathways by the Hsp90 inhibitor SNX-2112 in EGFR resistance models as a single agent or in combination with erlotinib.

Inhibition of Hsp90 has emerged as a therapeutic strategy to target NSCLC subtypes, which are refractory to epidermal growth factor receptor (EGFR) inhibitor-based treatment. We report on a novel small molecule inhibitor of Hsp90, SNX-2112, and an orally bioavailable prodrug analog, SNX-5422. In cellular models of wild-type or mutant EGFR (L858R and T790M mutations), SNX-2112 alone and in combination with erlotinib inhibited EGF activation of pAKT(473) and pSTAT3(705). pERK1/2 and pS6 were also potently inhibited by similar treatments. SNX-2112 reduced EGF cross-talk and activation of the c-Met receptor by causing c-Met degradation. In NCI-H1975 xenograft models, SNX-5422 showed activity as a single agent and in combination with erlotinib resulted in prolonged animal survival at reduced compound concentrations relative to either compound alone. These results support the advanced evaluation of SNX-5422 as a treatment for non-small cell lung cancer (NSCLC), especially in cases where the cancer is driven by c-Met amplification or mutated EGFR forms that are resistant to EGFR inhibitors.

Discovery of benzamide tetrahydro-4H-carbazol-4-ones as novel small molecule inhibitors of Hsp90.

Hsp90 maintains the conformational stability of multiple proteins implicated in oncogenesis and has emerged as a target for chemotherapy. We report here the discovery of a novel small molecule scaffold that inhibits Hsp90. X-ray data show that the scaffold binds competitively at the ATP site on Hsp90. Cellular proliferation and client assays demonstrate that members of the series are able to inhibit Hsp90 at nanomolar concentrations.

The use of high-content screening for the discovery and characterization of compounds that modulate mitotic index and cell cycle progression by differing mechanisms of action.

The advent of high-content screening has expanded the ability of researchers to identify and quantify compound effects on a number of cellular events in a manner that allows for the rapid screening of chemical libraries. We have validated an approach for characterizing inhibitors of Aurora kinase family members using high-content screening by determining compound effects on the levels of the mitotic marker phospho-histone H3 (Ser10). Analysis of the data from these experiments led us to the discovery of a series of related compounds that increased the level of cells staining positive for the mitotic marker, indicating a block of cell cycle progression at M-phase. We then reconfigured the Aurora kinase inhibition assay and validated a high-content approach to identify compounds that block progression through M-phase. We were able to take advantage of the flexibility within the high-content screening platform to measure DNA content, activation of apoptosis, and effects on beta-tubulin staining patterns, all of which directly led to the identification of the cellular target of this new class of compounds.