Integrating High-Dimensional Transcriptomics and Image Analysis Tools into Early Safety Screening: Proof of Concept for a New Early Drug Development Strategy.

During drug discovery and development, the early identification of adverse effects is expected to reduce costly late-stage failures of candidate drugs. As risk/safety assessment takes place rather late during the development process and due to the limited ability of animal models to predict the human situation, modern unbiased high-dimensional biology readouts are sought, such as molecular signatures predictive for in vivo response using high-throughput cell-based assays. In this theoretical proof of concept, we provide findings of an in-depth exploration of a single chemical core structure. Via transcriptional profiling, we identified a subset of close analogues that commonly downregulate multiple tubulin genes across cellular contexts, suggesting possible spindle poison effects. Confirmation via a qualified toxicity assay (in vitro micronucleus test) and the identification of a characteristic aggregate-formation phenotype via exploratory high-content imaging validated the initial findings. SAR analysis triggered the synthesis of a new set of compounds and allowed us to extend the series showing the genotoxic effect. We demonstrate the potential to flag toxicity issues by utilizing data from exploratory experiments that are typically generated for target evaluation purposes during early drug discovery. We share our thoughts on how this approach may be incorporated into drug development strategies.

Heteroaryl urea inhibitors of fatty acid amide hydrolase: structure-mutagenicity relationships for arylamine metabolites.

The structure-activity relationships for a series of heteroaryl urea inhibitors of fatty acid amide hydrolase (FAAH) are described. Members of this class of inhibitors have been shown to inactivate FAAH by covalent modification of an active site serine with subsequent release of an aromatic amine from the urea electrophile. Systematic Ames II testing guided the optimization of urea substituents by defining the structure-mutagenicity relationships for the released aromatic amine metabolites. Potent FAAH inhibitors were identified having heteroaryl amine leaving groups that were non-mutagenic in the Ames II assay.

Interlaboratory assessment of the GreenScreen HC GADD45a-GFP genotoxicity screening assay: an enabling study for independent validation as an alternative method.

Sixteen coded compounds were blind-tested at 4 laboratories using the recently described GADD45a-GFP genotoxicity assay. The compounds were chosen to include non-genotoxic compounds as well as weak and strong genotoxins. None of the compounds required metabolic activation in order to exhibit genotoxic effects. The participating laboratories included 2 global pharmaceutical companies, a global consumer goods company and the Gentronix laboratory in Manchester. Each compound was tested 4 times on different days following a protocol previously described. The tests were carried out after a 3-day training period from the parent lab (Manchester). Following the exclusion of data from tests with positive control failures and data series with 'spikes', 92% of assays gave the correct result: non-genotoxins giving negative results and genotoxins giving positive results. There were no randomly distributed problems suggesting that differences between the results from different sites reflected the use of different instruments, procedural differences and operator experience. In naïve operator laboratories the quality of data improved with operator practice. It was concluded that simple clarification of the protocol would provide the level of reliability required for widespread use of the assay in hazard assessment.

Interlaboratory evaluation of a flow cytometric, high content in vitro micronucleus assay.

An international, multi-lab trial was conducted to evaluate a flow cytometry-based method for scoring micronuclei in mouse lymphoma L5178Y cells [S.L. Avlasevich, S.M. Bryce, S.E. Cairns, S.D. Dertinger, In vitro micronucleus scoring by flow cytometry: differential staining of micronuclei versus apoptotic and necrotic chromatin enhances assay reliability, Environ. Mol. Mutagen. 47 (2006) 56-66]. A reference laboratory investigated the potential of six chemicals to induce micronuclei -- the genotoxicants mitomycin C (MMC), etoposide (ETOPO), and vinblastine (VB), and the non-genotoxicants sucrose (SUC), staurosporine (STS), and dexamethasone (DEX). The latter two non-genotoxicants were selected as extreme challenges to the assay because of their potent apoptogenic activity. Three collaborating laboratories were supplied with prototype In Vitro MicroFlow kits, and each was assigned one genotoxicant and one non-genotoxicant. Cells were treated continuously for 24h over a range of concentrations up to 5 mg/ml, or overtly cytotoxic concentrations. Micronuclei were scored via standard microscopy and flow cytometry. In addition to enumerating micronucleus frequencies, a cytotoxicity measurement that is simultaneously acquired with the flow cytometric micronucleus scoring procedure was evaluated (Flow-NBR). With this method, latex particles served as counting beads, and facilitated relative survival measurements that exclude the presence of dead/dying cells. For comparison purposes, additional cytotoxicity endpoints were measured, including several that are based on cell number, and others that reflect compromised membrane integrity, including dye permeability and/or phospholipid distribution. Key findings for this set of compounds include the following: (1) significant discrepancies in top concentration selection were found when cytotoxicity measurements were based on different methods, with the Flow-NBR approach tending to be the most sensitive, (2) both microscopy- and flow cytometry-based scoring methods detected concentration-dependent micronucleus formation for the three genotoxic agents studied, with good agreement between the reference laboratory and the collaborating laboratories, and (3) whereas flow cytometric analyses showed no significant increases for the non-genotoxicants when top concentration selection was based on Flow-NBR, significantly elevated micronucleus frequencies were observed for concentrations that were chosen based on less-sensitive cytotoxicity assays. Collectively, these results indicate that rapid assessment of genotoxicity can be accomplished with a relatively simple flow cytometric technique, and that the scoring system is transferable across laboratories. Furthermore, a concurrent assessment of cytotoxicity, Flow-NBR, may help reduce the occurrence of irrelevant positive results, as it may represent a more appropriate means for choosing top concentration levels. Finally, the data presented herein reinforce concerns about the manner in which cytotoxicity limits are described in guidance documents, since these recommendations tend to cite fixed cut-off values without reference to methodology.