Development and Validation of a Higher Throughput Screening Approach to Genotoxicity Testing Using the GADD45a-GFP GreenScreen HC Assay

Abstract

There is a pressing need to develop rapid yet accurate screening assays for the identification of genotoxic liability and for early hazard assessment in drug discovery. The GADD45a-GFP human cell-based genotoxicity assay (GreenScreen HC) has been reformatted to test 12 compounds per 96-well microplate in a higher throughput, automated screening mode and the protocol applied to the analysis of 1266 diverse, pharmacologically active compounds. Testing from a fixed starting concentration of 100 μM and over 3 serial dilutions, the hit rates for genotoxicity (7.3%) and cytotoxicity (33%) endpoints of the assay have been determined in a much wider chemical space than previously reported. The degree of interference from color, autofluorescence, and low solubility has also been assessed. The assay results have been compared to an in silico approach to genotoxicity assessment using Derek for Windows software. Where carcinogenicity data were available, GreenScreen HC demonstrated a higher specificity than in silico methods while identifying genotoxic species that were not highlighted for genotoxic liability in structure-activity relationship software. Higher throughput screening from a fixed, low concentration reduces sensitivity to less potent genotoxins, but the maintenance of the previously reported high specificity is essential in early hazard assessment where misclassification can lead to the needless rejection of potentially useful compounds in drug development. (Journal of Biomolecular Screening 2009:16-30)

High-throughput approaches for genotoxicity testing in drug development: recent advances

“…The technology has been validated using the Johnson and Johnson compound collection. 19 On the other hand, the gene-conversion assay is used to detect mitotic crossing over, mitotic gene conversions and reverse mutation by employing heteroallelic (ade2-40/ ade2-119 and trp5-12/trp5-27) and homoallelic (ilv1-92/ ilv1-92) gene loci. The presence of white colonies indicates original heteroallelic condition, whereas the presence of pink or red colonies (formed due to homologous genotypes) indicates the occurrence of mitotic crossover.…”

“…This gene is induced upon exposure to clastogens, aneugens, and mutagens, thereby leading to overexpression under genotoxic conditions. 19 The GreenScreen HC Assay uses the humanderived p53-competent TK6 cell line with a patented GFPreporter system that exploits the regulation of the GADD45A gene. The patented GFP fluorescence reporter comprises complex regulatory elements that recognize mutagens, aneugens, clastogens, and topoisomerase and polymerase inhibitors.…”

Genetic Toxicity:In VitroApproaches for Medicinal Chemists

“…This can be significantly improved using robotics and/or a reduced number of compound dilutions. In a recent study using this higher throughput assay format, a library of 1266 pharmacologically active compounds (LOPAC, Sigma-Aldrich Co. Ltd) was screened [68]. The potential throughput from this assay format could be as high as 36,000 compounds per year.…”

Genetic Toxicity: In Vitro Approaches for Hit and Lead Profiling

“…An operator can achieve a throughput of 80-100 compounds/week, though this can be significantly improved with a reformatted version of the assay in which the number of compounds tested per 96-well plate is increased. In a recent study using this higher-throughput assay format, a library of 1266 pharmacologically active compounds (LOPAC; Sigma-Aldrich Co. Ltd.) was screened [46]. The potential throughput from this assay format could be as high as 36 000 compounds/year.…”