The BlueScreen-384 Assay as an Indicator of Genotoxic Hazard Potential in Early-Stage Drug Discovery

Abstract

High-throughput cell-based techniques that permit early detection of compound-induced genotoxic damage have recently become available. Methods based on induction of the GADD45a promoter are attractive because multiple intracellular mechanisms that detect genetic damage intersect at this checkpoint gene. Consequently, assays such as GreenScreen HC, which uses p53-competant human TK6 lymphoblastoid cells and a GADD45a-GFP reporter, have been developed. GreenScreen HC allows weekly testing of dozens of compounds using 96-well microplates, with high interassay consistency. BlueScreen HC is a recent advancement, coupling GADD45a to Gaussia luciferase, with several advantages over GADD45a-GFP including the potential for miniaturization. Here we describe implementation of a 384-well BlueScreen assay. For drug discovery programs carrying out iterative analogue synthesis around a chemical lead series, these assays permit assessment of compound genotoxic potential in parallel to, rather than subsequent to, determination of activity at a therapeutic target. We demonstrate comparability of BlueScreen-384 to GreenScreen HC and illustrate the use of BlueScreen-384 to explore the structure-activity relationship around a genotoxic lead molecule to identify nongenotoxic analogues. BlueScreen-384 can reduce the need for costly and time-consuming analogue testing in more traditional genotoxicity tests, such as the Ames test.

Emerging metrology for high-throughput nanomaterial genotoxicology

“…When testing the the European Centre for the Validation of Alternative Methods (ECVAM) recommended lists of genotoxic and non-genotoxic chemicals for assessment of new genotoxicity tests, the GreenScreen HC assays showed good sensitivity and specificity (139). More recently, the BlueScreen HC, a luciferase-based version of the GADD45α reporter assay, was described (140), and this assay has also been developed into a 384-well version (141). GADD45α is a member of the growth arrest and DNA damage (GADD) proteins that are induced upon various cellular stresses, including nutrient deprivation, oxidative stress and DNA damage (142).…”

Novel Methods and Approaches for Safety Evaluation of Nanoparticle Formulations: A Focus Towards In Vitro Models and Adverse Outcome Pathways

“…GreenScreen HC assay is one of the most widely verified assays for NM genotoxicity research. Another BlueScreen HC, a luciferase-based version of GADD45α reporter assay in a 384-well plate, was developed ( Hughes et al, 2012 ; Simpson et al, 2013 ). GADD45α is a growth arresting, DNA damaging protein that gets activated upon different cell stresses ( Nelson et al, 2017 ).…”

A novel safety assessment strategy applied to non-selective extracts

“…State-of-theart conventional (in vitro) assays or alternative assays such as the BlueScreen HC assay (Hughes et al 2012) may therefore be considered instead. The BlueScreen HC assay is based on a human cell line and is sensitive for the three classic genotoxic endpoints; gene mutations, clastogenicity and aneugenicity, and is used as an indicator of genotoxic hazard in highthroughput screening for early-stage drug discovery (Simpson et al 2012).…”