Toxicity screening of a combinatorial library: Correlation of cytotoxicityand gene induction to compound structure

Combinatorial chemistry has increased the number of compounds available for efficacy and safety assessment by several orders of magnitude and has made high throughput assays essential. To test whether higher throughput toxici ty assays could be of utility in screening compounds in early development, a selected set of combinatorial chemistry compounds was screened for induct ion of 70-Kd heat shock protein (HSP70) and 45-Kd growth arrest and DNA dam age protein (GADD45) mRNA levels as well as cytotoxicity, in HepG2 cells, u sing a 96-well microtiter plate format. Both assays, the branched DNA (Quan tigene) assay for mRNA levels and MTT for cytotoxicity, were robust enough to be incorporated into a screening format using a single replicate and a s ingle concentration of compound. Significantly, a structure/toxicity correl ation was established with this set of compounds with cytotoxicity and gene induction patterns linked to compound structure. Therefore, this type of e arly screening may be useful in identifying toxic substituents, enabling th e design of libraries with less potential for toxicity. While structure/tox icity correlations were observed, no relationship was observed between GADD 45 gene induction and mutagenesis as measured by the Ames bacterial reverse mutation assay.