In Vitro Toxicology Society Annual Meeting

Here, we report an in vitro split luciferase based assay that can be used to identify modulators of apoptosome formation, a key step in programmed cell death. In this method, an N-terminal fragment of luciferase is fused to one protein and a C-terminal fragment is fused to a second protein. Luciferase activity is reconstituted if the two proteins interact and bring the fragments into close proximity with each other. In the current study, we demonstrate for the first time that split luciferase Apaf-1 reagents show dATP and cytochrome c dependent luciferase activity that was blocked by a known inhibitor of apoptosome formation (NS3694). This assay was then used to screen a library of toxicants and identify six new inhibitors of Apaf-1. As apoptosome formation is required for spermatogenesis in insects and mammals, this assay could be used to complement existing in vitro tests for reproductive toxicants.