Reporter gene-based assays are used extensively in high-throughput screening (HTS) to identify chemical modulators of cellular pathways for drug discovery and development. However, such screening frequently results in a large number of false “hits” due to interactions of screened compounds with reporter proteins, producing confounding results. Thus, validation of results using these assays often involves significant time and expense.

The inventors have developed an assay system that significantly improves detection of target-relevant active compounds by discriminating between signals arising from the target activity and those caused by reporter bias. This system utilizes simultaneous detection (also known as “coincidence detection”) of non-homologous reporter proteins with dissimilar properties, such as differing catalysis, light emission, or fluorescence characteristics; simultaneous observation of signals from these reporters indicates a high probability that it is a true target response. The reporters are cotranslationally expressed from a single RNA transcript, which ensures stable stoichiometry of the expressed proteins.