A novel high-throughput cellular screening assay for the discovery of HIV-1 integrase inhibitors.

The discovery of HIV-1 integrase inhibitors has been enabled by high-throughput screening and rational design of novel chemotypes. Traditionally, biochemical assays focusing on the strand transfer activity of integrase have been used to screen compound libraries for identification of novel inhibitors. In contrast, cellular screening assays enable a phenotypic or multi-target approach, and may result in identification of compounds inhibiting integrase in its natural context, the pre-integration complex. Furthermore, a cellular assay encompassing 3' processing, strand transfer and nuclear import may lead to the identification of compounds with novel mechanisms of action targeting cellular and viral factors. Therefore, a cellular screening assay was developed, which focused on integrase activity, where infection of MT4 cells with an HIV-1 based lentiviral vector was synchronized by temporary arrest at the reverse transcriptase step and subsequent release to enable integration. The assay was validated using a panel of antivirals and proved to be a robust cellular screening assay for the identification of novel integrase inhibitors.