High-throughput screening for the identification of dual inhibitors of BRD4 and RIPK3 toward the development of small-molecule medical countermeasure agents against arsenicals.

Warfare arsenicals are potent blistering agents and cause severe inflammation following their skin exposure. Data from our group (unpublished) show that these chemicals act by activating bromodomain-4 and RIPK signaling. To develop a dual inhibitor of the bromodomain-containing protein 4 (BRD4) and the receptor-interacting serine/threonine-protein kinase 3 (RIPK3), we conducted a high-throughput screening (HTS) campaign for inhibitors of BRD4 and RIPK3 activity to identify anti-inflammatory agent candidates that could alleviate arsenicals-induced injury. Our primary assays were adapted to 384-well microplates and used to screen a collection of 4,074 compounds consisting of FDA-approved drugs and other bioactive compounds. The BRD4 primary screen had an average Z' value of 0.93 and a signal-to-background (S/B) ratio of 3,018, while the RIPK3 primary screen had an average Z' value of 0.86 and S/B = 12.6. A counter screen assay was used to ensure activity was due to target engagement and not assay interference. Hits that inhibited BRD4 binding by > 54.6% and kinase activity by > 22.4% in the primary screen and were not statistical outliers in the counter screen assays, were confirmed in concentration-response format. Hits were also tested in a cell-based IL-6 assay to determine corresponding inflammatory inhibitory activity. Eighteen compounds were active in both BRD4 and RIPK3 assays, of which three displayed IC₅₀ values < 10 μM with promising IL-6 inhibition. These compounds could serve as good candidates for further chemical optimization for the development of small-molecule medical counter measure agents against arsenicals.