| A family of racemic 1,4-benzodiazepine-2,5-diones (BZDs) with potent pro-apoptotic activity has been identified. Systematic design and synthesis of analogs identified the functional groups critical for the cytotoxic activity. These studies culminated with the identification of BZD-29, which is representative of this class of compounds. BZD-29 is a potent cytotoxic agent, with selectivity for transformed T cells and certain neoplastic cells, over B cells and normal fibroblasts. Based on this in vitro activity profile coupled with its favorable drug-like profile, BZD-29 was evaluated in a mouse model of inflammation (Schistosoma mansoni infection), which in part, is driven by T cell signaling. Although BZD-29 reduced inflammation in this model, treatment was not associated with T cell cytotoxicity.;To elucidate the basis for the observed therapeutic effects, experiments were conducted to identify the molecular target of BZD-29 and investigate the mechanism by which BZD-29 modulates biological responses. These studies revealed that BZD-29 binds to and specifically inhibits Rho-associated kinase 2 (ROCKII). Inhibition is observed with purified recombinant enzyme and in whole cells. BZD-29 selectively inhibits ROCKII in preference to related kinases as well as a closely related isoform, ROCKI. Both enantiomers of BZD-29 inhibit ROCKII and were active in vivo. However, only the S-enantiomer was cytotoxic, which suggests that this compound binds to an additional target resulting in cell death.;To determine how inhibition of ROCKII decreases inflammation, studies were conducted in a murine model of acute inflammation. These studies revealed BZD-29 attenuates inflammation through several orthogonal pathways, targeting the function of both neutrophils and macrophages. Given the safety profile associated with ROCKI/II inhibition, BZD-29 serves as a lead compound for the design of novel anti-inflammatory therapeutics and as a tool compound to explore the biology of ROCKII. |
