Discovery of incednine as a potent modulator of the anti-apoptotic function of Bcl-xL from microbial origin

Anti-apoptotic oncoproteins Bcl-2 and BcI-xL are overexpressed in many cancers and play a crucial role in cancer initiation, progression, and resistance to chemotherapy. Therefore, the discovery of a functional inhibitor for these proteins and improved understanding of the molecular mechanisms of these proteins will be an aid to novel anti-tumor therapies. Here, using chemical-genetic cell-based screening, we have discovered a chemically and biologically unique substance, incednine, as a novel functional modulator of Bcl-2/Bcl-xL from the fermentation broth of Streptomyces sp. ML-693-90F3. This compound was isolated as a HCI salt by solvent extraction and using centrifugal liquid-liquid partition chromatography. Its structure was elucidated by spectroscopic analysis, X-ray crystallographic analysis, and computational studies. Incednine has a molecular formula, C42H63N3O8, and consists of a novel skeletal structure, enol-ether amide in a 24-membered macrolactam core, with two aminosugars. Bcl-xL-overexpressing Ms-1 cells displayed resistance to several anti-tumor agents; however, anti-tumor agent-induced cell death was observed only when cells were treated with incednine. Overexpression of BcI-xL inhibited cell death in Bax-overexpressing HEK293T cells by forming a complex with Bax, whereas Bcl-xL failed to inhibit cell death in the presence of incednine without affecting the heterodimerization of Bcl-xL and Bax. These findings suggest that incednine serves as a potent modulator of the anti-apoptotic Bcl-2/Bcl-xL distinct from the other known Bcl-2 inhibitors and could provide a chemical probe to study the underlying mechanisms of Bcl-2/Bcl-xL.