Discovery of Sanggenon G as a natural cell-permeable small-molecular weight inhibitor of X-linked inhibitor of apoptosis protein (XIAP)

• Discovery of a novel XIAP-inhibitory natural compound from Morus root bark (Sanggenon G).
• Sanggenon G binds specific to the BIR3 domain of XIAP in a low μM range.
• Sanggenon G interferes with XIAP-BIR3-substrate binding in living cells.
• Sanggenon G acts as chemosensitizer in tumor cell lines with high XIAP expression.

Defects in the regulation of apoptosis are one main cause of cancer development and may result from overexpression of anti-apoptotic proteins such as the X-linked inhibitor of apoptosis protein (XIAP). XIAP is frequently overexpressed in human leukemia and prostate and breast tumors. Inhibition of apoptosis by XIAP is mainly coordinated through direct binding to the initiator caspase-9 via its baculovirus-IAP-repeat-3 (BIR3) domain. XIAP inhibits caspases directly making it to an attractive target for anti-cancer therapy. In the search for novel, non-peptidic XIAP inhibitors in this study we focused on the chemical constituents of sāng bái pí (mulberry root bark). Most promising candidates of this plant were tested biochemically in vitro by a fluorescence polarization (FP) assay and in vivo via protein fragment complementation analysis (PCA). We identified the Diels Alder adduct Sanggenon G (SG1) as a novel, small-molecular weight inhibitor of XIAP. As shown by FP and PCA analyses, SG1 binds specifically to the BIR3 domain of XIAP with a binding affinity of 34.26 μM. Treatment of the transgenic leukemia cell line Molt3/XIAP with SG1 enhances caspase-8, -3 and -9 cleavage, displaces caspase-9 from XIAP as determined by immunoprecipitation experiments and sensitizes these cells to etoposide-induced apoptosis. SG1 not only sensitizes the XIAP-overexpressing leukemia cell line Molt3/XIAP to etoposide treatment but also different neuroblastoma cell lines endogenously expressing high XIAP levels. Taken together, Sanggenon G (SG1) is a novel, natural, non-peptidic, small-molecular inhibitor of XIAP that can serve as a starting point to develop a new class of improved XIAP inhibitors.