Many human cancers over-express B cell lymphoma 2 (Bcl-2) or X-linked inhibitor of apoptosis (IAP) proteins to evade cell death. The pro-apoptotic ARTS (Sept4_i2) protein binds directly to both Bcl-2 and XIAP and promotes apoptosis by stimulating their degradation via the ubiquitin-proteasome system (UPS). Here we describe a small molecule, A4, that mimics the function of ARTS. Microscale thermophoresis assays showed that A4 binds XIAP, but not cellular inhibitor of apoptosis protein 1 (cIAP1). A4 binds to a distinct ARTS binding pocket in the XIAP-BIR3 (baculoviral IAP repeat 3) domain. Like ARTS, A4 stimulated poly-ubiquitylation and UPS-mediated degradation of XIAP and Bcl-2, but not cIAP1, resulting in caspase-9 and -3 activation and apoptosis. In addition, over-expression of XIAP rescued HeLa cells from A4-induced apoptosis, consistent with the idea that A4 kills by antagonizing XIAP. On the other hand, treatment with the SMAC-mimetic Birinapant induced secretion of tumour necrosis factor-α (TNFα) and killed ~50% of SKOV-3 cells, and addition of A4 to Birinapant-treated cells significantly reduced secretion of TNFα and blocked Birinapant-induced apoptosis. This suggests that A4 acts by specifically targeting XIAP. The effect of A4 was selective as peripheral blood mononuclear cells and normal human breast epithelial cells were unaffected. Furthermore, proteome analysis revealed that cancer cell lines with high levels of XIAP were particularly sensitive to the killing effect of A4. These results provide proof of concept that the ARTS binding site in XIAP is “druggable”. A4 represents a novel class of dual-targeting compounds stimulating apoptosis by UPS-mediated degradation of important anti-apoptotic oncogenes.
Bibliographical noteFunding Information:
We thank Hermann Steller for thoughtful discussions of the manuscript. We thank Dr. Fabrice E. Benchetrit for his help with the initial screen of the compounds. We also thank Dr. Maya Lalzar for thorough proteome analysis, and Dr. Moran Jerabek for helpful suggestions, performing and analysing the MST binding assays (at Crelux a WuXi AppTech company Ltd, Germany). We are grateful to Drs. Joe Opferman, St. Jude, Memphis, TN, USA, and Dr. Reuven Stein, Tel-Aviv University, Israel for sharing with us the DKO BAK/BAX MEFs. D. M. is a recipient of a presidential scholarship from the University of Haifa. Medicinal Chemistry analysis by Dr. David Sheppard and MST assays (CreLuX Ltd) were funded by ARTSaVIT Ltd. This work was funded by Israel Science Foundation (ISF) Grant 822/12 (to S.L.) and by a generous grant award from the Hymen Milgrom Trust (to S.L.).
© 2020, The Author(s).
ASJC Scopus subject areas
- Cellular and Molecular Neuroscience
- Cell Biology
- Cancer Research