ARTS and ARTS mimetics promote cancer cell killing by degrading anti-apoptotic proteins

Many human cancers over-express Inhibitor of Apoptosis (IAP) proteins or Bcl-2 (B-cell lymphoma 2). Thus, they represent favorable therapeutic targets. ARTS (Sept4_i2) binds directly to Bcl-2 and XIAP and promotes apoptosis by stimulating the degradation of both proteins via the Ubiquitin Proteasome System (UPS). ARTS is required for bringing Bcl-2 into close proximity with XIAP forming a complex which promotes auto-ubiquitylation and degradation of the E3-ligase XIAP and of Bcl-2. ARTS binds directly to XIAP-BIR3 domain in a pocket which is distinct from that of SMAC/Diablo. We have used a structure-based computational screen to identify ARTS mimetic small molecules. These molecules were identified based on their specific docking to the unique binding site of ARTS within XIAP-BIR3. MST (Microscale Thremophoresis) binding assays identified one of these compounds which binds to XIAP but not cIAP1. This compound initiates UPS-mediated degradation of XIAP and Bcl-2, resulting in caspase 3 and 9 activation and apoptosis. Significantly, this compound decreases XIAP and Bcl-2 levels in MEFs from Sept4/ARTS deficient mice. Thus, it can substitute the function of ARTS. Furthermore, it also behaved as a functional XIAP antagonist and was able to counteract inhibition of cIAP1 in SKOV-3 cells. Importantly, cancer cells exhibiting high levels of XIAP were significantly more sensitive to treatment with this ARTS mimetic and high XIAP levels correlated with low IC50, efficient killing with this compound, with no effect on non-malignant PBMC (peripheral blood mononuclear cells). This provides a proof-of-concept that the ARTS distinct binding site in XIAP is “druggable’. Furthermore, that this first identified ARTS mimetic, represents a novel class of dual targeting compounds stimulating apoptosis by UPS-induced degradation.