title: Discovery of Sanggenon G as a natural cell-permeable small-molecular weight inhibitor of X-linked inhibitor of apoptosis protein (XIAP).
abstract: 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.
authors.affiliation: Department of Pediatrics I, Medical University Innsbruck, Anichstraße 35, A-6020 Innsbruck, Austria ; Tyrolean Cancer Research Institute, Innrain 66, A-6020 Innsbruck, Austria.
authors.forename: Maximilian A
authors.initials: MA
authors.lastname: Seiter
authors.affiliation: Department of Pediatrics II, Medical University Innsbruck, Anichstraße 35, A-6020 Innsbruck, Austria ; Tyrolean Cancer Research Institute, Innrain 66, A-6020 Innsbruck, Austria.
authors.forename: Stefan
authors.initials: S
authors.lastname: Salcher
authors.affiliation: Department of Pediatrics II, Medical University Innsbruck, Anichstraße 35, A-6020 Innsbruck, Austria ; Tyrolean Cancer Research Institute, Innrain 66, A-6020 Innsbruck, Austria.
authors.forename: Martina
authors.initials: M
authors.lastname: Rupp
authors.affiliation: Department of Pediatrics II, Medical University Innsbruck, Anichstraße 35, A-6020 Innsbruck, Austria ; Tyrolean Cancer Research Institute, Innrain 66, A-6020 Innsbruck, Austria.
authors.forename: Judith
authors.initials: J
authors.lastname: Hagenbuchner
authors.affiliation: Department of Pediatrics II, Medical University Innsbruck, Anichstraße 35, A-6020 Innsbruck, Austria.
authors.forename: Ursula
authors.initials: U
authors.lastname: Kiechl-Kohlendorfer
authors.affiliation: Freie Universität Berlin, Institute of Pharmacy, Department Pharmaceutical & Medicinal Chemistry, Koenigin-Luise-Straße 2, 14195 Berlin, Germany.
authors.forename: Jérémie
authors.initials: J
authors.lastname: Mortier
authors.affiliation: Freie Universität Berlin, Institute of Pharmacy, Department Pharmaceutical & Medicinal Chemistry, Koenigin-Luise-Straße 2, 14195 Berlin, Germany.
authors.forename: Gerhard
authors.initials: G
authors.lastname: Wolber
authors.affiliation: Institutes of Pharmacy/Pharmacognosy and Center for Molecular Biosciences Innsbruck, University of Innsbruck, Innrain 80-82, A-6020 Innsbruck, Austria.
authors.forename: Judith M
authors.initials: JM
authors.lastname: Rollinger
authors.affiliation: Department of Pediatrics II, Medical University Innsbruck, Anichstraße 35, A-6020 Innsbruck, Austria ; Tyrolean Cancer Research Institute, Innrain 66, A-6020 Innsbruck, Austria.
authors.forename: Petra
authors.initials: P
authors.lastname: Obexer
authors.affiliation: Department of Pediatrics I, Medical University Innsbruck, Anichstraße 35, A-6020 Innsbruck, Austria ; Tyrolean Cancer Research Institute, Innrain 66, A-6020 Innsbruck, Austria.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Ausserlechner
keywords: (FP-) assay, fluorescence polarization assay; ARPF-FAM, ARPF-K(5-Fam)-NH2-peptide; BIR-3, baculovirus-IAP-repeat-3; CC, column chromatography; Cell permeable; Kd, dissociation constant; Ki, binding affinity; MAC, methanol crude extract of mulberry root bark; Natural; PCA, protein fragment complementation analysis; RLU, relative luminescence units; SG1, sanggenon G; Sanggenon G; Small-molecular weight; XIAP inhibitor; XIAP, X-linked inhibitor of apoptosis protein