title: AD-O53.2--a novel recombinant fusion protein combining the activities of TRAIL/Apo2L and Smac/Diablo, overcomes resistance of human cancer cells to TRAIL/Apo2L.
abstract: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its receptors became promising molecules for selective targeting of tumor cells without affecting normal tissue. Unfortunately, cancer cells have developed a number of mechanisms that confer resistance to TRAIL\Apo2L-induced apoptosis, which substantiates the need for development of alternative therapeutic strategies. Here we present a recombinant variant of TRAIL\Apo2L peptide, named AD-O53.2, fused to the peptide-derived from Smac/Diablo protein-the natural inhibitor of the apoptotic X-linked IAP (XIAP) protein considered as a pro-apoptotic agent. The proposed mechanism of action for this construct involves specific targeting of the tumor by TRAIL\Apo2L followed by activation and internalization of pro-apoptotic peptide into the cancer cells. While in the cytoplasm , the Smac\Diablo peptide inhibits activity of X-linked IAP (XIAP) proteins and promotes caspase-mediated apoptosis. AD-O53.2 construct was expressed in E.coli and purified by Ion Exchange Chromatography (IEC). Derived protein was initially characterized by circular dichroism spectroscopy (CD), HPLC-SEC chromatography, surface plasmon resonance, protease activation and cell proliferation assays. Our Smac/Diablo-TRAIL fusion variant was tested against a panel of cancer cells (including lung, colorectal, pancreatic, liver, kidney and uterine) and showed a potent cytotoxic effect with the IC50 values in femtomolar range for the most sensitive cell lines, while it remained ineffective against non-transformed HUVEC cells as well as isolated normal human and rat hepatocytes. Importantly, the construct was well tolerated by animals and significantly reduced the rate of the tumor growth in colon and lung adenocarcinoma animal models.
authors.affiliation: Drug Discovery Department, Adamed Group, Pieńków 149, 05-152, Czosnów, Poland.
authors.forename: Jerzy S
authors.initials: JS
authors.lastname: Pieczykolan
authors.forename: Konrad
authors.initials: K
authors.lastname: Kubiński
authors.forename: Maciej
authors.initials: M
authors.lastname: Masłyk
authors.forename: Sebastian D
authors.initials: SD
authors.lastname: Pawlak
authors.forename: Anna
authors.initials: A
authors.lastname: Pieczykolan
authors.forename: Piotr K
authors.initials: PK
authors.lastname: Rózga
authors.forename: Michał
authors.initials: M
authors.lastname: Szymanik
authors.forename: Marlena
authors.initials: M
authors.lastname: Gałązka
authors.forename: Małgorzata
authors.initials: M
authors.lastname: Teska-Kamińska
authors.forename: Bartłomiej
authors.initials: B
authors.lastname: Żerek
authors.forename: Katarzyna
authors.initials: K
authors.lastname: Bukato
authors.forename: Katarzyna
authors.initials: K
authors.lastname: Poleszak
authors.forename: Albert
authors.initials: A
authors.lastname: Jaworski
authors.forename: Wojciech
authors.initials: W
authors.lastname: Strożek
authors.forename: Robert
authors.initials: R
authors.lastname: Świder
authors.forename: Rafał
authors.initials: R
authors.lastname: Zieliński
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology / Q000627:therapeutic use; D051017:Apoptosis Regulatory Proteins; D002352:Carrier Proteins / Q000494:pharmacology / Q000627:therapeutic use; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D015179:Colorectal Neoplasms / Q000188:drug therapy; D019008:Drug Resistance, Neoplasm / Q000187:drug effects*; D005260:Female; D022781:Hepatocytes / Q000187:drug effects; D061307:Human Umbilical Vein Endothelial Cells / Q000187:drug effects; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics; D008175:Lung Neoplasms / Q000188:drug therapy; D016513:Mice, SCID; D024101:Mitochondrial Proteins / Q000235:genetics; D009842:Oligopeptides / Q000235:genetics; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000494:pharmacology / Q000627:therapeutic use; D053221:TNF-Related Apoptosis-Inducing Ligand / Q000235:genetics / Q000494:pharmacology / Q000627:therapeutic use; D023041:Xenograft Model Antitumor Assays
chemical_list: C000599295:AD-O53.2 fusion protein; D000970:Antineoplastic Agents; D051017:Apoptosis Regulatory Proteins; D002352:Carrier Proteins; C412403:DIABLO protein, human; D047908:Intracellular Signaling Peptides and Proteins; D024101:Mitochondrial Proteins; D009842:Oligopeptides; D011993:Recombinant Fusion Proteins; C532637:SMAC peptide; D053221:TNF-Related Apoptosis-Inducing Ligand; C505653:TNFSF10 protein, human