title: Obatoclax is a direct and potent antagonist of membrane-restricted Mcl-1 and is synthetic lethal with treatment that induces Bim.
abstract: BACKGROUND
  Obatoclax is a clinical stage drug candidate that has been proposed to target and inhibit prosurvival members of the Bcl-2 family, and thereby contribute to cancer cell lethality. The insolubility of this compound, however, has precluded the use of many classical drug-target interaction assays for its study. Thus, a direct demonstration of the proposed mechanism of action, and preferences for individual Bcl-2 family members, remain to be established.
  
  
  METHODS
  Employing modified proteins and lipids, we recapitulated the constitutive association and topology of mitochondrial outer membrane Mcl-1 and Bak in synthetic large unilamellar liposomes, and measured bakdependent bilayer permeability. Additionally, cellular and tumor models, dependent on Mcl-1 for survival, were employed.
  
  
  RESULTS
  We show that regulation of bilayer permeabilization by the tBid - Mcl-1 - Bak axis closely resemblesthe tBid - Bcl-XL - Bax model. Obatoclax rapidly and completely partitioned into liposomal lipid but also rapidly exchanged between liposome particles. In this system, obatoclax was found to be a direct and potent antagonist of liposome-bound Mcl-1 but not of liposome-bound Bcl-XL, and did not directly influence Bak. A 2.5 molar excess of obatoclax relative to Mcl-1 overcame Mcl-1-mediated inhibition of tBid-Bak activation. Similar results were found for induction of Bak oligomers by Bim. Obatoclax exhibited potent lethality in a cellmodel dependent on Mcl-1 for viability but not in cells dependent on Bcl-XL. Molecular modeling predicts that the 3-methoxy moiety of obatoclax penetrates into the P2 pocket of the BH3 binding site of Mcl-1. A desmethoxy derivative of obatoclax failed to inhibit Mcl-1 in proteoliposomes and did not kill cells whose survival depends on Mcl-1. Systemic treatment of mice bearing Tsc2(+) (/) (-) Em-myc lymphomas (whose cells depend on Mcl-1 for survival) with obatoclax conferred a survival advantage compared to vehicle alone (median 31 days vs 22 days, respectively; p=0.003). In an Akt-lymphoma mouse model, the anti-tumor effects of obatoclax synergized with doxorubicin. Finally, treatment of the multiple myeloma KMS11 cell model (dependent on Mcl-1 for survival) with dexamethasone induced Bim and Bim-dependent lethality. As predicted for an Mcl-1 antagonist, obatoclax and dexamethasone were synergistic in this model.
  
  
  CONCLUSIONS
  Taken together, these findings indicate that obatoclax is a potent antagonist of membranerestricted Mcl-1. Obatoclax represents an attractive chemical series to generate second generation Mcl-1 inhibitors.
authors.affiliation: Department of Biochemistry, McGill University, Montreal, Québec, Canada. mai.nguyen@mcgill.ca.
authors.forename: Mai
authors.initials: M
authors.lastname: Nguyen
authors.affiliation: Department of Biochemistry, McGill University, Montreal, Québec, Canada. regina.cencic@mcgill.ca.
authors.forename: Regina
authors.initials: R
authors.lastname: Cencic
authors.affiliation: Department of Biochemistry, McGill University, Montreal, Québec, Canada. franziska.ertel@gmx.de.
authors.forename: Franziska
authors.initials: F
authors.lastname: Ertel
authors.affiliation: Goodman Cancer Research Center, McGill University, Montreal, Québec, Canada. cynthia.bernier@mcgill.ca.
authors.forename: Cynthia
authors.initials: C
authors.lastname: Bernier
authors.affiliation: Department of Biochemistry, McGill University, Montreal, Québec, Canada. jerry.pelletier@mcgill.ca.|Goodman Cancer Research Center, McGill University, Montreal, Québec, Canada. jerry.pelletier@mcgill.ca.
authors.forename: Jerry
authors.initials: J
authors.lastname: Pelletier
authors.affiliation: Goodman Cancer Research Center, McGill University, Montreal, Québec, Canada. anne.roulston@mcgill.ca.
authors.forename: Anne
authors.initials: A
authors.lastname: Roulston
authors.affiliation: Department of Biochemistry, McGill University, Montreal, Québec, Canada. john.silvius@mcgill.ca.
authors.forename: John R
authors.initials: JR
authors.lastname: Silvius
authors.affiliation: Department of Biochemistry, McGill University, Montreal, Québec, Canada. gordon.shore@mcgill.ca.|Goodman Cancer Research Center, McGill University, Montreal, Québec, Canada. gordon.shore@mcgill.ca.
authors.forename: Gordon C
authors.initials: GC
authors.lastname: Shore
mesh_terms: D000818:Animals; D051017:Apoptosis Regulatory Proteins / Q000378:metabolism*; D000072224:Bcl-2-Like Protein 11; D045744:Cell Line, Tumor; D004195:Disease Models, Animal; D004317:Doxorubicin / Q000008:administration & dosage; D004357:Drug Synergism; D006801:Humans; D007211:Indoles; D008223:Lymphoma / Q000188:drug therapy* / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism*; D051379:Mice; D064549:Myeloid Cell Leukemia Sequence 1 Protein / Q000037:antagonists & inhibitors* / Q000737:chemistry; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D011758:Pyrroles / Q000008:administration & dosage* / Q000494:pharmacology; D023041:Xenograft Model Antitumor Assays
chemical_list: D051017:Apoptosis Regulatory Proteins; C000606775:BCL2L11 protein, human; D000072224:Bcl-2-Like Protein 11; C000606776:Bcl2l11 protein, mouse; D007211:Indoles; C579095:MCL1 protein, human; D008565:Membrane Proteins; D064549:Myeloid Cell Leukemia Sequence 1 Protein; D011518:Proto-Oncogene Proteins; D011758:Pyrroles; D004317:Doxorubicin; C520962:obatoclax