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Obatoclax is a direct and potent antagonist of membrane-restricted Mcl-1 and is synthetic lethal with treatment that induces Bim.

Nguyen M, Cencic R, Ertel F, Bernier C, Pelletier J, Roulston A, Silvius JR, Shore GC - BMC Cancer (2015)

Bottom Line: 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.Similar results were found for induction of Bak oligomers by Bim.A desmethoxy derivative of obatoclax failed to inhibit Mcl-1 in proteoliposomes and did not kill cells whose survival depends on Mcl-1.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, McGill University, Montreal, Québec, Canada. mai.nguyen@mcgill.ca.

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.

No MeSH data available.


Related in: MedlinePlus

a Bak-dependent release of calcein from proteoliposomes is not activated by obatoclax or Noxa BH3 peptide. Assays ± tBid were conducted as described in Fig. 1d, in the presence of 1 μM obatoclax, Noxa BH3, or vehicle (0.05 % DMSO) alone. b As in Fig. 1d except proteoliposomes contained Bak and Mcl-1 derivatives and the assays conducted in the presence of 1 μM obatoclax or 1 μM des-methoxyl (DM) obatoclax. c As in Fig. 1d except that liposome-anchored Bcl-XL replaced Mcl-1 and ABT-737 was tested. Concentrations of assay constituents are provided in right panel. Shown are representative assays from at least 3 independent experiments
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Fig2: a Bak-dependent release of calcein from proteoliposomes is not activated by obatoclax or Noxa BH3 peptide. Assays ± tBid were conducted as described in Fig. 1d, in the presence of 1 μM obatoclax, Noxa BH3, or vehicle (0.05 % DMSO) alone. b As in Fig. 1d except proteoliposomes contained Bak and Mcl-1 derivatives and the assays conducted in the presence of 1 μM obatoclax or 1 μM des-methoxyl (DM) obatoclax. c As in Fig. 1d except that liposome-anchored Bcl-XL replaced Mcl-1 and ABT-737 was tested. Concentrations of assay constituents are provided in right panel. Shown are representative assays from at least 3 independent experiments

Mentions: Assay mixtures (100 μl) containing proteoliposomes (20 μg lipid) with encapsulated fluorescence reporter calcein (quenched) and surface anchored Bak (0.14 μM) were challenged with 0.04 μM caspase-8-cleaved recombinant human Bid (aa 1–195 cleaved to 7 kDa and 15 kDa tBid fragments). Calcein acquires spontaneous fluorescence emission upon transbilayer release from the liposome, quantified as the % total calcein emission that is observed by treating the proteoliposomes with detergent. Conditions were selected to provide a robust Bak-dependent release of calcein in response to tBid (Figs. 1d and 2a), while at the same time minimizing the spontaneous and concentration-dependent release of calcein by Bak in the absence of tBid (Fig. 2a), which was seen at higher ratios of Bak:lipid concentration (not shown). The sub-stoichiometric ratio of tBid:Bak that was needed to observe robust release of calcein is consistent with the “hit-and-run” mechanism proposed for Bax activation in liposomes [13]. In the presence of a 3 - fold molar excess of surface-anchored Mcl-1 relative to surface-anchored Bak, the release of calcein in response to tBid was blocked (Fig. 1d).Fig. 2


Obatoclax is a direct and potent antagonist of membrane-restricted Mcl-1 and is synthetic lethal with treatment that induces Bim.

Nguyen M, Cencic R, Ertel F, Bernier C, Pelletier J, Roulston A, Silvius JR, Shore GC - BMC Cancer (2015)

a Bak-dependent release of calcein from proteoliposomes is not activated by obatoclax or Noxa BH3 peptide. Assays ± tBid were conducted as described in Fig. 1d, in the presence of 1 μM obatoclax, Noxa BH3, or vehicle (0.05 % DMSO) alone. b As in Fig. 1d except proteoliposomes contained Bak and Mcl-1 derivatives and the assays conducted in the presence of 1 μM obatoclax or 1 μM des-methoxyl (DM) obatoclax. c As in Fig. 1d except that liposome-anchored Bcl-XL replaced Mcl-1 and ABT-737 was tested. Concentrations of assay constituents are provided in right panel. Shown are representative assays from at least 3 independent experiments
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4522062&req=5

Fig2: a Bak-dependent release of calcein from proteoliposomes is not activated by obatoclax or Noxa BH3 peptide. Assays ± tBid were conducted as described in Fig. 1d, in the presence of 1 μM obatoclax, Noxa BH3, or vehicle (0.05 % DMSO) alone. b As in Fig. 1d except proteoliposomes contained Bak and Mcl-1 derivatives and the assays conducted in the presence of 1 μM obatoclax or 1 μM des-methoxyl (DM) obatoclax. c As in Fig. 1d except that liposome-anchored Bcl-XL replaced Mcl-1 and ABT-737 was tested. Concentrations of assay constituents are provided in right panel. Shown are representative assays from at least 3 independent experiments
Mentions: Assay mixtures (100 μl) containing proteoliposomes (20 μg lipid) with encapsulated fluorescence reporter calcein (quenched) and surface anchored Bak (0.14 μM) were challenged with 0.04 μM caspase-8-cleaved recombinant human Bid (aa 1–195 cleaved to 7 kDa and 15 kDa tBid fragments). Calcein acquires spontaneous fluorescence emission upon transbilayer release from the liposome, quantified as the % total calcein emission that is observed by treating the proteoliposomes with detergent. Conditions were selected to provide a robust Bak-dependent release of calcein in response to tBid (Figs. 1d and 2a), while at the same time minimizing the spontaneous and concentration-dependent release of calcein by Bak in the absence of tBid (Fig. 2a), which was seen at higher ratios of Bak:lipid concentration (not shown). The sub-stoichiometric ratio of tBid:Bak that was needed to observe robust release of calcein is consistent with the “hit-and-run” mechanism proposed for Bax activation in liposomes [13]. In the presence of a 3 - fold molar excess of surface-anchored Mcl-1 relative to surface-anchored Bak, the release of calcein in response to tBid was blocked (Fig. 1d).Fig. 2

Bottom Line: 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.Similar results were found for induction of Bak oligomers by Bim.A desmethoxy derivative of obatoclax failed to inhibit Mcl-1 in proteoliposomes and did not kill cells whose survival depends on Mcl-1.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, McGill University, Montreal, Québec, Canada. mai.nguyen@mcgill.ca.

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.

No MeSH data available.


Related in: MedlinePlus