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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

Mcl-1 inhibits Bim BH3-dependent oligomerization of Bak, which is overcome by obatoclax and Noxa BH3. a Bim BH3 but not Noxa BH3 nor obatoclax induce Bak oligomerization. Bak was conjugated onto liposomes and the liposomes treated with the indicated concentration of BH3 peptides or obatoclax or vehicle (1 % DMSO, lane 1). Liposome-conjugated Bak proteins were crosslinked with 0.5 mM BS3 and the samples analyzed by immunoblot with anti-Bak antibody. Migration of molecular weight markers is indicated. (*) denotes oligomers of Bak. b, c Mcl-1 (3-fold molar excess over Bak) inhibits Bim BH3-induced Bak oligomerization. Treatments and analysis were as in (a), in the presence of Noxa BH3 (b) or obatoclax (c) or vehicle alone, as indicated. Shown are representative blots from at least 3 independent experiments
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Fig3: Mcl-1 inhibits Bim BH3-dependent oligomerization of Bak, which is overcome by obatoclax and Noxa BH3. a Bim BH3 but not Noxa BH3 nor obatoclax induce Bak oligomerization. Bak was conjugated onto liposomes and the liposomes treated with the indicated concentration of BH3 peptides or obatoclax or vehicle (1 % DMSO, lane 1). Liposome-conjugated Bak proteins were crosslinked with 0.5 mM BS3 and the samples analyzed by immunoblot with anti-Bak antibody. Migration of molecular weight markers is indicated. (*) denotes oligomers of Bak. b, c Mcl-1 (3-fold molar excess over Bak) inhibits Bim BH3-induced Bak oligomerization. Treatments and analysis were as in (a), in the presence of Noxa BH3 (b) or obatoclax (c) or vehicle alone, as indicated. Shown are representative blots from at least 3 independent experiments

Mentions: Bak, like Bax, undergoes auto-oligomerization in the mitochondrial outer membrane in response to tBid, to form predicted transmembrane pores [28]. Employing chemical cross-linking and immunoblot, we monitored Bak oligomerization (i.e., the formation of cross-linked Bak adducts) in proteoliposomes induced by a 25 aa peptide spanning the BH3 helix of the activator BH3-only protein Bim, and tested the influence of Noxa peptide and obatoclax. The results aligned well with the conclusions from the functional analyses derived by monitoring calcein release from the proteoliposomes. Bim, but neither Noxa peptide nor obatoclax, stimulated the formation of higher order Bak adducts (Fig. 3a). Excess Mcl-1 inhibited Bim-induced Bak oligomerization (Fig. 3b,c), and this inhibition was overcome by Noxa peptide (Fig. 3b) and by obatoclax (Fig. 3c).Fig. 3


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)

Mcl-1 inhibits Bim BH3-dependent oligomerization of Bak, which is overcome by obatoclax and Noxa BH3. a Bim BH3 but not Noxa BH3 nor obatoclax induce Bak oligomerization. Bak was conjugated onto liposomes and the liposomes treated with the indicated concentration of BH3 peptides or obatoclax or vehicle (1 % DMSO, lane 1). Liposome-conjugated Bak proteins were crosslinked with 0.5 mM BS3 and the samples analyzed by immunoblot with anti-Bak antibody. Migration of molecular weight markers is indicated. (*) denotes oligomers of Bak. b, c Mcl-1 (3-fold molar excess over Bak) inhibits Bim BH3-induced Bak oligomerization. Treatments and analysis were as in (a), in the presence of Noxa BH3 (b) or obatoclax (c) or vehicle alone, as indicated. Shown are representative blots 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

Fig3: Mcl-1 inhibits Bim BH3-dependent oligomerization of Bak, which is overcome by obatoclax and Noxa BH3. a Bim BH3 but not Noxa BH3 nor obatoclax induce Bak oligomerization. Bak was conjugated onto liposomes and the liposomes treated with the indicated concentration of BH3 peptides or obatoclax or vehicle (1 % DMSO, lane 1). Liposome-conjugated Bak proteins were crosslinked with 0.5 mM BS3 and the samples analyzed by immunoblot with anti-Bak antibody. Migration of molecular weight markers is indicated. (*) denotes oligomers of Bak. b, c Mcl-1 (3-fold molar excess over Bak) inhibits Bim BH3-induced Bak oligomerization. Treatments and analysis were as in (a), in the presence of Noxa BH3 (b) or obatoclax (c) or vehicle alone, as indicated. Shown are representative blots from at least 3 independent experiments
Mentions: Bak, like Bax, undergoes auto-oligomerization in the mitochondrial outer membrane in response to tBid, to form predicted transmembrane pores [28]. Employing chemical cross-linking and immunoblot, we monitored Bak oligomerization (i.e., the formation of cross-linked Bak adducts) in proteoliposomes induced by a 25 aa peptide spanning the BH3 helix of the activator BH3-only protein Bim, and tested the influence of Noxa peptide and obatoclax. The results aligned well with the conclusions from the functional analyses derived by monitoring calcein release from the proteoliposomes. Bim, but neither Noxa peptide nor obatoclax, stimulated the formation of higher order Bak adducts (Fig. 3a). Excess Mcl-1 inhibited Bim-induced Bak oligomerization (Fig. 3b,c), and this inhibition was overcome by Noxa peptide (Fig. 3b) and by obatoclax (Fig. 3c).Fig. 3

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