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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 KMS-11 cells were treated with the indicated combinations of siRNA. After 24 h, the percent cell death was determined and normalized to cells not treated with siRNA. Error bars show the mean of triplicate samples with SEM. b Western blot analysis of KMS-11 cells (expressing Bcl-2 to prevent death) treated with the indicated concentrations of dexamethasone (dex) in the presence of 40 μM zVAD-fmk for 48 h. c Western blot analysis of KMS-11 cells treated with Bim siRNA (siBim) or control (scrambled non-targeting) siRNA (siCtl) in the presence or absence of 100 nM dexamethasone for 72 h, and probed with antibodies against Bim and GAPDH (left panel). Cell death normalized to control siRNA (siCtl) was determined on the same samples (right panel). Error bars show the mean with SEM. d KMS-11 cells were pre-treated with dex for 72 h followed by exposure to obatoclax for 48 h. Viability for the combination treatments is expressed relative to samples without obatoclax in each dex dose group. Error bars represent the SD of triplicate samples. The table shows combination indices (CI) at the indicated concentrations of dex, calculated using COMPUSYN V.1.0 software according to the original method from Chou and Talalay [23]. e As in (D) except that viability was determined in the presence of control (siCtl) or siBim
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Fig6: a KMS-11 cells were treated with the indicated combinations of siRNA. After 24 h, the percent cell death was determined and normalized to cells not treated with siRNA. Error bars show the mean of triplicate samples with SEM. b Western blot analysis of KMS-11 cells (expressing Bcl-2 to prevent death) treated with the indicated concentrations of dexamethasone (dex) in the presence of 40 μM zVAD-fmk for 48 h. c Western blot analysis of KMS-11 cells treated with Bim siRNA (siBim) or control (scrambled non-targeting) siRNA (siCtl) in the presence or absence of 100 nM dexamethasone for 72 h, and probed with antibodies against Bim and GAPDH (left panel). Cell death normalized to control siRNA (siCtl) was determined on the same samples (right panel). Error bars show the mean with SEM. d KMS-11 cells were pre-treated with dex for 72 h followed by exposure to obatoclax for 48 h. Viability for the combination treatments is expressed relative to samples without obatoclax in each dex dose group. Error bars represent the SD of triplicate samples. The table shows combination indices (CI) at the indicated concentrations of dex, calculated using COMPUSYN V.1.0 software according to the original method from Chou and Talalay [23]. e As in (D) except that viability was determined in the presence of control (siCtl) or siBim

Mentions: Our earlier in silico studies of obatoclax docking into the P1 and P2 hydrophobic pockets of the BH3-binding groove of Mcl-1 predicted that the −3-methoxy moiety of obatoclax penetrated deep into the P2 pocket, driving hydrophobic binding [15]. Of note, and in contrast to obatoclax, the des- methoxy analog of obatoclax (Fig. 1a) did not overcome Mcl-1-mediated inhibition of tBid-induced, Bak-dependent release of calcein from proteoliposomes (Fig. 2b). Moreover, when assessed for cytotoxicity in KMS-11 cells, whose survival in standard cell culture depends upon Mcl-1 (see Fig. 6a), the des-methoxy analog was without toxicity compared to obatoclax (see Fig. 4a). Thus, the 3-methoxy moiety of obatoclax appears to be essential for its inhibitory activity against Mcl-1. Finally, replacing liposome-tethered Mcl-1 with liposome-tethered Bcl-XL did not allow obatoclax to overcome the inhibition by Bcl-XL of tBid-dependent Bak-mediated release of calcein, whereas the validated small molecule antagonist of Bcl-XL, ABT-737 [27], was active (Fig. 2c). This suggests that obatoclax exhibits a preference for Mcl-1 compared to Bcl-XL.


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 KMS-11 cells were treated with the indicated combinations of siRNA. After 24 h, the percent cell death was determined and normalized to cells not treated with siRNA. Error bars show the mean of triplicate samples with SEM. b Western blot analysis of KMS-11 cells (expressing Bcl-2 to prevent death) treated with the indicated concentrations of dexamethasone (dex) in the presence of 40 μM zVAD-fmk for 48 h. c Western blot analysis of KMS-11 cells treated with Bim siRNA (siBim) or control (scrambled non-targeting) siRNA (siCtl) in the presence or absence of 100 nM dexamethasone for 72 h, and probed with antibodies against Bim and GAPDH (left panel). Cell death normalized to control siRNA (siCtl) was determined on the same samples (right panel). Error bars show the mean with SEM. d KMS-11 cells were pre-treated with dex for 72 h followed by exposure to obatoclax for 48 h. Viability for the combination treatments is expressed relative to samples without obatoclax in each dex dose group. Error bars represent the SD of triplicate samples. The table shows combination indices (CI) at the indicated concentrations of dex, calculated using COMPUSYN V.1.0 software according to the original method from Chou and Talalay [23]. e As in (D) except that viability was determined in the presence of control (siCtl) or siBim
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Fig6: a KMS-11 cells were treated with the indicated combinations of siRNA. After 24 h, the percent cell death was determined and normalized to cells not treated with siRNA. Error bars show the mean of triplicate samples with SEM. b Western blot analysis of KMS-11 cells (expressing Bcl-2 to prevent death) treated with the indicated concentrations of dexamethasone (dex) in the presence of 40 μM zVAD-fmk for 48 h. c Western blot analysis of KMS-11 cells treated with Bim siRNA (siBim) or control (scrambled non-targeting) siRNA (siCtl) in the presence or absence of 100 nM dexamethasone for 72 h, and probed with antibodies against Bim and GAPDH (left panel). Cell death normalized to control siRNA (siCtl) was determined on the same samples (right panel). Error bars show the mean with SEM. d KMS-11 cells were pre-treated with dex for 72 h followed by exposure to obatoclax for 48 h. Viability for the combination treatments is expressed relative to samples without obatoclax in each dex dose group. Error bars represent the SD of triplicate samples. The table shows combination indices (CI) at the indicated concentrations of dex, calculated using COMPUSYN V.1.0 software according to the original method from Chou and Talalay [23]. e As in (D) except that viability was determined in the presence of control (siCtl) or siBim
Mentions: Our earlier in silico studies of obatoclax docking into the P1 and P2 hydrophobic pockets of the BH3-binding groove of Mcl-1 predicted that the −3-methoxy moiety of obatoclax penetrated deep into the P2 pocket, driving hydrophobic binding [15]. Of note, and in contrast to obatoclax, the des- methoxy analog of obatoclax (Fig. 1a) did not overcome Mcl-1-mediated inhibition of tBid-induced, Bak-dependent release of calcein from proteoliposomes (Fig. 2b). Moreover, when assessed for cytotoxicity in KMS-11 cells, whose survival in standard cell culture depends upon Mcl-1 (see Fig. 6a), the des-methoxy analog was without toxicity compared to obatoclax (see Fig. 4a). Thus, the 3-methoxy moiety of obatoclax appears to be essential for its inhibitory activity against Mcl-1. Finally, replacing liposome-tethered Mcl-1 with liposome-tethered Bcl-XL did not allow obatoclax to overcome the inhibition by Bcl-XL of tBid-dependent Bak-mediated release of calcein, whereas the validated small molecule antagonist of Bcl-XL, ABT-737 [27], was active (Fig. 2c). This suggests that obatoclax exhibits a preference for Mcl-1 compared to Bcl-XL.

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