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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

Obatoclax is active in cells whose survival depends on Mcl-1. a Viability of KMS-11 myeloma cells was measured in the presence of 0.5 μM and 1 μM obatoclax (gray bars) or the des-methoxy derivative of obatoclax (white bars) or vehicle (1 % DMSO) for 48 h. The % cell death was normalized to vehicle control. Error bars show the mean with SEM. b TE671 cells depend on both Mcl-1 and Bcl-XL for survival. Cells were transfected with the indicated combinations of siRNA for 48 h. Cell death was determined and expressed as percentage of cells treated with control siRNA (siCtl) (upper panel). Western blot analysis of the siRNA treated cells (lower panel). c Knocking down of Bcl-XL enhances death of TE671 cells by obatoclax. TE671 cells were transfected with control siRNA or Bcl-XL siRNA for 24 h followed by 48-hr treatment with 200 nM obatoclax. Cell death was determined and expressed as percentage of transfected cells treated with DMSO. d Obatoclax induced caspase activation in Tsc2+/−Eμ-Myc lymphoma cells. Cells were treated with 1 μM obatoclax for the indicated time and DEVDase activity was determined. e Flow cytometry analysis of GFP-positive Tsc2+/−Eμ-Myc lymphoma cells as readout for cell survival of cells transduced with shFLuc.1309 or shMcl-1.1334. Cells were transduced once with the indicated constructs and flow cytometry analysis performed 12 h after transduction (t = 0) as well as 15 h later (t = 15). Error bars indicate SEM (n = 3)
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Fig4: Obatoclax is active in cells whose survival depends on Mcl-1. a Viability of KMS-11 myeloma cells was measured in the presence of 0.5 μM and 1 μM obatoclax (gray bars) or the des-methoxy derivative of obatoclax (white bars) or vehicle (1 % DMSO) for 48 h. The % cell death was normalized to vehicle control. Error bars show the mean with SEM. b TE671 cells depend on both Mcl-1 and Bcl-XL for survival. Cells were transfected with the indicated combinations of siRNA for 48 h. Cell death was determined and expressed as percentage of cells treated with control siRNA (siCtl) (upper panel). Western blot analysis of the siRNA treated cells (lower panel). c Knocking down of Bcl-XL enhances death of TE671 cells by obatoclax. TE671 cells were transfected with control siRNA or Bcl-XL siRNA for 24 h followed by 48-hr treatment with 200 nM obatoclax. Cell death was determined and expressed as percentage of transfected cells treated with DMSO. d Obatoclax induced caspase activation in Tsc2+/−Eμ-Myc lymphoma cells. Cells were treated with 1 μM obatoclax for the indicated time and DEVDase activity was determined. e Flow cytometry analysis of GFP-positive Tsc2+/−Eμ-Myc lymphoma cells as readout for cell survival of cells transduced with shFLuc.1309 or shMcl-1.1334. Cells were transduced once with the indicated constructs and flow cytometry analysis performed 12 h after transduction (t = 0) as well as 15 h later (t = 15). Error bars indicate SEM (n = 3)

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)

Obatoclax is active in cells whose survival depends on Mcl-1. a Viability of KMS-11 myeloma cells was measured in the presence of 0.5 μM and 1 μM obatoclax (gray bars) or the des-methoxy derivative of obatoclax (white bars) or vehicle (1 % DMSO) for 48 h. The % cell death was normalized to vehicle control. Error bars show the mean with SEM. b TE671 cells depend on both Mcl-1 and Bcl-XL for survival. Cells were transfected with the indicated combinations of siRNA for 48 h. Cell death was determined and expressed as percentage of cells treated with control siRNA (siCtl) (upper panel). Western blot analysis of the siRNA treated cells (lower panel). c Knocking down of Bcl-XL enhances death of TE671 cells by obatoclax. TE671 cells were transfected with control siRNA or Bcl-XL siRNA for 24 h followed by 48-hr treatment with 200 nM obatoclax. Cell death was determined and expressed as percentage of transfected cells treated with DMSO. d Obatoclax induced caspase activation in Tsc2+/−Eμ-Myc lymphoma cells. Cells were treated with 1 μM obatoclax for the indicated time and DEVDase activity was determined. e Flow cytometry analysis of GFP-positive Tsc2+/−Eμ-Myc lymphoma cells as readout for cell survival of cells transduced with shFLuc.1309 or shMcl-1.1334. Cells were transduced once with the indicated constructs and flow cytometry analysis performed 12 h after transduction (t = 0) as well as 15 h later (t = 15). Error bars indicate SEM (n = 3)
© Copyright Policy - open-access
Related In: Results  -  Collection

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Fig4: Obatoclax is active in cells whose survival depends on Mcl-1. a Viability of KMS-11 myeloma cells was measured in the presence of 0.5 μM and 1 μM obatoclax (gray bars) or the des-methoxy derivative of obatoclax (white bars) or vehicle (1 % DMSO) for 48 h. The % cell death was normalized to vehicle control. Error bars show the mean with SEM. b TE671 cells depend on both Mcl-1 and Bcl-XL for survival. Cells were transfected with the indicated combinations of siRNA for 48 h. Cell death was determined and expressed as percentage of cells treated with control siRNA (siCtl) (upper panel). Western blot analysis of the siRNA treated cells (lower panel). c Knocking down of Bcl-XL enhances death of TE671 cells by obatoclax. TE671 cells were transfected with control siRNA or Bcl-XL siRNA for 24 h followed by 48-hr treatment with 200 nM obatoclax. Cell death was determined and expressed as percentage of transfected cells treated with DMSO. d Obatoclax induced caspase activation in Tsc2+/−Eμ-Myc lymphoma cells. Cells were treated with 1 μM obatoclax for the indicated time and DEVDase activity was determined. e Flow cytometry analysis of GFP-positive Tsc2+/−Eμ-Myc lymphoma cells as readout for cell survival of cells transduced with shFLuc.1309 or shMcl-1.1334. Cells were transduced once with the indicated constructs and flow cytometry analysis performed 12 h after transduction (t = 0) as well as 15 h later (t = 15). Error bars indicate SEM (n = 3)
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