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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

Noxa BH3 peptide and membrane-restricted obatoclax (OBX) directly antagonize the ability of Mcl-1 to inhibit Bak-mediated calcein release from proteoliposomes. a Left. Model for the regulation of liposome bilayer permeabilization by the tBid-Bak-Mcl-1 axis. Membrane anchoring of Mcl-1 and Bak is achieved by replacing their C-terminal TM segments with chemical functionalities (blue circle) that interact with modified head groups (red circle) of liposome phospholipids. Bilayer permeabilization is assayed by the acquisition of calcein (Cn) fluorescence upon its release from liposomes induced by tBid. Right. Chemical structures of obatoclax and des-methoxy obatoclax. b Obatoclax binds avidly to lipid vesicles. Addition of lipid vesicles (0–40 μM) to obatoclax (0.15 μM) in buffer at 37 °C leads to rapid obatoclax partitioning into vesicle bilayers and enhancement of obatoclax fluorescence (λex/λem = 540/575 nm, slitwidths = 10/10 nm); obatoclax half-maximally associates with bilayers at 13 ± 1 μM lipid (mean/half-range of two experiments). c Obatoclax transfers rapidly between distinct bilayers. Obatoclax (0.3 μM) added to lipid vesicles (10 μM) incorporating NBD-PE causes rapid energy transfer-mediated quenching of NBD-PE fluorescence (λex/λem = 470/538 nm, slitwidths = 10/10 nm) as obatoclax partitions into the vesicle bilayers (first arrow). On subsequent addition of sonicated vesicles lacking NBD-PE (20 μM; second arrow), obatoclax transfers from NBD-incorporating to NBD-PE-free vesicles, partially restoring NBD-PE fluorescence, over a time scale of seconds. d Proteolipsomes harboring membrane-anchored Mcl-1 and/or Bak derivatives (BakΔC*; Mcl-1ΔC*) were challenged with tBid in the presence or absence of obatoclax or Noxa BH3 peptide. Shown are representative fluorimetric assays from 3 independent experiments of calcein release from liposome in response to 40 nM tBid over time (right panel). Concentrations of assay constituents are given in the left panel
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Fig1: Noxa BH3 peptide and membrane-restricted obatoclax (OBX) directly antagonize the ability of Mcl-1 to inhibit Bak-mediated calcein release from proteoliposomes. a Left. Model for the regulation of liposome bilayer permeabilization by the tBid-Bak-Mcl-1 axis. Membrane anchoring of Mcl-1 and Bak is achieved by replacing their C-terminal TM segments with chemical functionalities (blue circle) that interact with modified head groups (red circle) of liposome phospholipids. Bilayer permeabilization is assayed by the acquisition of calcein (Cn) fluorescence upon its release from liposomes induced by tBid. Right. Chemical structures of obatoclax and des-methoxy obatoclax. b Obatoclax binds avidly to lipid vesicles. Addition of lipid vesicles (0–40 μM) to obatoclax (0.15 μM) in buffer at 37 °C leads to rapid obatoclax partitioning into vesicle bilayers and enhancement of obatoclax fluorescence (λex/λem = 540/575 nm, slitwidths = 10/10 nm); obatoclax half-maximally associates with bilayers at 13 ± 1 μM lipid (mean/half-range of two experiments). c Obatoclax transfers rapidly between distinct bilayers. Obatoclax (0.3 μM) added to lipid vesicles (10 μM) incorporating NBD-PE causes rapid energy transfer-mediated quenching of NBD-PE fluorescence (λex/λem = 470/538 nm, slitwidths = 10/10 nm) as obatoclax partitions into the vesicle bilayers (first arrow). On subsequent addition of sonicated vesicles lacking NBD-PE (20 μM; second arrow), obatoclax transfers from NBD-incorporating to NBD-PE-free vesicles, partially restoring NBD-PE fluorescence, over a time scale of seconds. d Proteolipsomes harboring membrane-anchored Mcl-1 and/or Bak derivatives (BakΔC*; Mcl-1ΔC*) were challenged with tBid in the presence or absence of obatoclax or Noxa BH3 peptide. Shown are representative fluorimetric assays from 3 independent experiments of calcein release from liposome in response to 40 nM tBid over time (right panel). Concentrations of assay constituents are given in the left panel

Mentions: In this study, large unilamellar proteoliposomes were created that recapitulate the constitutive integral association that native Mcl-1 and Bak make with the MOM in intact cells. To that end, lipids were employed that reflect both the composition and relative abundance found in the MOM [12], but which also included low amounts of the modified lipids N-(4-maleimidobutyroyl)-PEG3-POPE (Mal-PEG3-PE) and/or the tris-(nitrilotriacetic acid)-modified lipid DOD-tris-(NTA(Ni2+)). Recombinant forms of human full length Bak and Mcl-1 were created in which the C-terminal TM segment was replaced with 6 His residues followed by a unique terminal Cys, and the proteins were linked to the ecto-surface of liposomes either covalently through the Cys residue (via Mal-PEG3-PE) or through high-affinity coordination of the His6 sequence to bilayer-incorporated DOD-tris-(NTA(Ni2+)) (Fig. 1a), thereby overcoming the otherwise difficult challenge to express and properly anchor the proteins via their native TM segment. In all experiments reported here, proteoliposomes were recovered free of unattached Mcl-1 or Bak prior to functional analyses. As reported below and in ref [1], the basic tenets that have been elucidated for the tBid - Bcl-XL - Bax model for execution and regulation of permeabilization of liposomal membranes by Bax, appear also to apply to the tBid - Mcl-1 - Bak axis, and are outlined in Fig. 1a (left).Fig. 1


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)

Noxa BH3 peptide and membrane-restricted obatoclax (OBX) directly antagonize the ability of Mcl-1 to inhibit Bak-mediated calcein release from proteoliposomes. a Left. Model for the regulation of liposome bilayer permeabilization by the tBid-Bak-Mcl-1 axis. Membrane anchoring of Mcl-1 and Bak is achieved by replacing their C-terminal TM segments with chemical functionalities (blue circle) that interact with modified head groups (red circle) of liposome phospholipids. Bilayer permeabilization is assayed by the acquisition of calcein (Cn) fluorescence upon its release from liposomes induced by tBid. Right. Chemical structures of obatoclax and des-methoxy obatoclax. b Obatoclax binds avidly to lipid vesicles. Addition of lipid vesicles (0–40 μM) to obatoclax (0.15 μM) in buffer at 37 °C leads to rapid obatoclax partitioning into vesicle bilayers and enhancement of obatoclax fluorescence (λex/λem = 540/575 nm, slitwidths = 10/10 nm); obatoclax half-maximally associates with bilayers at 13 ± 1 μM lipid (mean/half-range of two experiments). c Obatoclax transfers rapidly between distinct bilayers. Obatoclax (0.3 μM) added to lipid vesicles (10 μM) incorporating NBD-PE causes rapid energy transfer-mediated quenching of NBD-PE fluorescence (λex/λem = 470/538 nm, slitwidths = 10/10 nm) as obatoclax partitions into the vesicle bilayers (first arrow). On subsequent addition of sonicated vesicles lacking NBD-PE (20 μM; second arrow), obatoclax transfers from NBD-incorporating to NBD-PE-free vesicles, partially restoring NBD-PE fluorescence, over a time scale of seconds. d Proteolipsomes harboring membrane-anchored Mcl-1 and/or Bak derivatives (BakΔC*; Mcl-1ΔC*) were challenged with tBid in the presence or absence of obatoclax or Noxa BH3 peptide. Shown are representative fluorimetric assays from 3 independent experiments of calcein release from liposome in response to 40 nM tBid over time (right panel). Concentrations of assay constituents are given in the left panel
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4522062&req=5

Fig1: Noxa BH3 peptide and membrane-restricted obatoclax (OBX) directly antagonize the ability of Mcl-1 to inhibit Bak-mediated calcein release from proteoliposomes. a Left. Model for the regulation of liposome bilayer permeabilization by the tBid-Bak-Mcl-1 axis. Membrane anchoring of Mcl-1 and Bak is achieved by replacing their C-terminal TM segments with chemical functionalities (blue circle) that interact with modified head groups (red circle) of liposome phospholipids. Bilayer permeabilization is assayed by the acquisition of calcein (Cn) fluorescence upon its release from liposomes induced by tBid. Right. Chemical structures of obatoclax and des-methoxy obatoclax. b Obatoclax binds avidly to lipid vesicles. Addition of lipid vesicles (0–40 μM) to obatoclax (0.15 μM) in buffer at 37 °C leads to rapid obatoclax partitioning into vesicle bilayers and enhancement of obatoclax fluorescence (λex/λem = 540/575 nm, slitwidths = 10/10 nm); obatoclax half-maximally associates with bilayers at 13 ± 1 μM lipid (mean/half-range of two experiments). c Obatoclax transfers rapidly between distinct bilayers. Obatoclax (0.3 μM) added to lipid vesicles (10 μM) incorporating NBD-PE causes rapid energy transfer-mediated quenching of NBD-PE fluorescence (λex/λem = 470/538 nm, slitwidths = 10/10 nm) as obatoclax partitions into the vesicle bilayers (first arrow). On subsequent addition of sonicated vesicles lacking NBD-PE (20 μM; second arrow), obatoclax transfers from NBD-incorporating to NBD-PE-free vesicles, partially restoring NBD-PE fluorescence, over a time scale of seconds. d Proteolipsomes harboring membrane-anchored Mcl-1 and/or Bak derivatives (BakΔC*; Mcl-1ΔC*) were challenged with tBid in the presence or absence of obatoclax or Noxa BH3 peptide. Shown are representative fluorimetric assays from 3 independent experiments of calcein release from liposome in response to 40 nM tBid over time (right panel). Concentrations of assay constituents are given in the left panel
Mentions: In this study, large unilamellar proteoliposomes were created that recapitulate the constitutive integral association that native Mcl-1 and Bak make with the MOM in intact cells. To that end, lipids were employed that reflect both the composition and relative abundance found in the MOM [12], but which also included low amounts of the modified lipids N-(4-maleimidobutyroyl)-PEG3-POPE (Mal-PEG3-PE) and/or the tris-(nitrilotriacetic acid)-modified lipid DOD-tris-(NTA(Ni2+)). Recombinant forms of human full length Bak and Mcl-1 were created in which the C-terminal TM segment was replaced with 6 His residues followed by a unique terminal Cys, and the proteins were linked to the ecto-surface of liposomes either covalently through the Cys residue (via Mal-PEG3-PE) or through high-affinity coordination of the His6 sequence to bilayer-incorporated DOD-tris-(NTA(Ni2+)) (Fig. 1a), thereby overcoming the otherwise difficult challenge to express and properly anchor the proteins via their native TM segment. In all experiments reported here, proteoliposomes were recovered free of unattached Mcl-1 or Bak prior to functional analyses. As reported below and in ref [1], the basic tenets that have been elucidated for the tBid - Bcl-XL - Bax model for execution and regulation of permeabilization of liposomal membranes by Bax, appear also to apply to the tBid - Mcl-1 - Bak axis, and are outlined in Fig. 1a (left).Fig. 1

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