Chapter 22 Synthesis and Biophysical Characterization of Stabilized α‐Helices of BCL‐2 Domains

Bird, Gregory H.; Bernal, Federico; Pitter, Kenneth L.; Walensky, Loren D.

doi:10.1016/s0076-6879(08)01622-4

Cited by 90 publications

(112 citation statements)

References 38 publications

Supporting

Mentioning

110

Contrasting

Order By: Relevance

“…SAHBs were synthesized, derivatized, purified, stored, reconstituted, and characterized by circular dichroism as described in detail previously (20,47). All peptides were purified by liquid chromatography/mass spectrometry to more than 95% purity and quantified by amino acid analysis.…”

Section: Methodsmentioning

confidence: 99%

A stapled BIM peptide overcomes apoptotic resistance in hematologic cancers

et al. 2012

Self Cite

View full text Add to dashboard Cite

Cancer cells subvert the natural balance between cellular life and death, achieving immortality through pathologic enforcement of survival pathways and blockade of cell death mechanisms. Pro-apoptotic BCL-2 family proteins are frequently disarmed in relapsed and refractory cancer through genetic deletion or interactionbased neutralization by overexpressed antiapoptotic proteins, resulting in resistance to chemotherapy and radiation treatments. New pharmacologic strategies are urgently needed to overcome these formidable apoptotic blockades. We harnessed the natural killing activity of BCL-2-interacting mediator of cell death (BIM), which contains one of the most potent BH3 death domains of the BCL-2 protein family, to restore BH3-dependent cell death in resistant hematologic cancers. A hydrocarbon-stapled peptide modeled after the BIM BH3 helix broadly targeted BCL-2 family proteins with high affinity, blocked inhibitory antiapoptotic interactions, directly triggered proapoptotic activity, and induced dose-responsive and BH3 sequence-specific cell death of hematologic cancer cells. The therapeutic potential of stapled BIM BH3 was highlighted by the selective activation of cell death in the aberrant lymphoid infiltrates of mice reconstituted with BIM-deficient bone marrow and in a human AML xenograft model. Thus, we found that broad and multimodal targeting of the BCL-2 family pathway can overcome pathologic barriers to cell death. IntroductionThe BCL-2 family interaction network lies at the crossroads of the cell's life-death decision (1, 2). Among the oncogenic signals that drive the development and maintenance of cancer, the pathologic reprogramming of the BCL-2 family interaction network has emerged as a formidable barrier to modern day anticancer treatment. Antiapoptotic members of the BCL-2 family contain a surface hydrophobic groove that can bind, sequester, and neutralize the critical BH3 death domains of proapoptotic members (3). Small molecules, such as ABT-737 and ABT-263, that selectively inhibit the antiapoptotic proteins BCL-2 and BCL-X L induce apoptosis of tumors that are especially dependent upon this subset of survival proteins (4, 5). However, the compounds are rendered ineffective by cellular expression of alternate antiapoptotic proteins, such as MCL-1 and BFL1/A1, which lie outside the molecules' range of binding specificity or by the relative or absolute absence of proapoptotic effectors, such as BAX and BAK (6, 7). Thus, a pharmacologic quest is underway to develop nextgeneration agents that simulate broader spectrum BH3-dependent killing activities and effectively deactivate the deflector shields of relapsed and refractory cancers.Chief among the killer BH3-only proteins, Bcl-2-interacting mediator of cell death (BIM) exhibits the most broad-ranging BCL-2 protein interactions, engaging all of the antiapoptotic proteins with high affinity (8, 9) and also directly triggering death effectors, such as BAX (10,11). A recent genetic analysis elegantly demonstrated that only BIM BH3, but not oth...

show abstract

Section: Methodsmentioning

confidence: 99%

A stapled BIM peptide overcomes apoptotic resistance in hematologic cancers

et al. 2012

Self Cite

View full text Add to dashboard Cite

show abstract

“…Peptide synthesis, hydrocarbon stapling by olefin metathesis, and derivatization with FITC and biotin were performed according to our established methods (54,55). All peptides were purified by LC/MS to >95% purity and quantified by amino acid analysis.…”

Section: Methodsmentioning

confidence: 99%

Direct activation of full-length proapoptotic BAK

Leshchiner

Braun

Bird

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

143

151

View full text Add to dashboard Cite

Proapoptotic B-cell lymphoma 2 (BCL-2) antagonist/killer (BAK) and BCL-2-associated X (BAX) form toxic mitochondrial pores in response to cellular stress. Whereas BAX resides predominantly in the cytosol, BAK is constitutively localized to the outer mitochondrial membrane. Select BCL-2 homology domain 3 (BH3) helices activate BAX directly by engaging an α1/α6 trigger site. The inability to express full-length BAK has hampered full dissection of its activation mechanism. Here, we report the production of full-length, monomeric BAK by mutagenesisbased solubilization of its C-terminal α-helical surface. Recombinant BAK autotranslocates to mitochondria but only releases cytochrome c upon BH3 triggering. A direct activation mechanism was explicitly demonstrated using a liposomal system that recapitulates BAKmediated release upon addition of BH3 ligands. Photoreactive BH3 helices mapped both triggering and autointeractions to the canonical BH3-binding pocket of BAK, whereas the same ligands crosslinked to the α1/α6 site of BAX. Thus, activation of both BAK and BAX is initiated by direct BH3-interaction but at distinct trigger sites. These structural and biochemical insights provide opportunities for developing proapoptotic agents that activate the death pathway through direct but differential engagement of BAK and BAX.apoptosis | BCL-2 family | SAHB | stapled peptide | photocrosslinking

show abstract

“…Peptides were dissolved in 5 mM potassium phosphate (pH 7.5) to a final concentration of 50 μM. Precise concentrations were determined by amino acid analysis and used to calculate percent α-helical content as described previously (40).…”

Section: Methodsmentioning

confidence: 99%

Mucosal delivery of a double-stapled RSV peptide prevents nasopulmonary infection

Bird¹,

Boyapalle²,

Wong³

et al. 2014

J. Clin. Invest.

Self Cite

View full text Add to dashboard Cite

Respiratory syncytial virus (RSV) infection accounts for approximately 64 million cases of respiratory disease and 200,000 deaths worldwide each year, yet no broadly effective prophylactic or treatment regimen is available. RSV deploys paired, self-associating, heptad repeat domains of its fusion protein, RSV-F, to form a fusogenic 6-helix bundle that enables the virus to penetrate the host cell membrane. Here, we developed hydrocarbon double-stapled RSV fusion peptides that exhibit stabilized α-helical structure and striking proteolytic resistance. Pretreatment with double-stapled RSV peptides that specifically bound to the RSV fusion bundle inhibited infection by both laboratory and clinical RSV isolates in cells and murine infection models. Intranasal delivery of a lead double-stapled RSV peptide effectively prevented viral infection of the nares. A chitosan-based nanoparticle preparation markedly enhanced pulmonary delivery, further preventing progression of RSV infection to the lung. Thus, our results provide a strategy for inhibiting RSV infection by mucosal and endotracheal delivery of double-stapled RSV fusion peptides.

show abstract

Chapter 22 Synthesis and Biophysical Characterization of Stabilized α‐Helices of BCL‐2 Domains

Cited by 90 publications

References 38 publications

A stapled BIM peptide overcomes apoptotic resistance in hematologic cancers

A stapled BIM peptide overcomes apoptotic resistance in hematologic cancers

Direct activation of full-length proapoptotic BAK

Mucosal delivery of a double-stapled RSV peptide prevents nasopulmonary infection

Contact Info

Product

Resources

About