Cathepsin-Cleavable BIM BH3 Peptide Amphiphiles Are Potent Inducers of Cellular Apoptosis

Joseph Bellairs, Ravand Samaeekia, Handan Acar, Matthew Tirrell and James LaBelle


The BCL-2 family of proteins forms a complex interaction network that regulates cellular life and death decisions and contributes to cancer development, maintenance, and chemoresistance. BH3 only member proteins (e.g. BIM) serve as cellular stress sentinels and, when triggered, signal irreversible activation of apoptosis through their α-helical BH3 death domains. These pro-apoptotic signals are normally held in check by the multidomain anti-apoptotic proteins (e.g. BCL-XL, MCL-1) but when they are unable to do so the multidomain pro-apoptotic proteins BAX and BAK induce cell death through pore formation in the mitochondrial outer membrane. Therapeutic manipulation of the BCL-2 family with BH3 mimetics (including small molecules and synthetic peptides) is an emerging paradigm in cancer treatment and immune modulation. The design of next-generation therapeutics based on the BIM BH3 helix offers the unique advantage of recapitulating BIM's natural capacity to directly target the full complement of anti- and pro-apoptotic BCL-2 proteins. Here, we utilize the highly active BH3 domain of BIM as part of a peptide amphiphile nanostructure designed to overcome malignant cell death blockade. Peptide amphiphiles consist of bioactive peptides linked to hydrophobic lipid-like tail groups. In aqueous solutions, amphiphiles spontaneously assemble into micelles. Micelle-based peptide delivery provides several advantages: single micelles deliver high concentration of peptides into cells, they stabilize peptide secondary structure(s), and they have the potential for combinatorial synthesis using multiple bioactive moieties targeting non-redundant cell death escape pathways. While the exact mechanism behind cellular uptake of peptide amphiphiles remains controversial, recent work has shown that peptide amphiphiles intracellularly traffic through lysosomes and endosomes. In order to prevent the bioactive peptides from being sequestered within these structures, a system of escape is needed. Lysosomes contain many well-characterized proteases, and cathepsin B has previously been utilized to release chemotherapeutics in the context of targetable antibody-based treatments. Here, we generate peptide amphiphiles with BIM BH3 peptides and show that these nanostructures are able to specifically bind recombinant BCL-2 proteins, are stable at physiologic temperatures and pH, quickly enter into cells, and induce dose-responsive apoptosis in malignant hematologic cancers as measured by viability and caspase 3/7 activation. We further demonstrate that incorporating a cathepsin B-cleavable linker between the BIM BH3 peptide and the hydrophobic tail within individual amphiphiles results in increased binding to recombinant BCL-2 proteins while also allowing for increased cellular uptake and mitochondrial localization leading to faster and more potent dose-dependent cytotoxicity and caspase activation in malignant cells. Thus, we have developed a modular and potentially targetable nanostructure that represents a new promising strategy for BCL-2 family modulation and apoptosis induction in cancer.

Disclosures No relevant conflicts of interest to declare.

  • * Asterisk with author names denotes non-ASH members.