Mechanistic insights into the pH-dependent membrane peptide ATRAM

Abstract

pH-responsive peptides are promising therapeutic molecules that can specifically target the plasma membrane in the acidified extracellular medium that bathes cells in tumors. We designed the acidity-triggered rational membrane (ATRAM) peptide to have a pH-responsive membrane interaction. At physiological pH, ATRAM binds to the membrane surface in a largely unstructured conformation, while in acidic conditions it inserts into lipid bilayers forming a transmembrane helix. However, the molecular mechanism ATRAM uses to target and insert into tumor cells remains poorly understood. Here, we determined that ATRAM inserts into cancer cells with a preferential membrane orientation, where the C-terminus of the peptide traverses the plasma membrane and explores the cytoplasm. Using biophysical techniques, we determined that the membrane interaction of ATRAM is contingent on the concentration of the peptide. Kinetic studies showed that membrane insertion occurs in at least three steps, where only the first step was affected by the membrane density of ATRAM. These observations, combined with membrane binding and leakage data, indicate that the interaction of ATRAM with lipid membranes is dependent on its oligomerization state. SPECT/CT imaging in mice revealed that ATRAM accumulates in the blood pool, where it has a prolonged circulation time (> 4 hours). Since fast peptide clearance and degradation in circulation are major problems for clinical development, we studied the mechanism ATRAM uses to remain in the blood stream. Using binding and transfer assays, we determined that ATRAM binds reversibly to human serum albumin. We propose that ATRAM uses albumin as a carrier in the blood stream to evade clearance and proteolysis before interacting with the plasma membrane of cancer cells. We also show that ATRAM is able to be deliver liposomes to cells in a pH dependent way. Our data highlight the potential of ATRAM as a specific therapeutic agent for diseases that lead to acidic tissues, including cancer.

pH-responsive high stability polymeric nanoparticles for targeted delivery of anticancer therapeutics

“…Interestingly, ATRAM exhibits a longer circulation half-life compared to peptides of similar molecular weight 27 . Moreover, ATRAM was shown to accumulate within acidic tumor tissue in mice 55 .…”

“…In order to specifically target tumor cells, the BSA-PLGA NPs were functionalized with the ATRAM peptide, which inserts into lipid bilayers under acidic conditions 26 . The measured membrane insertion pK a of ATRAM is 6.5 (see Supplementary Note 3) [26][27][28] , making the peptide ideal for targeting cancer cells in the acidic microenvironment of solid tumors 45,53 , which results from the high glycolytic rates of tumor cells 54 . Indeed, ATRAM was shown to efficiently target tumors in mice 55 .…”

“…We previously established that ATRAM inserts into membranes via its C-terminus 27 . Therefore we conjugated ATRAM to the surface of the BSA-PLGA NPs by covalently coupling the remaining lysine residues of BSA in the NPs to the N-terminal amino group of the peptide via carbodiimide chemistry 56 , and the attachment was evaluated by release of the urea byproduct at 232 nm ( Supplementary Fig.…”

“…To address these challenges, we have designed hybrid NPs that consist of a polylactic-co-glycolic acid (PLGA) core 'wrapped' with a cross-linked bovine serum albumin (BSA) shell that is functionalized with the acidity-triggered rational membrane (ATRAM) peptide [26][27][28] . The pH-responsive hybrid ATRAM-BSA-PLGA NPs are a promising cancer drug delivery platform that combines high stability with effective tumor targeting and triggered release of chemotherapeutic agents in cancer cells.…”

Transmembrane Peptides as Inhibitors of Protein-Protein Interactions: An Efficient Strategy to Target Cancer Cells?

“…Rather, they exert their function as small molecules by a widespread distribution in the body. However, TM domain peptides can be combined with targeting moieties attached to nanocarriers to address this point and produce drugs with a more selective action on a given cell type (35). While the development of formulations compatible with a clinical use remains to be fully achieved, the recent development in the production of TM domain peptides with pH sensitive membrane interaction is opening interesting opportunities both in term of solubility or activity.…”