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A series of cyclic peptides containing a number of tryptophan (W) and glutamic acid (E) residues were synthesized and evaluated as pH-sensitive agents for targeting of acidic tissue and pH-dependent cytoplasmic delivery of molecules. Biophysical studies revealed the molecular mechanism of peptides action and localization within the lipid bilayer of the membrane at high and low pHs. The symmetric, c[(WE) 4 WC] and asymmetric, c[E 4 W 5 C], cyclic peptides translocated amanitin, a polar cargo molecule of similar size, across the lipid bilayer and induced cell death in a pH- and concentration-dependent manner. Fluorescently-labelled peptides were evaluated for targeting of acidic 4T1 mammary tumors in mice. The highest tumor to muscle ratio (5.6) was established for asymmetric cyclic peptide, c[E 4 W 5 C], at 24 hours after intravenous administration. pH-insensitive cyclic peptide c[R 4 W 5 C], where glutamic acid residues (E) were replaced by positively charged arginine residues (R), did not exhibit tumor targeting. We have introduced a novel class of cyclic peptides, which can be utilized as a new pH-sensitive tool in investigation or targeting of acidic tissue.

To advance mechanistic understanding of membrane-associated peptide folding and insertion, we have studied the kinetics of three single tryptophan pHLIP (pH-Low Insertion Peptide) variants, where tryptophan residues are located near the N terminus, near the middle, and near the inserting C-terminal end of the pHLIP transmembrane helix. Single-tryptophan pHLIP variants allowed us to probe different parts of the peptide in the pathways of peptide insertion into the lipid bilayer (triggered by a pH drop) and peptide exit from the bilayer (triggered by a rise in pH). By using pH jumps of different magnitudes, we slowed down the processes and established the intermediates that helped us to understand the principles of insertion and exit. The obtained results should also aid the applications in medicine that are now entering the clinic.

The pH-low insertion peptide (pHLIP) and pH-low insertion cycle (pHLIC) have been shown to target cancer cells and inflammation due to the acidic environment present at those sites. It has been demonstrated that pHLIP’s and pHLIC’s pH dependent behavior stems from the protonation and deprotonation of aspartic acid (Asp) and glutamic acid (Glu) residues. A decrease in pH leads to the protonation of Asp/Glu located in membrane-inserting part of peptides, which increases the overall hydrophobicity of pHLIP and pHLIC and triggers the insertion across a lipid bilayer.Despite similarity of pHLIP and pHLIC ability to sense pH at cell surfaces the mechanisms of peptides insertion into membrane is different. pHLIP, which is a flexible polymer in solution at high pH, undergoes pH-triggered folding in membrane to transition from coil to transmembrane helix. pHLIC, which is a rigid cyclic peptide, undergoes pH-triggered partition into membrane without changes of its structure.pHLIP peptide insertion occurs in several steps, with a rapid interfacial helix formation (folding) completed within 100 ms followed by the rate limiting step of peptide insertion across membrane to form a transmembrane helix. Exit from the bilayer and unfolding is triggered by deprotonation of Asp/Glu residues induced by pH raise. The reverse process of unfolding and exit proceeds through different intermediate states. The detailed kinetics study of pHLIP variants pH-triggered insertion and exit from the membrane of liposomes allowed to elucidate the molecular mechanism of membrane-associated folding and unfolding, and design and test new pHLIP variants with tunable pH-dependent properties.Biophysical investigation of several pH-sensitive and pH-insensitive cyclic peptides led to the selection of best pHLIC candidate for targeting and imaging of neuroinflammation, which is associated with development of variety of neurodegenerative diseases.