pHLIP Variants Research Articles

Abstract 9919: PH-low Insertion Peptide (pHLIP) Targets Ischemic Myocardium

pHLIP molecule (∼3 kD) inserts into a lipid bilayer when ambient pH is low. Originally, this property was used for targeted delivery of small molecules, liposomes and nanoparticles conjugated with pHLIP to visualize and treat tumors. Since cardiac ischemia causes a decrease in tissue pH we hypothesized that pHLIP can selectively bind to ischemic myocardium. METHODS. We studied pH-sensitive (Var7) and a pH-insensitive (kVar7) variants of pHLIP conjugated with fluorescent dye Alexa488 in three models. (1) Local myocardial ischemia was induced by repetitively occluding (10 min) and reperfusing (5 s) left coronary artery of anesthetized mice for 2 hr; 100 μL of 80 μM pHLIP were administered IV 3 min prior to the first occlusion. In the end, hearts were excised and retrogradely perfused with Evans Blue to delineate the area at risk. Hearts were frozen and sectioned to measure mean intensity of fluorescence (F). (2) Low flow (5% of normal flow rate) global ischemia was induced in Langendorff-perfused mouse hearts paced at 5 Hz and perfused with 1 μM pHLIP for 15 min. (3) Effects of 10 μM pHLIP on transmembrane potential and developed force were studied in isolated preparations of paced (4 Hz) murine left atrial appendages (AA) at normal and low (6.5) pH. RESULTS. In the model of local ischemia, when pH-sensitive Var7 was used, area of bright fluorescence coincided with the area at risk. F in the area at risk normalized to F in intact myocardium was higher with Var7 (3.89±.43, n=4) than with pH-insensitive kVar7 (1.11±.09, n=5, p.05). In global ischemia, F was higher in the hearts perfused with Var7 vs either kVar7 or Var7 in the absence of ischemia (Var7: 76±8, n=5; kVar7: 25±5, n=4; Var7 + no ischemia: 12±4, n=3, p<.05). In AA exposed to pH 6.5 (but not at pH 7.4), F was higher with Var7 than with kVar7 (76±9 vs 28±3, n=5; p<.05). Neither at the normal or low pH, pHLIP variants affected parameters of transmembrane potential or developed force. CONCLUSIONS. pH-sensitive pHLIP selectively binds to ischemic myocardium without affecting either ectrical or mechanical activity. Based on analogy with applications in oncology, pHLIP can be used for targeted delivery of contrasting agents and drugs to ischemic regions of the heart.

Modulation of the pHLIP Transmembrane Helix Insertion Pathway

The membrane-associated folding/unfolding of pH (low) insertion peptide (pHLIP) provides an opportunity to study how sequence variations influence the kinetics and pathway of peptide insertion into bilayers. Here, we present the results of steady-state and kinetics investigations of several pHLIP variants with different numbers of charged residues, with attached polar cargoes at the peptide's membrane-inserting end, and with three single-Trp variants placed at the beginning, middle, and end of the transmembrane helix. Each pHLIP variant exhibits a pH-dependent interaction with a lipid bilayer. Although the number of protonatable residues at the inserting end does not affect the ultimate formation of helical structure across a membrane, it correlates with the time for peptide insertion, the number of intermediate states on the folding pathway, and the rates of unfolding and exit. The presence of polar cargoes at the peptide's inserting end leads to the appearance of intermediate states on the insertion pathway. Cargo polarity correlates with a decrease of the insertion rate. We conclude that the existence of intermediate states on the folding and unfolding pathways is not mandatory and, in the simple case of a polypeptide with a noncharged and nonpolar inserting end, the folding and unfolding appears as an all-or-none transition. We propose a model for membrane-associated insertion/folding and exit/unfolding and discuss the importance of these observations for the design of new delivery agents for direct translocation of polar therapeutic and diagnostic cargo molecules across cellular membranes.

Roles of Carboxyl Groups in the Transmembrane Insertion of Peptides

We have used pHLIP® [ pH ( low) insertion peptide] to study the roles of carboxyl groups in transmembrane (TM) peptide insertion. pHLIP binds to the surface of a lipid bilayer as a disordered peptide at neutral pH; when the pH is lowered, it inserts across the membrane to form a TM helix. Peptide insertion is reversed when the pH is raised above the characteristic p K a (6.0). A key event that facilitates membrane insertion is the protonation of aspartic acid (Asp) and/or glutamic acid (Glu) residues, since their negatively charged side chains hinder membrane insertion at neutral pH. In order to gain mechanistic understanding, we studied the membrane insertion and exit of a series of pHLIP variants where the four Asp residues were sequentially mutated to nonacidic residues, including histidine (His). Our results show that the presence of His residues does not prevent the pH-dependent peptide membrane insertion at ∼ pH 4 driven by the protonation of carboxyl groups at the inserting end of the peptide. A further pH drop leads to the protonation of His residues in the TM part of the peptide, which induces peptide exit from the bilayer. We also find that the number of ionizable residues that undergo a change in protonation during membrane insertion correlates with the pH-dependent insertion into the lipid bilayer and exit from the lipid bilayer, and that cooperativity increases with their number. We expect that our understanding will be used to improve the targeting of acidic diseased tissue by pHLIP.

Correlation between Properties of pHLIP Peptide-Lipid Interaction and Tumor Targeting In Vivo

We have discovered a way to target acidity in vivo, which is a hallmark of many pathological states. The tumor targeting is based on the pH-dependent transmembrane insertion and folding of the water-soluble membrane peptide, pHLIP - pH (Low) Insertion Peptide. Here we present the result of the sequence variation study of pHLIP, which was carried out with the main goal to improve blood clearance and tumor targeting at low pH. We have investigated more than 10 pHLIP variants with various mutations in transmembrane and C-terminal parts, including peptides with significantly truncated transmembrane part. Currently our library of pHLIP variants, contains peptides inserting into bilayer with pKa ranging from 4.5 to 6.5, which have different affinity to lipid bilayer at neutral and low pHs. Kinetics measurements indicate that all investigated variants containing truncated or no C-terminal flanking sequence demonstrate fast insertion into lipid bilayer. The results of biophysical studies are in excellent agreement with the tumor targeting and blood clearance data obtained in vivo. Our data contribute in understanding of main principles of peptide-lipid interactions.