Dhammika Weerakkody

Family of pH (low) insertion peptides for tumor targeting

Cancer is a complex disease with a range of genetic and biochemical markers within and among tumors, but a general tumor characteristic is extracellular acidity, which is associated with tumor growth and development. Acidosis could be a universal marker for cancer imaging and the delivery of therapeutic molecules, but its promise as a cancer biomarker has not been fully realized in the clinic. We have discovered a unique approach for the targeting of acidic tissue using the pH-sensitive folding and transmembrane insertion of pH (low) insertion peptide (pHLIP). The essence of the molecular mechanism has been elucidated, but the principles of design need to be understood for optimal clinical applications. Here, we report on a library of 16 rationally designed pHLIP variants. We show how the tuning of the biophysical properties of peptide–lipid bilayer interactions alters tumor targeting, distribution in organs, and blood clearance. Lead compounds for PET/single photon emission computed tomography and fluorescence imaging/MRI were identified, and targeting specificity was shown by use of noninserting variants. Finally, we present our current understanding of the main principles of pHLIP design.

pH (low) insertion peptide (pHLIP) inserts across a lipid bilayer as a helix and exits by a different path

What are the molecular events that occur when a peptide inserts across a membrane or exits from it? Using the pH-triggered insertion of the pH low insertion peptide to enable kinetic analysis, we show that insertion occurs in several steps, with rapid (0.1 sec) interfacial helix formation, followed by a much slower (100 sec) insertion pathway to give a transmembrane helix. The reverse process of unfolding and peptide exit from the bilayer core, which can be induced by a rapid rise of the pH from acidic to basic, proceeds approximately 400 times faster than folding/insertion and through different intermediate states. In the exit pathway, the helix–coil transition is initiated while the polypeptide is still inside the membrane. The peptide starts to exit when about 30% of the helix is unfolded, and continues a rapid exit as it unfolds inside the membrane. These insights may guide understanding of membrane protein folding/unfolding and the design of medically useful peptides for imaging and drug delivery.

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 peptides 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 peptides 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 pK(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.

Novel pH-Sensitive Cyclic Peptides

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)4WC], and asymmetric, c[E4W5C], 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[E4W5C], at 24 hours after intravenous administration. pH-insensitive cyclic peptide c[R4W5C], 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.

Bilayer Thickness and Curvature Influence Binding and Insertion of a pHLIP Peptide

The physical properties of lipid bilayers, such as curvature and fluidity, can affect the interactions of polypeptides with membranes, influencing biological events. Additionally, given the growing interest in peptide-based therapeutics, understanding the influence of membrane properties on membrane-associated peptides has potential utility. pH low insertion peptides (pHLIPs) are a family of water-soluble peptides that can insert across cell membranes in a pH-dependent manner, enabling the use of pH to follow peptide-lipid interactions. Here we study pHLIP interactions with liposomes varying in size and composition, to determine the influence of several key membrane physical properties. We find that pHLIP binding to bilayer surfaces at neutral pH is governed by the ease of access to the membranes hydrophobic core, which can be facilitated by membrane curvature, thickness, and the cholesterol content of the membrane. After surface binding, if the pH is lowered, the kinetics of pHLIP folding to form a helix and subsequent insertion across the membrane depends on the fluidity and energetic dynamics of the membrane. We showed that pHLIP is capable of forming a helix across lipid bilayers of different thicknesses at low pH. However, the kinetics of the slow phase of insertion corresponding to the translocation of C-terminal end of the peptide across lipid bilayer, vary approximately twofold, and correlate with bilayer thickness and fluidity. Although these influences are not large, local curvature variations in membranes of different fluidity could selectively influence surface binding in mixed cell populations.

Molecular Mechanism of Polypeptide Insertion into Bilayer and Exit

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.

Insertion into lipid bilayer of truncated pHLIP®peptide

The investigation of pH-dependent membrane-associated folding has both fundamental interest and practical applications for targeting of acidic tumors and specific delivery of therapeutic molecules across membrane of cancer cells. We and others investigated molecular mechanism and medical uses of class of water soluble membrane peptides, pH (Low) Insertion Peptides (pHLIP® peptides). Here we employed optical spectroscopy methods to study interactions of the truncated pHLIP® peptide (Short pHLIP®) with lipid bilayer of membrane. Tryptophan fluorescence, CD and OCD data indicate on pH-triggered formation of transmembrane helical structure. Dual quenching and FRET assays demonstrated that Short pHLIP® peptide spans lipid bilayer of membrane similar to Long pHLIP® peptides. Truncated pHLIP® peptides with multiple charged and protonatable residues in their sequences potentially can make these peptides to be less hydrophobic compared to Long pHLIP® peptides, and might have utility in tumor imaging, and potentially, in pH-regulated cytoplasmic delivery of moderately hydrophobic drugs.

Abstract C2: Aspartic acid residues drive the membrane translocation of pHLIP, a therapeutic and imaging agent for solid tumors

The pHLIP peptide is a new therapeutic and imaging agent for cancer treatment. The pHLIP (pH-Low-Insertion Peptide) is soluble in solution but interestingly, it is able to interact with the plasma membrane and insert as a monomeric transmembrane helix (pKa=6.0). The insertion requires an acidic extracellular environment, such as that found in most solid tumors and inflammation sites. The pHLIP, which is nontoxic in mice, is specifically distributed to tumors (with minor labeling of kidney), as imaged by PET and near-infrared fluorescence. The insertion of pHLIP is unidirectional, as the C-terminus is translocated across the membrane, while the N-terminus remains exposed to the extracellular medium. The insertion energy can be employed to translocate into the cytoplasm membrane-impermeable molecules. As a proof of principle, we showed that the polar toxin phalloidin (which binds to F-actin, inhibiting its depolymerization) is translocated in a pH-dependent fashion into HeLa, JC, and M4A4 cancer cells, inhibiting cell growth and causing cytoeskeletal immobilization and multinucleation. Here we study the molecular determinants of pHLIP membrane insertion. The pH-mediated translocation is thought to be triggered by the protonation (loss of negative charge) of carboxyl groups present in pHLIP. Accordingly, the four aspartic acid (Asp) residues of pHLIP were sequentially mutated, and the efficacy of the membrane insertion and exit was studied in a liposome system. We found a correlation between the number and location of Asp with the peptides ability in insert into and exit from the membrane in a pH-dependent manner. We also observed that the number of Asp in the peptide determines the insertion properties (pKa and the cooperativity), suggesting that the tumor labeling properties of pHLIP can be specifically tuned to better match the physiological acidity of solid tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr C2.

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.