pH-(Low) Insertion Peptides (pHLIPs)
The Engelman Lab and colleagues are pursuing applications of the novel pH-responsive transmembrane peptides, pHLIPs, for translational and basic research applications in membrane biophysics and medicine.
pH Dependence of pHLIPs' Membrane Insertion Activity
The discovery of pHLIPs is an example illustrating how basic research can lead to translational innovations. In the 1990s, the Engelman lab was investigating transmembrane peptide interactions using a seven-helix membrane-spanning protein, Bacteriorhodopsin. When one helix, isolated from the protein, failed to form a transmembrane helix, the team noted that its transmembrane domain contains two aspartic acid residues, which would bear negative charges near neutral pH. As hoped, when the reaction conditions were made acidic, these residues became protonated and the peptide formed a transmembrane helix (Hunt, et al. 1997). With the realization that the pH at which this transition occurred coincided with the acidity of cancerous tumors, the bacteriorhodopsin C-helix became the basis of what would become an important new tool for targeting such pathologies in vivo.
"Wild-type" pHLIP originally derived from the C-helix of bacteriorhodopsin, contains polar ends and a central transmembrane domain, containing two aspartic acid residues, which impart its pH-dependent activity.
pHLIPs exhibit three distinct states in vivo:
State I - pHLIPs are largely unstructured as soluble monomers or low-order multimers in aqueous solution
State II - In the presence of membranes, pHLIPs remain largely unstructured at neutral and basic pHand bind reversibly to the outer leaflet of the membrane as monomers
State III - In acidic conditions (below pH ~6) pHLIPs form stable, monomeric transmembrane alpha-helixes, inserting their C-termini into the lumen of liposomes or into the cytosol of cells
pHLIPs Target Acidic Pathologies
At normal physiological pH, ~7.4, pHLIPs exist in States I and II, interacting with the outside surface of cells but exchanging with the aqueous surroundings. A number of pathological conditions produce a more acidic extracellular environment due to the affects of hypoxia, ischemia, or abnormal metabolic processes. A significant example of such acidity is in solid tumors. Due to their heightened metabolic activity, their compromised blood supply, membrane bound carbonic anhydrase activity, and to the Warburg effect, cancerous tumors produce a significantly acidic extracellular environment of around pH 6. In a serendipitous coincidence, this pH is sufficiently low to protonate the aspartic acid residues in pHLIPs' transmembrane domain, causing pHLIPs to insert into cells, where they are relatively stable and thereby accumulate in the cells of acidic tumors.
Applications of pHLIP Targeting
pHLIP insertion occurs directionally and with a favorable Gibbs free energy change. Therefore, cargos associated with pHLIPs' N-terminus will be localized to the targeted tissues and upon insertion will decorate the cell surface. Such surface binding has been used to localize imaging agents, such as fluorophores (Andreev, et al. 2007, Segala, et al. 2009, Reshetnyak, et al. 2011), nanogold particles (Yao, et al. 2013), and PET or SPECT tracers (Vavere, et al. 2009, Macholl, et al. 2012, Emmetiere, et al. 2013), as well as to deliver liposomes loaded with therapeutic cargos (Wijesinghe, et al. 2013, Yao, et al. 2013) to the extracellular environment of tumors, in vivo.
The insertion of the C-terminus occurs favorably enough to facilitate the delivery of large molecular cargos bound to the C-termini of pHLIPs. Using biologically labile reversible linkages, such as disulfide bonds between cysteine residues in the pHLIP C-terminus and thiols in the cargo, cytosolic delivery has been achieved for a number of cargoes, including peptides, small molecules, and even large peptide-nucleic acids. These approaches represent a new mode of targeted drug-delivery, directly into the cytosol of the targeted cells without the use of disruptive carriers, such as cell-penetrating peptides (CPPs).
Intracellular Delivery of Therapeutic Cargos
Lateral association of
Membrane protein folding and oligomerization often involve interactions between lipid solvated monomeric alpha-helices, and further associations of the helices into higher order states. The two states of the simplest example of this equilibrium, monomers and a homodimer, are cartooned below.
The formation of the dimer of helices results in an increase of helix-helix and
lipid-lipid interactions and a loss of helix-lipid interactions. The entropy of
the lipids is expected to increase, as depicted by the blue lipids released upon
dimerization; the entropy of the helices is expected to decrease. The value of
the equilibrium constant will depend on the magnitudes of these entropic terms
and on the enthalpic terms that arise from the detailed helix-helix, helix-lipid
and lipid-lipid contacts. Further, chnges in helix associations, for example by ratations to a different helix interface, may be a part of receptor signaling, channel gating, or other functions in membranes.
Therefore, we have worked to define features and energies that arise in helix interactions, including packing, hydrogen bonding, and lipid effects. We have found motifs, such as GxxxG, developed methods, such as TOXCAT and GALLEX, and exploited computational modleing. We continue to use a range of biophysical and biochemical methods in our studies.
Selected Current Research
Ongoing work in the lab is focusing on multiple applications of the pHLIP delivery platform:
pHLIP Targeting of Acidic Tissues
Work continues using Alexa Fluor-conjugated pHLIPs to investigate additional pathological and physiological targets of pHLIP, in vivo. By expanding the potential applications of pHLIP in health and disease, we hope to find new ways for pHLIPs to contribute to human health. Imaging experiments using PET are also under way, in collaborations at Sloan Kettering and the University of Rhode Island.
pHLIP-FIRE (Fluorescent Insertion REporter)
Taking advantage of the reducing environment inside of the cell, we are working towards improving imaging contrast by delivering quenched imaging probes on the pHLIP C-terminus, where they can be inserted into targeted cells and their fluorescence activated by the reduction of disulfide bond. Work in this area is in collaboration with the laboratories of Reschetnyak and Andreev at the University of Rhode Island.
pHLIP-DIRECT (Drug Insertion and Reductive Escape in the Cytosol of Tumors)
Traditional chemotherapeutic treatments, while vital to the fight against cancers, carry with them serious side effects due to drug uptake by healthy cells. We are using pHLIPs' low-pH-selective insertion properties to impart tumor specificity on traditional chemotherapeutic agents in an attempt to improve the efficacy and decrease the side effects of chemotherapy. Additionally, pHLIP is able to translocate normally cell-impermeable cargos into cells, even large and polar molecules such as peptide nucleic acids. Ongoing studies are investigating the delivery of non-traditional therapeutic molecules, such as oncogenic microRNA-targeted peptide nucleic acid therapies in several mouse models of cancer. Work in this area is also in collaboration with the Rhode Island groups.
Computational Simulations of Transmembrane Peptide Properties
In collaboration with the DiMaio lab at the Yale School of Medicine and others, the Engelman lab is investigating the interactions between the transmembrane domains of signaling proteins using Molecular Dynamics simulations and Rosetta Membrane modeling. We also aim to apply these technologies toward further investigations of pHLIP insertion activity.