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

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.

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).