Towards PHLIP Insertion in the Plasmamembrane of Cancer Cells at Physiological Tumor Acidity

Abstract

The pH-(Low) Insertion Peptide (pHLIP) can target cancer cells based on their low extracellular pH (pHe). Under slightly acidic conditions, pHLIP inserts into membrane, forming a transmembrane (TM) helix. When cargo is attached to pHLIP's C-terminus, it is carried across the membrane during pHLIP insertion. Therefore, pHLIP is also a drug carrier that can deliver cargo directly into the cell cytoplasm. Imaging studies have shown that pHLIP can target acidic tumors in mice (and the signal stayed in the tumor for more than a week). We ask whether such targeting and long residence time arise from pHLIP insertion into the plasmamembrane of cancer cells or preferential binding to cell surface without TM insertion (followed by endocytosis and insertion in the endosomal membrane). To quantify pHLIP insertion in cells, we developed fluorescently self-quenched pHLIP probes that upon interactions with cells become more fluorescent. The turn-on fluorescence (i.e. dequenching) results from cleavage of a C-terminal disulfide bond that would only occur in cells. Our data suggest that even at pH 6.2 (which is more acidic than tumor pHe), pHLIP insertion is highly cell line dependent. When the experiment was carried out at 4°C instead of 37°C, about 50% of the gain in fluorescence signal is lost, suggesting at least half of pHLIP insertion occurred in endosomes. Further, compared to the ‘WT’ pHLIP, the D25E pHLIP variant inserts more readily at plasmamembrane. Using Structure-Activity-Relationship (SAR) studies (i.e. by changing pHLIP sequence and/or side-chain structures), we searched for new pHLIP variants that would (a) insert in plasmamembrane more readily at the tumor acidity of pH 6.5-7.0, and (b) with insertion occurring over a narrower pH range. The results of these SAR efforts will also be discussed.