The pH-Low Insertion Peptide (pHLIP) offers the potential to deliver drugs selectively to the cytoplasm of cancer cells based on tumor acidosis. The WT pHLIP inserts into membrane with a pH50 of 6.1 while most solid tumors have extracellular pH (pHe) of 6.5-7.0. To close this gap, a SAR study was carried out to search for pHLIP variants with improved pH-response. We learned that (a) replacing Asp25 with α-aminoadipic acid (Aad) adjusts the pH50 to 6.74, matching average tumor acidity, and (b) replacing Asp14 with γ-carboxyglutamic acid (Gla) increases the sharpness of pH-response (i.e. transition over 0.5 instead of 1 pH unit). These effects are additive — the Asp14Gla/Asp25Aad double variant shows a pH50 of 6.79, with sharper transition than Asp25Aad. Further, the advantage of the double variant over WT pHLIP in terms of cargo delivery was demonstrated in turn-on fluorescence assays and anti-proliferation studies (using paclitaxel as cargo) in A549 lung cancer cells at pH 6.6.

The pH-Low Insertion Peptide (pHLIP) offers the potential to deliver drugs selectively to the cytoplasm of cancer cells based on tumor acidosis. The WT pHLIP inserts into membrane with a pH 50 of 6.1 while most solid tumors have extracellular pH (pH e ) of 6.5-7.0. To close this gap, a ** We thank Prof. Dr. Donald M. Engelman (Yale) for discussion and support; Prof. Dr. Eriks Rozners and Prof. Dr. Susan Bane (both of SUNY-Binghamton) for frequent use of their fluorimeter and plate reader, respectively. We also thank Raemer J. Lapid (for assistance in processing data), Rebecca A. Chandler, Meghan M. Bell, Vladyslav Nazarenko, Ilana G. Bandler (for assistance in cell assays) and Emma A. Gordon (for assistance in synthesis). This work was supported by SUNY-Binghamton University (BU) (Start-up funds to M.A. and L.Y., various financial supports to J.O., L.K. and M.C., HHMI-BU undergraduate summer fellowships to C-H. E. and R. L.), NIH (R01-GM073857 for training of M.A. and initial work), and NSF grant CHE-0922815 for the Regional NMR Facility (600 MHz instrument) at SUNY-Binghamton.

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