Noncanonical Amino Acids to Improve the pH Response of pHLIP Insertion at Tumor Acidity

Abstract

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.

pH-Selective Cytotoxicity of pHLIP-Antimicrobial Peptide Conjugates

“…Remarkably, unlike other drug delivery system strategies, pHLIP alone can target tumors, carry cargo and translocate the payload across the plasma membrane without relying on cell receptor interactions or by the formation of pores within the membrane1920. pHLIP has been shown to translocate a plethora of cargo molecules across the plasma membrane into the cytoplasm of cancer cells212223242526, and to target solid tumors as well as metastasis foci in mice26272829. We have also recently shown that pHLIP can target the potent antimitotic monomethyl auristatin E to breast cancer xenographs in mice23.…”

Design, synthesis and evaluation of anti-CD123 antibody drug conjugates

“…In order to examine the cleavage of disulfide linker on CD123-CPT conjugate, we incubated CD123-CPT with GSH, the most abundant thiol compound in cells, 21,22 since previous studies reported that GSH was able to react with the disulfide linker. 23 After 4 h incubation, the corresponding molecular peak found in the mass spectrum (Fig. S3A) indicated that CPT was successfully released from CD123-CPT conjugates owing to the disulfide linker cleavage.…”

“…23 50 μL of the CD123-CPT (11.8 μM) was incubated with 97.5 μL of GSH (18.15 mM in DPBS) at the final concentration of CD123-CPT and GSH is 4 μM and 12 mM. Four hours later, the MW of the mixture was measured with ESI-MS (LTQ Orbitrap, Thermo Fisher Scientific, USA).…”

Organic Nanocarriers for Delivery and Targeting of Therapeutic Agents for Cancer Treatment

“…They also showed that the replacement of Asp14 with ⊥ ‐carboxyglutamic acid (Gla) increased the sharpness of pH response and the Asp14Gla/Asp25Aad double variant showed pH of 6.79. Additionally, they performed turn‐on fluorescence assays and anti‐proliferation studies using fluorescence labeled and Taxol conjugated Asp14Gla/Asp25Aad variant respectively and showed that the double variant had better cargo delivery capability than WT pHLIP in cancer cells at pH 6.6 . Weerakkody et al.…”