Antiproliferative effect of pHLIP-amanitin

Biochemistry. 2013 Feb 19;52(7):1171-8. doi: 10.1021/bi301647y. Epub 2013 Feb 8.


Toxins could be effective anticancer drugs, if their selective delivery into cancer cells could be achieved. We have shown that the energy of membrane-associated folding of water-soluble membrane peptides of the pHLIP (pH low insertion peptide) family could be used to move cell-impermeable cargo across the lipid bilayer into the cytoplasm of cancer cells. Here we present the results of a study of pHLIP-mediated cellular delivery of a polar cell-impermeable toxin, α-amanitin, an inhibitor of RNA polymerase II. We show that pHLIP can deliver α-amanitin into cells in a pH-dependent fashion and induce cell death within 48 h. Translocation capability could be tuned by conjugating amanitin to the C-terminus of pHLIP via linkers of different hydrophobicities that could be cleaved in the cytoplasm. pHLIP-SPDP-amanitin, which exhibits 4-5 times higher antiproliferative ability at pH 6 than at pH 7.4, was selected as the best construct. The major mechanism of amanitin delivery is direct translocation (flip) across a membrane by pHLIP and cleavage of the S-S bond in the cytoplasm. The antiproliferative effect was monitored on four different human cancer cell lines. pHLIP-mediated cytoplasmic delivery of amanitin could create great opportunities to use the toxin as a potent pH-selective anticancer agent, which predominantly targets highly proliferative cancer cells at low extracellular pH values.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alpha-Amanitin / chemistry
  • Alpha-Amanitin / pharmacology*
  • Amino Acid Sequence
  • Antineoplastic Agents / administration & dosage
  • Antineoplastic Agents / chemistry
  • Antineoplastic Agents / pharmacology*
  • Cell Death / drug effects
  • Cell Line, Tumor
  • Cell Membrane / chemistry
  • Cell Proliferation / drug effects
  • Drug Carriers / chemistry
  • Drug Carriers / pharmacology*
  • Drug Delivery Systems / methods
  • Drug Screening Assays, Antitumor
  • HeLa Cells / drug effects
  • Humans
  • Hydrogen-Ion Concentration
  • Hydrophobic and Hydrophilic Interactions
  • Kinetics
  • Membrane Proteins / chemistry*
  • Membrane Proteins / pharmacology
  • Molecular Sequence Data
  • Protein Transport


  • Alpha-Amanitin
  • Antineoplastic Agents
  • Drug Carriers
  • Membrane Proteins
  • pHLIP protein