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Lysine to arginine mutagenesis of chlorotoxin enhances its cellular uptake
Author(s) -
Ojeda Paola G.,
Henriques Sónia Troeira,
Pan Yijun,
Nicolazzo Joseph A.,
Craik David J.,
Wang Conan K.
Publication year - 2017
Publication title -
peptide science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.556
H-Index - 125
eISSN - 1097-0282
pISSN - 0006-3525
DOI - 10.1002/bip.23025
Subject(s) - chemistry , hela , peptide , mutant , internalization , cell penetrating peptide , biochemistry , protein engineering , cell , arginine , cancer cell , microbiology and biotechnology , amino acid , biology , cancer , gene , genetics , enzyme
Chlorotoxin (CTX), a disulfide‐rich peptide from the scorpion Leiurus quinquestriatus , has several promising biopharmaceutical properties, including preferential affinity for certain cancer cells, high serum stability, and cell penetration. These properties underpin its potential for use as a drug design scaffold, especially for the treatment of cancer; indeed, several analogs of CTX have reached clinical trials. Here, we focus on its ability to internalize into cells—a trait associated with a privileged subclass of peptides called cell‐penetrating peptides—and whether it can be improved through conservative substitutions. Mutants of CTX were made using solid‐phase peptide synthesis and internalization into human cervical carcinoma (HeLa) cells was monitored by fluorescence and confocal microscopy. CTX_M1 (ie, [K15R/K23R]CTX) and CTX_M2 (ie, [K15R/K23R/Y29W]CTX) mutants showed at least a twofold improvement in uptake compared to CTX. We further showed that these mutants internalize into HeLa cells largely via an energy‐dependent mechanism. Importantly, the mutants have high stability, remaining intact in serum for over 24 h; thus, retaining the characteristic stability of their parent peptide. Overall, we have shown that simple conservative substitutions can enhance the cellular uptake of CTX, suggesting that such type of mutations might be useful for improving uptake of other peptide toxins.