Lysine to arginine mutagenesis of chlorotoxin enhances its cellular uptake

Abstract

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.

Peptides to Tackle Leishmaniasis: Current Status and Future Directions

“…The latter peptide had stronger activity than the all-lysine one against both promastigotes and amastigotes of the different species/strains of Leishmania parasites tested (27 to 71 µM for promastigotes; 16 to 18 µM for amastigotes); moreover, p–Ac1R7 displayed a faster parasite membrane-permeation action than p–Ac1 [ 42 ]. These observations are in line with studies involving antimicrobial and anticancer peptides that have shown a greater capacity to penetrate cells when having a high density of arginine residues, as compared to analogs of the same length containing lysines instead [ 85 , 86 ].…”

Cell-Penetrating Peptides Derived from Animal Venoms and Toxins

“…Accordingly, cellular uptake of TM601 in U373 glioma cells was a rapid concentration-and time-dependent process, which was affected by chlorpromazine, a pharmacological inhibitor of clathrin-mediated endocytosis, involved in the transport of coated pits. Interestingly, as in the case of lycosin-I from spider venom, the replacement of Lys with Arg in the CTX sequence, as observed with the synthetic analogs [K15R/K23R]CTX and [K15R/K23R/Y29W], enhanced its cellular uptake, as demonstrated in human cervical carcinoma (HeLa) cells with fluorescent derivatives [95].…”

A C-Terminal Fragment of Chlorotoxin Retains Bioactivity and Inhibits Cell Migration

“…However, given that CTX-3 binds to both a tumor cell line and a normal cell line, in contrast to previous studies on CTX, it appears likely that this region is not sufficient by itself to account for the selective binding to tumor cells. It is possible the cell surface binding observed for CTX-3 is non-specific membrane binding mediated by the presence of the high proportion of positively charged (two lysine and one arginine) residues within a seven-residue peptide as reported for other highly charged peptides (Futaki et al., 2007; Ojeda et al., 2017). Although no cell surface binding was observed for CTX-4, it was the only fragment that appeared to be specifically internalized into the glioma cells at 37°C.…”