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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., (© 2017 Wiley Periodicals, Inc.)

Cyclotides are disulfide rich macrocyclic plant peptides that are defined by their unique topology in which a head-to-tail cyclized backbone is knotted by the interlocking arrangement of three disulfide bonds. This cyclic cystine knot motif gives the cyclotides exceptional resistance to thermal, chemical, or enzymatic degradation. Over 100 cyclotides have been reported and display a variety of biological activities, including a cytoprotective effect against HIV infected cells. It has been hypothesized that cyclotides from one subfamily, the Möbius subfamily, may be more appropriate than bracelet cyclotides as drug candidates given their lower toxicity to uninfected cells. Here, we report the anti-HIV and cytotoxic effects of three cyclotides, including two from the Möbius subfamily. We show that Möbius cyclotides have comparable inhibitory activity against HIV infection to bracelet cyclotides and that they are generally less cytotoxic to the target cells. To explore the structure activity relationships (SARs) of the 29 cyclotides tested so far for anti-HIV activity, we modeled the structures of the 21 cyclotides whose structures have not been previously solved. We show that within cyclotide subfamilies there is a correlation between hydrophobicity of certain loop regions and HIV inhibition. We also show that charged residues in these loops impact on the activity of the cyclotides, presumably by modulating membrane binding. In addition to providing new SAR data, this report is a mini-review that collates all cyclotide anti-HIV information reported so far and provides a resource for future studies on the therapeutic potential of cyclotides as natural anti-HIV agents., ((c) 2007 Wiley Periodicals, Inc.)

The cyclotides are a recently discovered family of miniproteins that contain a head-to-tail cyclized backbone and a knotted arrangement of disulfide bonds. They are approximately 30 amino acids in size and are present in high abundance in plants from the Violaceae, Rubiaceae, and Cucurbitaceae families, with individual plants containing a suite of up to 100 cyclotides. They have a diverse range of biological activities, including uterotonic, anti-HIV, antitumor, and antimicrobial activities, although their natural function is likely that of defending their host plants from pathogens and pests. This review focuses on the structural aspects of cyclotides, which may be thought of as a natural combinatorial peptide template in which a wide range of amino acids is displayed on a compact molecular core made up of the cyclic cystine knot structural motif. Cyclotides are exceptionally stable and are resistant to denaturation via thermal, chemical, or enzymatic treatments. The structural features that contribute to their remarkable stability are described in this review., (Copyright 2006 Wiley Periodicals, Inc.)