Chlorotoxin is the key toxin found in the venom of Leiurus quinquestriatus. The 36-amino acid long peptide weighing 3,996 Da contains four disulfide bridges, as well as a single tyrosine residue. The presence of the four disulfide bridges, made from eight cysteine residues, gives chlorotoxin its highly folded and condensed structure, allowing the peptide to be delivered via the narrow extracellular matrix (Lippens et al., 1995). The tyrosine residue allows for radio-labelling using iodine isotopes (Mamelak et al., 2007).
Mode of Action
Chlorotoxin is the first ligand reported to inhibit the chloride ion channel. The binding was found to follow first-order binding kinetics, with a single chlorotoxin molecule being sufficient to block one chloride ion channel with high affinity (DeBin et al., 1991).
Chlorotoxin was also found to bind selectively to matrix metalloproteinase-2 (MMP-2), the expression of which is particularly elevated in tumour cells, including those of gliomas, primary prostate carcinoma, and rhabdomyosarcoma (Veiseh et al., 2007). Upon binding to the enzyme, the chlorotoxin-MMP-2 complex is endocytosed into the tumour cell, leading to the inhibition of enzymatic activity and reduction in the surface expression of MMP-2 (Deshane, et al., 2002). Additionally, metastasis is disrupted due to the reduction in MMP-2 expression, as MMP-2 is required to break through the extracellular matrix during tumour cell invasion (University of Washington, Apr. 2009).
Furthermore, by combining chlorotoxin with nanoparticles, it was found that anti-cancer therapy could be improved. While the attachment of nanoparticles allowed chlorotoxin to remain active for longer in the body, increasing its probability of reaching the tumour cell, the combination also led to the formation of chlorotoxin clusters around each nanoparticle. As an average of ten chlorotoxin molecules can congregate in one such cluster, numerous MMP-2 proteins can be inactivated simultaneously (University of Washington, Apr. 2009).
Potential therapeutic use
Due to chlorotoxin’s unique ability to target tumour cells while showing no binding to healthy cells, it is currently being studied as a potential treatment and means to identify gliomas, the most widespread of brain tumours. One such study, conducted by researchers at TransMolecular, Inc. (Cambridge, Massachusetts), successfully attached the radioactive iodine-131 isotope to synthetic chlorotoxin, TM-601. When the radio-labelled peptide was injected into the bloodstream, it was found to bind directly to tumour cells. Once bonded, the radiation destroyed the tumour cell without harming any surrounding healthy cells (Wu et al., 2010). The tumour-targeting drug received FDA approval in 2002 and was granted Orphan Drug Designation in 2008.
A novel method of detecting tumours with chlorotoxin is Tumour Paint, a CTX:Cy5.5 biconjugate consisting of chlorotoxin attached to the fluorescent dye, Cy5.5. The process of accurately identifying tumours is often an arduous one, relying on subtle visual cues, such as colour and texture (Veiseh, et al., 2007). By attaching the fluorescent dye to chlorotoxin, tumours can be illuminated, thus facilitating their localisation in clinical practice. The new visualisation technique was not only found to increase the contrast between tumour tissue and healthy tissue, but also found to improve the resolution significantly, thus that smaller tumours could be detected in earlier treatments (University of Washington, Aug. 2009).