Cl Channel Inhibitors: Chlorotoxin

Introduction of CTX

CTX was first isolated from the venom of the Israeli scorpion Leiurus quinquestriatus in 1993. It was initially considered to be a chloride channel inhibitor. It holds great promise for the treatment of glioblastoma multiforme (GBM) and other solid tumors.

The primary structure of CTX consists of 36 amino acids, which is maintained by four disulfide bonds that connect the eight cysteine residues present in the sequence. So CTX is classified as a short-chain, disulfide-containing peptide. The secondary structure of CTX comprises an α-helix and three β-sheets. Scorpion toxins are considered promising cancer drug candidates, mainly due to the discovery of CTX, which can preferentially bind to cancer cells.

The amount of CTX present in venom is limited. Fortunately, CTX has been successfully synthesized and folded in vitro, using classical molecular biology laboratory techniques and a solid-phase peptide synthesis (SPPS).

Fig.1 Amino acid sequence and 3D representation of CTX. (Dardevet, 2015)

Biological Activities of CTX

• Insecticidal Activity

CTX was named for its selective paralytic activity on insects and other invertebrates. For example, in crayfish, CTX caused a failure of motor control, which progressed to rigid paralysis. These paralytic effects are also observed in American cockroaches, and the degree of paralysis has been shown to depend on the dosage of a peptide.

• Tumor Binding Activity

One of the clinical effects of CTX is tumor binding activity. In the previous study, ¹²⁵I-labeled peptide (¹²⁵I-CTX) was bound to glioma cells but was unable to bind normal rat astrocytes and Te671, a human rhabdomyosarcoma cell line. CTX was also found to bind tumors of neuroectodermal origin, and unable to bind samples that presented other, non-tumorigenic, neurological diseases. The specific binding of CTX to tumors has been further confirmed in vivo by using chlorotoxin conjugated to a fluorescent dye.

• Antiangiogenic Activity

CTX also displays antiangiogenic properties. Using a Transwell migration assay, the migration of U251MG (glioma) cells and human umbilical vein endothelial cells (HUVECs) were inhibited by CTX. Moreover, CTX (100 μM) administration did not show any toxic effects, suggesting that inhibition of migration of HUVECs was due to a mechanism other than cell death.

Therapeutic Applications of CTX

CTX has been widely used as a Cl channel blocker to analyze Cl channels and has been considered as a potential specific marker for the diagnosis and treatment of glioma.

• Targeted Imaging

Many applications of CTX have been proposed. One of the most promising applications is in optical imaging. “Tumor paint” - a fluorescent molecular probe (CTX: Cy5.5) is an example. This probe was proven to be the most sensitive probe for intraoperative visualization of GBM foci. Molecular-imaging techniques have integrated venom-based moieties into numerous applications for tumor visualization and/or delivery of therapeutic anti-tumor agents. Labeling these agents with potential optical, magnetic resonance, or nuclear medicine imaging moieties allows for validation of the therapeutic profile in vivo along with directly determining the bio-distribution of the material.

Many applications have used CTX-based strategies for developing novel therapeutic and diagnostic techniques for detecting glioma therapeutic anti-tumor agents. The CTX-based nuclear imaging, CTX-based MRI/Optical imaging, and US/Optoacoustic imaging of Glioma are examples.

• Act as Therapeutic Targeting Peptides

CTX is also used as a targeting peptide to precisely deliver a therapeutic agent or radionuclides. It serves as an optimal “magic bullet”, specifically targeting the GBM cells while posing no danger to normal cells of the brain or the body. Using its cancer-targeting properties, many applications have emerged, although its mechanism of action is far from being fully elucidated. In addition, it has been used to enhance the efficacy of already existing anti-tumor molecules and conjugated to active drugs to create new, more effective drugs.

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Reference

1. Dardevet, L.; et al. Chlorotoxin: a helpful natural scorpion peptide to diagnose glioma and fight tumor invasion.Toxins (Basel). 2015, 7: 1079-101.