Chemotherapy, radiation, and surgery are the conventional treatment modalities for cancer. The success achieved with these approaches has been limited due to several factors like chemoresistance to drugs, non-specificity leading to peripheral toxicity, and non-resectable tumors. To combat these problems, the concept of targeted therapy using immunotoxins was developed. Immunotoxins are chimeric proteins with a cell-selective ligand chemically linked or genetically fused to a toxin moiety and can target cancer cells overexpressing tumor-associated antigens, membrane receptors, or carbohydrate antigens. Ligands for these receptors or monoclonal antibodies or single chain variable fragments directed against these antigens are fused with bacterial or plant toxins and are made use of as immunotoxins. Pseudomonas exotoxin, anthrax toxin, and diphtheria toxin are the commonly used bacterial toxins. Ricin, saporin, gelonin, and poke weed antiviral protein are the plant toxins utilized in immunotoxin constructs. Several such fusion proteins are in clinical trials, and denileukin difitox is a FDA-approved fusion protein. In spite of the promise shown by bacterial- and plant toxin-based chimeric proteins, their clinical application is hampered by several factors like immunogenicity of the toxin moiety and non-specific toxicity leading to vascular leak syndrome. In order to overcome these problems, a novel generation of immunotoxins in which the cytotoxic moiety is an endogenous protein of human origin like proapoptotic protein or RNase has been developed. This review summarizes the advances in this new class of fusion protein and the future directions to be explored.

Humanized immunotoxins: A new generation of immunotoxins for targeted cancer therapy

Ribosome-inactivating proteins (RIPs) are EC3.2.32.22 N-glycosidases that recognize a universally conserved stem-loop structure in 23S/25S/28S rRNA, depurinating a single adenine (A4324 in rat) and irreversibly blocking protein translation, leading finally to cell death of intoxicated mammalian cells. Ricin, the plant RIP prototype that comprises a catalytic A subunit linked to a galactose-binding lectin B subunit to allow cell surface binding and toxin entry in most mammalian cells, shows a potency in the picomolar range. The most promising way to exploit plant RIPs as weapons against cancer cells is either by designing molecules in which the toxic domains are linked to selective tumor targeting domains or directly delivered as suicide genes for cancer gene therapy. Here, we will provide a comprehensive picture of plant RIPs and discuss successful designs and features of chimeric molecules having therapeutic potential.

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Ribosome-Inactivating Proteins: From Plant Defense to Tumor Attack

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1016/j.febslet.2010.04.008

Protein toxins from plants and bacteria: probes for intracellular transport and tools in medicine.

Protein toxins confer a defense against predation/grazing or a superior pathogenic competence upon the producing organism. Such toxins have been perfected through evolution in poisonous animals/plants and pathogenic bacteria. Over the past five decades, a lot of effort has been invested in studying their mechanism of action, the way they contribute to pathogenicity and in the development of antidotes that neutralize their action. In parallel, many research groups turned to explore the pharmaceutical potential of such toxins when they are used to efficiently impair essential cellular processes and/or damage the integrity of their target cells. The following review summarizes major advances in the field of toxin based therapeutics and offers a comprehensive description of the mode of action of each applied toxin.

Toxin-based therapeutic approaches.

Immunotoxins for leukemia

Delonix elata, Enicostemma axillare, Merremia tridentata, Mollugo cerviana and Solanum incanum are medicinal plants used in traditional Indian medicine for the treatment of various ailments. These plants were selected to evaluate their potential antibacterial activity. To determine antibacterial activity and phytochemicals in the crude extracts of five medicinal plants used in traditional Indian medicine for the treatment of various ailments like rheumatism, piles fever, skin diseases and snake bite. The antibacterial activity of organic solvent extracts of these plants were determined by disc diffusion and broth dilution techniques against grampositive bacterial strains (Bacillus subtilis, Staphylococcus aureus) and gram-negative bacterial strains (Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa). Results revealed that the chloroform and methanol extracts of D. elata and methanol extracts of M. cerviana exhibited significant antibacterial activity against gram-positive and gram-negative strains with minimum bactericidal concentration (MBC) ranging from 1.5 to 100 mg/ml. Methanol extracts of M. tridentata exhibited activity only against gram-positive bacterial strains with MBC ranging from 12.5 to 100 mg/ml. Extracts of E. axillare and S. incanum showed activity only against B. subtilis and were not bactericidal at 100 mg/ml. The most susceptible organism to the organic extracts from all the studied plants was B. subtilis and the most resistant organism was P. aeruginosa. The presence of phytochemicals such as alkaloids, tannins, triterpenoids, steroids and glycosides in the extracts of these plants supports their traditional uses as medicinal plants for the treatment of various ailments. The present study reveals potential use of these plants for developing new antibacterial compounds against pathogenic microorganisms. Key words: Antibacterial, Enicostemma axillare, Merremia tridentata, Mollugo cerviana, Solanum incanum

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Antibacterial activity of plants used in Indian herbal medicine

Targeted therapy of cancer using diphtheria toxin-derived immunotoxins

Modified DT–IL2 fusion toxin targeting uniquely IL2Rα expressing leukemia cell lines – Construction and characterization

The current treatment strategies, chemotherapy and radiation therapy being used for the management of cancer are deficient in targeted approach leading to treatment related toxicities and relapse. Contrarily, fusion toxins exhibit remarkable tumor specificity thus emerging as an alternative therapy for the treatment of cancer. Diphtheria toxin-HN-1 peptide (DT/HN-1) is a fusion toxin designed to target the head and neck squamous cell carcinoma (HNSCC). The aim of this study was to construct, characterize, and evaluate the cytotoxicity and specificity of DT/HN-1 fusion toxin against the HNSCC cells. The purified DT/HN-1 fusion toxin was characterized by SDS-PAGE and western blotting. Refolding of purified fusion toxins was monitored by fluorescence spectra and circular dichroism spectra. The activity of DT/HN-1 fusion toxin was demonstrated on various HNSCC cell lines by cell viability assay, cell proliferation assay, protein synthesis inhibition assay, apoptosis and cell cycle analysis. The fusion toxin DT/HN-1 demonstrated remarkably high degree of cytotoxicity specific to the HNSCC cells. The IC50 of DT/HN-1 fusion toxin was ~1 to 5 nM in all the three HNSCC cell lines. The percentage apoptotic cells in DT/HN-1 treated UMB-SCC-745 cells are 16% compared to 4% in untreated. To further demonstrate the specific toxicity of DT/HN-1 fusion toxin towards the HNSCC cells we constructed, characterized and evaluated the efficacy of DT protein. The DT protein coding for only a fragment of diphtheria toxin without its native receptor binding domain failed to exhibit any cytotoxicity on all the cell lines used in this study thus establishing the importance of a ligand in achieving targeted toxicity. To evaluate the translocation ability of HN-1 peptide, an additional construct DTΔT/HN-1 was constructed, characterized and evaluated for its cytotoxic activity. The fusion toxin DTΔT/HN-1 deficient of the translocation domain of diphtheria toxin showed no cytotoxicity on all the cell lines clearly indicating the inability of HN-1 peptide to translocate catalytic domain of the toxin into the cytosol.

Targeting head and neck squamous cell carcinoma using a novel fusion toxin-diphtheria toxin/HN-1

Fusion toxins are an emerging class of targeted therapeutics for the treatment of cancer. Diphtheria toxin-stem cell factor (DT-SCF) is one such novel fusion toxin designed to target malignancies expressing c-kit. Since, c-kit overexpression has been reported on many types of cancers, it appeared to be a reasonably good molecule to target. In the present study, we report construction, expression, purification, and characterization of DT-SCF. DT-SCF gene coding for 1-387 amino acids of diphtheria toxin, His-Ala linker, 2-141 amino acids of SCF was cloned into expression vector with C terminal His tag. The induced DT-SCF protein was exclusively expressed in insoluble fraction. Purification of DT-SCF was achieved by inclusion body isolation and metal affinity chromatography under denaturing and reducing conditions. Purified DT-SCF was renatured partially on-column by gradually reducing denaturant concentration followed by complete refolding through rapid dilution technique. Cell viability assay provided the evidence that DT-SCF is a potent cytotoxic agent selective to cells expressing c-kit. The novelty of this study lies in employing SCF as a ligand in construction of fusion toxin to target wide range of malignancies expressing c-kit. Efficacy of DT-SCF fusion toxin was demonstrated over a range of malignancies such as chronic myeloid leukemia (K562), acute lymphoblastic leukemia (MOLT4), pancreatic carcinoma (PANC-1), and cervical carcinoma (HeLa 229). This is the first study reporting specificity and efficacy of DT-SCF against tumor cells expressing c-kit. There was significant correlation (P = 0.007) between c-kit expression on cells and their sensitivity to DT-SCF fusion toxin.

Sirisha Potala

Papers

Antibacterial activity of plants used in Indian herbal medicine

Targeted therapy of cancer using diphtheria toxin-derived immunotoxins

Modified DT–IL2 fusion toxin targeting uniquely IL2Rα expressing leukemia cell lines – Construction and characterization

Targeting head and neck squamous cell carcinoma using a novel fusion toxin-diphtheria toxin/HN-1

A Novel Fusion Protein Diphtheria Toxin–Stem Cell Factor (DT-SCF)—Purification and Characterization