Natural protein toxins can be divided into plant, bacterial and fungal protein toxin families based on their origin. Among them, plant protein toxins include toxins with double-stranded structures and single-stranded ribosome inactivating proteins. It generally has N-glycosidase activity and can catalyze the depurination of the conserved site of ribosomal 28S RNA, thereby inactivating the ribosomal 60S subunit. Bacterial protein toxins have structural diversity and can inactivate elongation factor 2 (EF-2) or activate G protein, causing cellular dysfunction. Toxins generally enter cells through receptor-mediated endocytosis and are transported and translocated by the retrosecretory pathway. Natural protein toxins have anti-tumor and anti-viral effects and have good clinical application prospects. One of these applications is the development of antibody-drug conjugates (ADCs), which combine the specificity of antibodies with the cytotoxicity of protein toxins.
In nature, many animals, plants, bacteria and fungi can produce large-molecule protein toxins. In the long journey of biological evolution, the existence of toxic proteins has given these biological strains a unique selective advantage. On the one hand, toxic proteins can protect against external pathogens invading organisms, and at the same time, they can resist assimilation between species. The study of toxins begins with some toxic compounds found in nature. For example: tubocurarine and toxocarpine, etc., their chemical nature is alkaloid small molecule organic compounds. In the past 50 years, with the advancement of modern organic chemistry, biochemistry and molecular biology technology, a wide variety of toxin molecules have been isolated from animals, plants, marine animals and plants, bacteria and fungi, forming animal toxins (such as snake venom, scorpion venom), plant toxins (such as ricin, acacia toxin), marine toxins (such as tetrodotoxin, conotoxin), bacterial toxins (such as diphtheria toxin, cholera toxin, microcystin) and mycotoxins (such as α-Sarcin) and other new toxin research fields. At the same time, the chemical structure of toxin molecules has also been further expanded. In addition to alkaloids, they can also be terpenes, polyethers, cyclic peptides, peptides and proteins. Due to the wide variety of toxins and their specific action targets, the research field on toxins is very broad. Among them, protein toxins are rich in sources and have good anti-tumor and anti-viral effects. Therefore, the molecular structure, mechanism of action and clinical application prospects of protein toxins have attracted widespread attention from researchers.
Fig. 1. Structural and schematic models of Shiga toxin and ricin (FEBS Letters. 2010, 584: 2626-2634).
Cholera toxin B subunit (CTB) specifically binds to ganglioside GM1 and can be used to specifically label lipid raft structures on cell membranes, label neuronal cell membranes, and retrograde tracing of cells.
Clostridium difficile toxin B (TcdB) has an active region of glycosyltransferase and targets the Rho protein family (Rho, Rac, Cdc42). It can inactivate the GTPases of the Rho family, thereby affecting normal physiological activities in cells.
Diphtheria toxin is one of the toxin molecules commonly used to construct immunotoxins. It blocks cell protein synthesis by inactivating peptide chain elongation factor II (EF-2) of eukaryotic cells, leading to cell death. Diphtheria toxin toxoid is a mutant of diphtheria toxin in which the 52nd amino acid is mutated from Gly to Glu, resulting in a change in the active site of the diphtheria toxin enzyme, making it unable to produce toxic effects on cells. Although it has lost its enzymatic activity and toxicity, its antigenicity and immunogenicity are still consistent with those of natural diphtheria toxin.
Ricin is a naturally occurring, highly potent toxin derived from the castor plant. It is a plant protein with two peptide chains that inhibits protein synthesis.
Conotoxins are a series of bioactive peptides secreted by the venom ducts and venom glands on the inner wall of the venom sac of the marine gastropod mollusc conus snail. Conotoxins have strong biological activity and can specifically bind to ion channels and neuroreceptors on cell membranes. The targets of conotoxins discovered so far mainly include acetylcholine receptors, sodium, potassium, and calcium ion channels, N-methyl-D-aspartate receptors, hormone receptors, etc. Among them, the most conotoxins act on acetylcholine receptors, sodium ion and calcium ion channels, while less conotoxins act on other ion channels and receptors.
ADC drugs are a new type of tumor treatment method that combine targeted antibodies with cytotoxic drugs to accurately attack tumor cells and reduce damage to normal cells. A variety of protein toxins have been successfully used in ADCs, including:
Ricin is composed of two subunits, A and B, where the A subunit is the active component of the enzyme responsible for inhibiting protein synthesis in cells. Ricin and its derivatives have been extensively studied and used to develop ADCs. The process of creating ADCs using ricin involves conjugating ricin or its derivatives to mAbs via an ADC linker molecule. mAbs specifically target cancer cells that express specific antigens, allowing the ADC to selectively bind to these cells. Once bound, the ADC is internalized into cancer cells, where the linker molecule is cleaved, releasing the ricin payload. Ricin then inhibits protein synthesis within the cancer cells, causing cell death.
Pseudomonas exotoxin A (PE), composed of 638 amino acids, is the most toxic and immunogenic virulence factor of the Gram-negative bacterium Pseudomonas aeruginosa. After processing, it is converted into a mature toxin that is a single-chain polypeptide of 613 amino acids in length. The active form of this PE has ribosylation activity that inactivates elongation factor-2 (EF2), thereby inhibiting host cell protein synthesis leading to cell death. PE38 is a truncated form of PE that is less immunogenic. Intact or truncated PE can be used to construct immunotoxins (ITs) and be used for targeted cancer therapy. Many PE38-containing scFv ITs are in clinical trials, targeting antigens such as CD22, mesothelin, CD25, etc. Lumoxit, the FDA-approved ADC drug developed by AstraZeneca, uses PE38 as a payload and is the first drug to treat hairy cell leukemia in more than two decades.
Gelonin is a ribosome-inactivating protein (RIP) derived from the plant Gelonium multiflorum. It has been extensively studied for its potential application in the development of ADCs. Gelonin can be used as a payload or cytotoxic agent in ADCs. It has potent ribosome-inactivating activity, meaning it inhibits protein synthesis within target cells, leading to cell death. Gelonin can be conjugated to antibodies via a linker that is stable in circulation but releases a cytotoxic payload upon internalization by target cells.
Shiga-like toxin (Stx) is an exotoxin secreted by bacteria of the genus Shigella and is composed of α and β subunits. It is located in the prophage segment of the bacterial genome. Stx has been used in ADC drug research, for example, MT-5111 is a novel HER2-targeted ADC in clinical development that binds to a different epitope on HER2 than trastuzumab or pertuzumab, using the ribosome inhibitor Shiga toxin as payload.
Fig. 2. Endocytic uptake and retrograde transport of Shiga toxin (FEBS Letters. 2010, 584: 2626-2634).
Protein toxins are powerful molecules with different sources and mechanisms of action. They found many applications in medicine, especially in the development of ADCs. By combining the specificity of antibodies with the cytotoxic effects of protein toxins, ADC provides a targeted method for the treatment of cancer while minimizing systemic toxicity. Representative compounds of protein toxins, such as ricin, Pseudomonas exotoxin A, white tree toxins and Shiga-like toxins, have shown good results in preclinical and clinical studies. Continuous research and development in this field has great potential for improving cancer treatment options and patient prognosis.