Ribosome-Inactivating Proteins: From Plant Defense to Tumor Attack

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

Hosts for Hostile Protein Production: The Challenge of Recombinant Immunotoxin Expression

For the recombinant expression of toxin-based drugs, a crucial step lies not only in the choice of the production host(s) but also in the accurate design of the protein chimera. These issues are particularly important since such products may be toxic to the expressing host itself. To avoid or limit the toxicity to productive cells while obtaining a consistent yield in chimeric protein, several systems from bacterial to mammalian host cells have been employed. In this review, we will discuss the development of immunotoxin (IT) expression, placing special emphasis on advantages and on potential drawbacks, as one single perfect host for every chimeric protein toxin or ligand does not exist

Plant Ribosome-Inactivating Proteins: Progesses, Challenges and Biotechnological Applications (and a Few Digressions)

Plant ribosome-inactivating protein (RIP) toxins are EC3.2.2.22 N-glycosidases, found among most plant species encoded as small gene families, distributed in several tissues being endowed with defensive functions against fungal or viral infections. The two main plant RIP classes include type I (monomeric) and type II (dimeric) as the prototype ricin holotoxin from Ricinus communis that is composed of a catalytic active A chain linked via a disulphide bridge to a B-lectin domain that mediates efficient endocytosis in eukaryotic cells. Plant RIPs can recognize a universally conserved stem-loop, known as the α-sarcin/ ricin loop or SRL structure in 23S/25S/28S rRNA. By depurinating a single adenine (A4324 in 28S rat rRNA), they can irreversibly arrest protein translation and trigger cell death in the intoxicated mammalian cell. Besides their useful application as potential weapons against infected/tumor cells, ricin was also used in bio-terroristic attacks and, as such, constitutes a major concern. In this review, we aim to summarize past studies and more recent progresses made studying plant RIPs and discuss successful approaches that might help overcoming some of the bottlenecks encountered during the development of their biomedical applications

From Immunotoxins to Suicide Toxin Delivery Approaches: Is There a Clinical Opportunity?

Suicide gene therapy is a relatively novel form of cancer therapy in which a gene coding for enzymes or protein toxins is delivered through targeting systems such as vesicles, nanoparticles, peptide or lipidic co-adjuvants. The use of toxin genes is particularly interesting since their catalytic activity can induce cell death, damaging in most cases the translation machinery (ribosomes or protein factors involved in protein synthesis) of quiescent or proliferating cells. Thus, toxin gene delivery appears to be a promising tool in fighting cancer. In this review we will give an overview, describing some of the bacterial and plant enzymes studied so far for their delivery and controlled expression in tumor models

Strategies to Improve the Clinical Utility of Saporin-Based Targeted Toxins

Plant Ribosome-inactivating proteins (RIPs) including the type I RIP Saporin have been used for the construction of Immunotoxins (ITxs) obtained via chemical conjugation of the toxic domain to whole antibodies or by generating genetic fusions to antibody fragments/targeting domains able to direct the chimeric toxin against a desired sub-population of cancer cells. The high enzymatic activity, stability and resistance to conjugation procedures and especially the possibility to express recombinant fusions in yeast, make Saporin a well-suited tool for anti-cancer therapy approaches. Previous clinical work on RIPs-based Immunotoxins (including Saporin) has shown that several critical issues must be taken into deeper consideration to fully exploit their therapeutic potential. This review focuses on possible combinatorial strategies (chemical and genetic) to augment Saporin-targeted toxin efficacy. Combinatorial approaches may facilitate RIP escape into the cytosolic compartment (where target ribosomes are), while genetic manipulations may minimize potential adverse effects such as vascular-leak syndrome or may identify T/B cell epitopes in order to decrease the immunogenicity following similar strategies as those used in the case of bacterial toxins such as Pseudomonas Exotoxin A or as for Type I RIP Bouganin. This review will further focus on strategies to improve recombinant production of Saporin-based chimeric toxins

Structural and solution chemistry, antiproliferative effects, and serum albumin binding of three pseudohalide derivatives of auranofin

Three pseudohalide analogues of the established gold drug auranofin (AF hereafter), of general formula Au(PEt3)X, i.e. Au(PEt3)CN, Au(PEt3)SCN and Au(PEt3)N3 (respectively denoted as AFCN, AFSCN and AFN3), were prepared and characterized. The crystal structure was solved for Au(PEt3)SCN highlighting the classical linear geometry of the 2-coordinate gold(I) center. The solution behaviour of the compounds was then comparatively analysed through 31PNMR providing evidence for an acceptable stability under physiological-like conditions. Afterward, the reaction of these gold compounds with bovine serum albumin (BSA) and consequent adduct formation was investigated by 31PNMR. For all the studied gold compounds, the [Au(PEt3)]+ moiety was identified as the reactive species in metal/protein adducts formation. The cytotoxic effects of the complexes were subsequently measured in comparison to AF against a representative colorectal cancer cell line and found to be still relevant and roughly similar in the three cases though far weaker than those of AF. These results show that the nature of the anionic ligand can modulate importantly the pharmacological action of the gold-triethylphosphine moiety, affecting the cytotoxic potency. These aspects may be further explored to improve the pharmacological profiles of this family of metal complexes