E-N-(2-acetyl-phenyl)-3-phenyl-acrylamide targets abrin and ricin toxicity: Hitting two toxins with one stone.

The efficacy of small molecule inhibitors (SMIs) against the enzymatic activity of Shiga toxin prompted the evaluation of their efficacy on related toxins viz. ricin and abrin. Ricin, like Shiga toxin, is listed as a category B bioweapon and belongs to the type II family of ribosome inactivating proteins (RIPs). Abrin though structurally and functionally similar to ricin, is considerably more toxic. In the present study, 35 compounds were evaluated in A549 cells in in vitro assays, of which 5 offered protection against abrin and 2 against ricin, with IC50 values ranging between 30.5-1379 µM and 300-341 µM, respectively. These findings are substantiated by fluorescence based thermal shift assay. Moreover, the binding of the promising compounds to the toxin components has been validated by Surface Plasmon Resonance assay and in vitro protein synthesis assay. In vivo studies reveal complete protection of mice with compound 4 E-N-(2-acetyl-phenyl)-3-phenyl-acrylamide against orally administered lethal doses of, both, abrin and ricin. The present study thus proposes the emergence of E-N-(2-acetyl-phenyl)-3-phenyl-acrylamide as a lead compound against RIPs.

Unraveling the Roots of Selectivity of Peptide Affinity Reagents for Structurally Similar Ribosomal Inactivating Protein Derivatives.

Peptide capture agents have become increasingly useful tools for a variety of sensing applications due to their ease of discovery, stability, and robustness. Despite the ability to rapidly discover candidates through biopanning bacterial display libraries and easily mature them to Protein Catalyzed Capture (PCC) agents with even higher affinity and selectivity, an ongoing challenge and critical selection criteria is that the peptide candidates and final reagent be selective enough to replace antibodies, the gold-standard across immunoassay platforms. Here, we have discovered peptide affinity reagents against abrax, a derivative of abrin with reduced toxicity. Using on-cell Fluorescence Activated Cell Sorting (FACS) assays, we show that the peptides are highly selective for abrax over RiVax, a similar derivative of ricin originally designed as a vaccine, with significant structural homology to abrax. We rank the newly discovered peptides for strongest affinity and analyze three observed consensus sequences with varying affinity and specificity. The strongest (Tier 1) consensus was FWDTWF, which is highly aromatic and hydrophobic. To better understand the observed selectivity, we use the XPairIt peptide-protein docking protocol to analyze binding location predictions of the individual Tier 1 peptides and consensus on abrax and RiVax. The binding location profiles on the two proteins are quite distinct, which we determine is due to differences in pocket size, pocket environment (including hydrophobicity and electronegativity), and steric hindrance. This study provides a model system to show that peptide capture candidates can be quite selective for a structurally similar protein system, even without further maturation, and offers an in silico method of analysis for understanding binding and down-selecting candidates.

Mechanistic insights into the neutralization of cytotoxic abrin by the monoclonal antibody D6F10.

Abrin, an A/B toxin obtained from the Abrus precatorius plant is extremely toxic and a potential bio-warfare agent. Till date there is no antidote or vaccine available against this toxin. The only known neutralizing monoclonal antibody against abrin, namely D6F10, has been shown to rescue the toxicity of abrin in cells as well as in mice. The present study focuses on mapping the epitopic region to understand the mechanism of neutralization of abrin by the antibody D6F10. Truncation and mutational analysis of abrin A chain revealed that the amino acids 74-123 of abrin A chain contain the core epitope and the residues Thr112, Gly114 and Arg118 are crucial for binding of the antibody. In silico analysis of the position of the mapped epitope indicated that it is present close to the active site cleft of abrin A chain. Thus, binding of the antibody near the active site blocks the enzymatic activity of abrin A chain, thereby rescuing inhibition of protein synthesis by the toxin in vitro. At 1∶10 molar concentration of abrin:antibody, the antibody D6F10 rescued cells from abrin-mediated inhibition of protein synthesis but did not prevent cell attachment of abrin. Further, internalization of the antibody bound to abrin was observed in cells by confocal microscopy. This is a novel finding which suggests that the antibody might function intracellularly and possibly explains the rescue of abrin's toxicity by the antibody in whole cells and animals. To our knowledge, this study is the first report on a neutralizing epitope for abrin and provides mechanistic insights into the poorly understood mode of action of anti-A chain antibodies against several toxins including ricin.

Nucleic acid aptamers against biotoxins: a new paradigm toward the treatment and diagnostic approach.

Nucleic acid aptamers are short single-stranded DNA or RNA oligonucleotides that can bind to their targets with very high affinity and specificity, and are generally selected by a process referred to as systematic evolution of ligands by exponential enrichment. Conventional antibody-based therapeutic and diagnostic approach currently employed against biotoxins pose major limitations such as the requirement of a live animal for the in vivo enrichment of the antibody species, decreased stability, high production cost, and side effects. Aptamer technology is a viable alternative that can be used to combat these problems. Fully sequestered in vitro, aptamers eliminate the need for a living host. Furthermore, one of the key advantages of using aptamers instead of antibodies is that they can be selected against very weakly immunogenic and cytotoxic substances. In this review, we focus on nucleic acid aptamers developed against various biotoxins of plant, microorganism, or animal origin and show how these can be used in diagnostics (e.g., biosensors) and therapy.

Chemical inactivation of protein toxins on food contact surfaces.

We compared the kinetics and efficacies of sodium hypochlorite, peracetic acid, phosphoric acid-based detergent, chlorinated alkaline detergent, quaternary ammonium-based sanitizer, and peracetic acid-based sanitizer for inactivating the potential bioterrorism agents ricin and abrin in simple buffers, food slurries (infant formula, peanut butter, and pancake mix), and in dried food residues on stainless steel. The intrinsic fluorescence and cytotoxicity of purified ricin and abrin in buffers decreased rapidly in a pH- and temperature-dependent manner when treated with sodium hypochlorite but more slowly when treated with peracetic acid. Cytotoxicity assays showed rapid and complete inactivation of ricin and crude abrin in food slurries and dried food residues treated 0-5 min with sodium hypochlorite. Toxin epitopes recognized by ELISA decayed more gradually under these conditions. Higher concentrations of peracetic acid were required to achieve comparable results. Chlorinated alkaline detergent was the most effective industrial agent tested for inactivating ricin in dried food residues.

A neutralizing antibody to the a chain of abrin inhibits abrin toxicity both in vitro and in vivo.

Plant ribosome-inactivating proteins (RIPs) are RNA N-glycosidases that inhibit protein synthesis in cells. Abrin, a type II RIP, is an AB type toxin, which is one of the most lethal types of toxin known. The B chain facilitates the entry of the molecule into the cell, whereas the A chain exerts the toxic effect. We have generated hybridomas secreting antibodies of the immunoglobulin G class specific to the recombinant A chain of abrin. One monoclonal antibody, namely, D6F10, rescued cells from abrin toxicity. Importantly, the antibody also protected mice from lethal doses of the toxin. The neutralizing effect of the antibody was shown to be due to interference with abrin attachment to the cell surface.

Ribosome-inactivating protein and apoptosis: abrin causes cell death via mitochondrial pathway in Jurkat cells.

Abrin belongs to the type II family of ribosome-inactivating proteins comprising a galactose-binding B chain coupled with a toxic A chain through a single disulphide linkage. Apart from its RNA-N-glycosidase activity, another role that has been recently ascribed to abrin was the induction of apoptosis. Studies were undertaken to determine the kinetics of these two activities. In the present study, we report that the signal for apoptosis is triggered at a time point later than the inhibition of protein synthesis. This apoptotic pathway induced by abrin is caspase 3-dependent but caspase 8-independent and involves mitochondrial membrane potential damage and reactive oxygen species production. Overexpression of B-cell lymphocytic-leukaemia proto-oncogene 2 was found to block this apoptotic pathway.

Examination of the toxicity of several protein toxins of plant origin using bovine pulmonary endothelial cells.

The bovine pulmonary endothelial (BPE) cell line was examined as a model to study the toxicity of ricin and abrin toxins currently under investigation. The BPE cell line was examined because ricin has been shown to bind to endothelial cells. Cell viability was assessed using several different biochemical parameters including growth (DNA by binding of gentian violet stain), mitochondrial function (succinate dehydrogenase activity) using MTT and lysosomal integrity (neutral red retention assay). In order to compare toxicities and investigate potential protective compounds, concentrations of toxins causing death of 50% and 70% of the (control) cell population (LC50 and LC70, respectively) were determined. It is concluded that while ricin and abrin share a common mechanism of action ricin is slightly less toxic than abrin. BPE cells are a good model for future mechanistic studies and particularly for initial phase screening of potentially therapeutic compounds. Carbohydrates were used in an attempt to examine which receptor types were involved in the binding and uptake of ricin and abrin by the cell line. It was found that only high concentrations of galactose prevented lethality while mannose apparently had no effect. Furthermore, the molar excess of carbohydrate to toxin required in order to achieve protection indicated that this would be an impractical approach to adopt in vivo.

Cholera toxin B-subunit protects mammalian cells from ricin and abrin toxicity.

The glycoproteins ricin and abrin intoxicate cells by inhibiting protein synthesis. Pretreatment of HeLa cells with cholera toxin partially protects them from ricin and abrin activity. The involvement in this phenomenon of the various effects of cholera toxin, namely, redistribution of membrane receptors elicited from protomer B and increasing cyclic AMP concentrations induced by protomer A, were studied. Substances able to enhance cyclic AMP concentrations do not affect ricin and abrin activity, while protomer B alone protects cells. In addition, the effects of several lectins on ricin or abrin toxicity were examined. Almost complete prevention of ricin or abrin activity was obtained using concanavalin A (Con A) and wheat germ agglutinin (WGA). Conversely, neither succinyl Con A nor Ulex europeus agglutinin (UEA) affected the cellular response. Both protomer B of cholera toxin and Con A did not alter the binding of ricin or abrin; they seem to protect cells by altering membrane structure.

Abrogation of the non-specific toxicity of abrin conjugated to anti-lymphocyte globulin.

A covalent conjugate of abrin and anti-human lymphocyte globulin (AHLG) was prepared in an endeavour to create a cytotoxic agent with specificity for human lymphoid cells. The AHLG--abrin conjugate was found to be around 10-fold better able to inhibit 3H-leucine uptake by the human lymphoblastoid cell line, Daudi, in tissue culture than was the control conjugate comprising abrin and normal IgG (nIgG). Both materials were less potent than native abrin. Galactose, which is known competitively to antagonize the binding of abrin to cells, strongly inhibited the toxicities of abrin and the nIgG--abrin conjugate whereas that of ALG--abrin was unimpaired. Thus, at least for Daudi cells in tissue culture, abrin can be made selectively toxic, by linkage to AHLG, towards cells bearing antigens to which the antibody moiety of the conjugate can attach.

Toxicity, distribution and elimination of the cancerostatic lectins abrin and ricin after parenteral injection into mice.

The survival time of mice after i.v. injection of the cancerostatic lectins, abrin and ricin was recorded. The LD50 dose was found to be 10-13 ng and 55-65 ng per mouse for abrin and ricin, respectively. Increasing amounts of toxin reduced the survival time, reaching a minimum of about 10 h. Lactose injected with ricin, provided partial protection against ricin, as measured by the survival time. Abrin and ricin labelled with 125I, and shown to retain their full toxic activity, were injected into mice. Most of the radioactivity found in the organs was present in the form of intact toxins, at least up to 5 h after injection. After i.v. injection the highest concentration/g tissue was found in spleen, followed by kidneys, heart, liver and thymus. The relative concentration in liver was considerably higher for ricin than for abrin. Similar results were found after i.p. injection. When lactose was administered together with ricin, almost 80% of the ricin injected was found in the liver after 30 min, compared to 48% without lactose, and the amount in other organs was concurrently reduced. The elimination of total radioactivity was much faster for ricin than abrin. The radioactivity found in the urine was largely present in non-trichloroacetic acid precipitable form, indicating that the toxins were extensively degraded before excretion.