A fluorescence anisotropy-based competition assay to identify inhibitors against ricin and Shiga toxin ribosome interactions.

Ricin is one of the most toxic substances known and a type B biothreat agent. Shiga toxins (Stxs) produced by E. coli (STEC) and Shigella dysenteriae are foodborne pathogens. There is no effective therapy against ricin or STEC and there is an urgent need for inhibitors. Ricin toxin A subunit (RTA) and A1 subunit of Stx2a (Stx2A1) bind to the C-terminal domain (CTD) of the ribosomal P-stalk proteins to depurinate the sarcin/ricin loop. Modulation of toxin-ribosome interactions has not been explored as a strategy for inhibition. Therefore, development of assays that detect inhibitors targeting toxin-ribosome interactions remains a critical need. Here we describe a fluorescence anisotropy (FA)-based competitive binding assay using a BODIPY-TMR labeled 11-mer peptide (P11) derived from the P-stalk CTD to measure the binding affinity of peptides ranging from 3 to 11 amino acids for the P-stalk pocket of RTA and Stx2A1. Comparison of the affinity with the surface plasmon resonance (SPR) assay indicated that although the rank order was the same by both methods, the FA assay could differentiate better between peptides that show nonspecific interactions by SPR. The FA assay detects only interactions that compete with the labeled P11 and can validate inhibitor specificity and mechanism of action.

The Search for Antidotes Against Ricin.

The castor plant (Ricinus communis) is primarily known for its seeds, which contain a unique fatty acid called ricinoleic acid with several industrial and commercial applications. Castor seeds also contain ricin, a toxin considered a chemical and biological warfare agent. Despite years of investigation, there is still no effective antidote or vaccine available. However, some progress has been made, and the development of an effective treatment may be on the horizon. To provide an updated overview of this issue, we have conducted a comprehensive review of the literature on the current state of research in the fight against ricin. This review is based on the reported research and aims to address the challenges faced by researchers, as well as highlight the most successful cases achieved thus far. Our goal is to encourage the scientific community to continue their efforts in this critical search.

Structure-based design and optimization of a new class of small molecule inhibitors targeting the P-stalk binding pocket of ricin.

Ricin, a category-B agent for bioterrorism, and Shiga toxins (Stxs), which cause food poisoning bind to the ribosomal P-stalk to depurinate the sarcin/ricin loop. No effective therapy exists for ricin or Stx intoxication. Ribosome binding sites of the toxins have not been targeted by small molecules. We previously identified CC10501, which inhibits toxin activity by binding the P-stalk pocket of ricin toxin A subunit (RTA) remote from the catalytic site. Here, we developed a fluorescence polarization assay and identified a new class of compounds, which bind P-stalk pocket of RTA with higher affinity and inhibit catalytic activity with submicromolar potency. A lead compound, RU-NT-206, bound P-stalk pocket of RTA with similar affinity as a five-fold larger P-stalk peptide and protected cells against ricin and Stx2 holotoxins for the first time. These results validate the P-stalk binding site of RTA as a critical target for allosteric inhibition of the active site.

Synthesis and Structural Characterization of Ricin Inhibitors Targeting Ribosome Binding Using Fragment-Based Methods and Structure-Based Design.

Ricin toxin A subunit (RTA) is the catalytic subunit of ricin, which depurinates an adenine from the sarcin/ricin loop in eukaryotic ribosomes. There are no approved inhibitors against ricin. We used a new strategy to disrupt RTA-ribosome interactions by fragment screening using surface plasmon resonance. Here, using a structure-guided approach, we improved the affinity and inhibitory activity of small-molecular-weight lead compounds and obtained improved compounds with over an order of magnitude higher efficiency. Four advanced compounds were characterized by X-ray crystallography. They bind at the RTA-ribosome binding site as the original compound but in a distinctive manner. These inhibitors bind remotely from the catalytic site and cause local conformational changes with no alteration of the catalytic site geometry. Yet they inhibit depurination by ricin holotoxin and inhibit the cytotoxicity of ricin in mammalian cells. They are the first agents that protect against ricin holotoxin by acting directly on RTA.

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.

Ricin Antibodies' Neutralizing Capacity against Different Ricin Isoforms and Cultivars.

Ricin, a highly toxic protein from Ricinus communis, is considered a potential biowarfare agent. Despite the many data available, no specific treatment has yet been approved. Due to their ability to provide immediate protection, antibodies (Abs) are an approach of choice. However, their high specificity might compromise their capacity to protect against the different ricin isoforms (D and E) found in the different cultivars. In previous work, we have shown the neutralizing potential of different Abs (43RCA-G1 (anti ricin A-chain) and RB34 and RB37 (anti ricin B-chain)) against ricin D. In this study, we evaluated their protective capacity against both ricin isoforms. We show that: (i) RB34 and RB37 recognize exclusively ricin D, whereas 43RCA-G1 recognizes both isoforms, (ii) their neutralizing capacity in vitro varies depending on the cultivar, and (iii) there is a synergistic effect when combining RB34 and 43RCA-G1. This effect is also demonstrated in vivo in a mouse model of intranasal intoxication with ricin D/E (1:1), where approximately 60% and 40% of mice treated 0 and 6 h after intoxication, respectively, are protected. Our results highlight the importance of evaluating the effectiveness of the Abs against different ricin isoforms to identify the treatment with the broadest spectrum neutralizing effect.

Ligand-Based Virtual Screening, Molecular Docking, Molecular Dynamics, and MM-PBSA Calculations towards the Identification of Potential Novel Ricin Inhibitors.

Ricin is a toxin found in the castor seeds and listed as a chemical weapon by the Chemical Weapons Convention (CWC) due to its high toxicity combined with the easiness of obtention and lack of available antidotes. The relatively frequent episodes of usage or attempting to use ricin in terrorist attacks reinforce the urge to develop an antidote for this toxin. In this sense, we selected in this work the current RTA (ricin catalytic subunit) inhibitor with the best experimental performance, as a reference molecule for virtual screening in the PubChem database. The selected molecules were then evaluated through docking studies, followed by drug-likeness investigation, molecular dynamics simulations and Molecular Mechanics Poisson-Boltzmann Surface Area (MM-PBSA) calculations. In every step, the selection of molecules was mainly based on their ability to occupy both the active and secondary sites of RTA, which are located right next to each other, but are not simultaneously occupied by the current RTA inhibitors. Results show that the three PubChem compounds 18309602, 18498053, and 136023163 presented better overall results than the reference molecule itself, showing up as new hits for the RTA inhibition, and encouraging further experimental evaluation.

Post-Exposure Anti-Ricin Treatment Protects Swine Against Lethal Systemic and Pulmonary Exposures.

Ricin, a plant-derived toxin originating from the seeds of Ricinus communis (castor bean plant), is one of the most lethal toxins known. To date, there is no approved post-exposure therapy for ricin exposures. This work demonstrates for the first time the therapeutic efficacy of equine-derived anti-ricin F(ab')2 antibodies against lethal pulmonary and systemic ricin exposures in swine. While administration of the antitoxin at 18 h post-exposure protected more than 80% of both intratracheally and intramuscularly ricin-intoxicated swine, treatment at 24 h post-exposure protected 58% of the intramuscular-exposed swine, as opposed to 26% of the intratracheally exposed animals. Quantitation of the anti-ricin neutralizing units in the anti-toxin preparations confirmed that the disparate protection conferred to swine subjected to the two routes of exposure stems from variance between the two models. Furthermore, dose response experiments showed that approximately 3 times lesser amounts of antibody are needed for high-level protection of the intramuscularly compared to the intratracheally intoxicated swine. This study, which demonstrates the high-level post-exposure efficacy of anti-ricin antitoxin at clinically relevant time-points in a large animal model, can serve as the basis for the formulation of post-exposure countermeasures against ricin poisoning in humans.

A Humanized Monoclonal Antibody Cocktail to Prevent Pulmonary Ricin Intoxication.

PB10 IgG1, a monoclonal antibody (MAb) directed against an immunodominant epitope on the enzymatic subunit (RTA) of ricin toxin (RT), has been shown to passively protect mice and non-human primates from an aerosolized lethal-dose RT challenge. However, it was recently demonstrated that the therapeutic efficacy of PB10 IgG1 is significantly improved when co-administered with a second MAb, SylH3, targeting RT's binding subunit (RTB). Here we report that the PB10/SylH3 cocktail is also superior to PB10 alone when used as a pre-exposure prophylactic (PrEP) in a mouse model of intranasal RT challenge. The benefit of the PB10/SylH3 cocktail prompted us to engineer a humanized IgG1 version of SylH3 (huSylH3). The huPB10/huSylH3 cocktail proved highly efficacious in the mouse model, thereby opening the door to future testing in non-human primates.

Regulation of endo-lysosomal pathway and autophagic flux by broad-spectrum antipathogen inhibitor ABMA.

The endo-lysosome system is involved in endocytosis, protein sorting, and degradation as well as autophagy. Numerous toxins and pathogens exploit this system to enter host cells and exert their deleterious effects. Modulation of host endo-lysosome pathway may restrict multiple toxins intoxication as well as pathogen infection. ABMA, selected from a high-throughput screening against the cytotoxicity of ricin toxin, exhibits a broad-spectrum antitoxin and antipathogen activity. Here, we show that ABMA selectively retains endocytosed protein and toxin to late endosomes and thus delaying their intracellular trafficking. It also impairs the autophagic flux by excessive fusion of late endosomes and autophagosomes. Its exclusive action on late endosomes and corresponding consequences on the endo-lysosomal pathway and autophagic flux are distinct from known inhibitors such as bafilomycin A1, EGA, or chloroquine. Hence, besides being a broad-spectrum inhibitor of endocytosed toxins and pathogens, ABMA may serve as a molecular tool to dissect endo-lysosome system-related cellular physiology and mechanisms of pathogenesis.

Inhibition of ricin A-chain (RTA) catalytic activity by a viral genome-linked protein (VPg).

Ricin is a plant derived protein toxin produced by the castor bean plant (Ricinus communis). The Centers for Disease Control (CDC) classifies ricin as a Category B biological agent. Currently, there is neither an effective vaccine that can be used to protect against ricin exposure nor a therapeutic to reverse the effects once exposed. Here we quantitatively characterize interactions between catalytic ricin A-chain (RTA) and a viral genome-linked protein (VPg) from turnip mosaic virus (TuMV). VPg and its N-terminal truncated variant, VPg1-110, bind to RTA and abolish ricin's catalytic depurination of 28S rRNA in vitro and in a cell-free rabbit reticulocyte translational system. RTA and VPg bind in a 1 to 1 stoichiometric ratio, and their binding affinity increases ten-fold as temperature elevates (5 °C to 37 °C). RTA-VPg binary complex formation is enthalpically driven and favored by entropy, resulting in an overall favorable energy, ΔG = -136.8 kJ/mol. Molecular modeling supports our experimental observations and predicts a major contribution of electrostatic interactions, suggesting an allosteric mechanism of downregulation of RTA activity through conformational changes in RTA structure, and/or disruption of binding with the ribosomal stalk. Fluorescence anisotropy studies show that heat affects the rate constant and the activation energy for the RTA-VPg complex, Ea = -62.1 kJ/mol. The thermodynamic and kinetic findings presented here are an initial lead study with promising results and provides a rational approach for synthesis of therapeutic peptides that successfully eliminate toxicity of ricin, and other cytotoxic RIPs.

Determining the Relative Binding Affinity of Ricin Toxin A Inhibitors by Using Molecular Docking and Nonequilibrium Work.

Ricin is a ribosome-inactivating protein (RIP type 2) consisting of two subunits, ricin toxin A (RTA) and ricin toxin B (RTB). Because of its cytotoxicity, ricin has worried world authorities for its potential use as a chemical weapon; therefore, its inhibition is of great biotechnological interest. RTA is the target for inhibitor synthesis, and pterin derivatives are promising candidates to inhibit it. In this study, we used a combination of the molecular docking approach and fast steered molecular dynamics (SMD) to assess the correlation between nonequilibrium work, ⟨ W⟩, and the IC50 for six RTA inhibitors. The results showed that molecular docking is a powerful tool to predict good bioactive poses of RTA inhibitors, and ⟨ W⟩ presented a strong correlation with IC50 ( R2 = 0.961). Such a profile ranked the RTA inhibitors better than the molecular docking approach. Therefore, the combination of docking and fast SMD simulation was shown to be a promising tool to distinguish RTA-active inhibitors from inactive ones and could be used as postdocking filtering approach.

Cell toxicity by ricin and elucidation of mechanism of Ricin inactivation.

Castor cake is a by-product of the extraction of oil from from seeds of castor plants (Ricinus communis). This by-product contains high levels of proteins, but a toxic protein, ricin, limits its use as an animal feed. Ricin can be efficiently inactivated by treatment with calcium oxide (CaO), which can be evaluated by a cytotoxicity assay using LLC-MK2 cells. The mechanism by which the CaO treatment inactivates ricin, however, is unclear. We report the structural changes responsible for ricin inactivation. Purified ricin was treated with 0.6% CaO and then analyzed by mass spectrometry. This treatment degraded the ricin at preferential sites. The aqueous CaO solution had a pH >12, which preferentially cleaved asparagine residues, followed by glutamine, serine and glycine residues. The alkaline pH affected the tertiary structure of the ricin, cleaving its polypeptide chains and thereby eliminating its cytotoxic activity.

ABMA, a small molecule that inhibits intracellular toxins and pathogens by interfering with late endosomal compartments.

Intracellular pathogenic microorganisms and toxins exploit host cell mechanisms to enter, exert their deleterious effects as well as hijack host nutrition for their development. A potential approach to treat multiple pathogen infections and that should not induce drug resistance is the use of small molecules that target host components. We identified the compound 1-adamantyl (5-bromo-2-methoxybenzyl) amine (ABMA) from a cell-based high throughput screening for its capacity to protect human cells and mice against ricin toxin without toxicity. This compound efficiently protects cells against various toxins and pathogens including viruses, intracellular bacteria and parasite. ABMA provokes Rab7-positive late endosomal compartment accumulation in mammalian cells without affecting other organelles (early endosomes, lysosomes, the Golgi apparatus, the endoplasmic reticulum or the nucleus). As the mechanism of action of ABMA is restricted to host-endosomal compartments, it reduces cell infection by pathogens that depend on this pathway to invade cells. ABMA may represent a novel class of broad-spectrum compounds with therapeutic potential against diverse severe infectious diseases.

Improved antibody-based ricin neutralization by affinity maturation is correlated with slower off-rate values.

While potent monoclonal antibodies against ricin were introduced over the years, the question whether increasing antibody affinity enables better toxin neutralization was not fully addressed yet. The aim of this study was to characterize the contribution of antibody affinity to the ricin neutralization potential of the antibody. cHD23 monoclonal antibody that targets the toxin B-subunit and interferes with its binding to membranal receptors, was isolated. In order to create antibody clones with improved affinity toward ricin, a scFv-phage display library containing mutated versions of the variable regions of cHD23 was constructed and clones with improved binding of ricin were isolated. Structural modeling of these mutants suggests that the inserted mutations may increase the antibody conformational flexibility thus improving its ability to bind ricin. While it was found that the selected clones exhibited improved neutralization of ricin, the correlation between the KD values and potency was only minor (r = 0.55). However, a positive correlation (r = 0.84) exist between the off-rate values (koff) of the affinity matured clones and their ability to neutralize ricin. As cell membranes display inordinately large amounts of potential surface binding sites for ricin, it is suggested that antibodies with improved off-rate values block the ability of the toxin to bind to target receptors, in a highly efficient manner. Currently, antibody-based therapy is the most effective treatment for ricin intoxication and it is anticipated that the findings of this study will provide useful information and a possible strategy to design an improved antibody-based therapy for the toxin.

Structural Analysis of Single Domain Antibodies Bound to a Second Neutralizing Hot Spot on Ricin Toxin's Enzymatic Subunit.

Ricin toxin is a heterodimer consisting of RTA, a ribosome-inactivating protein, and RTB, a lectin that facilitates receptor-mediated uptake into mammalian cells. In previous studies, we demonstrated that toxin-neutralizing antibodies target four spatially distinct hot spots on RTA, which we refer to as epitope clusters I-IV. In this report, we identified and characterized three single domain camelid antibodies (VHH) against cluster II. One of these VHHs, V5E1, ranks as one of the most potent ricin-neutralizing antibodies described to date. We solved the X-ray crystal structures of each of the three VHHs (E1, V1C7, and V5E1) in complex with RTA. V5E1 buries a total of 1,133 Å2 of surface area on RTA and makes primary contacts with α-helix A (residues 18-32), α-helix F (182-194), as well as the F-G loop. V5E1, by virtue of complementarity determining region 3 (CDR3), may also engage with RTB and potentially interfere with the high affinity galactose-recognition element that plays a critical role in toxin attachment to cell surfaces and intracellular trafficking. The two other VHHs, E1 and V1C7, bind epitopes adjacent to V5E1 but display only weak toxin neutralizing activity, thereby providing structural insights into specific residues within cluster II that may be critical contact points for toxin inactivation.

Inhibitors of retrograde trafficking active against ricin and Shiga toxins also protect cells from several viruses, Leishmania and Chlamydiales.

Medical countermeasures to treat biothreat agent infections require broad-spectrum therapeutics that do not induce agent resistance. A cell-based high-throughput screen (HTS) against ricin toxin combined with hit optimization allowed selection of a family of compounds that meet these requirements. The hit compound Retro-2 and its derivatives have been demonstrated to be safe in vivo in mice even at high doses. Moreover, Retro-2 is an inhibitor of retrograde transport that affects syntaxin-5-dependent toxins and pathogens. As a consequence, it has a broad-spectrum activity that has been demonstrated both in vitro and in vivo against ricin, Shiga toxin-producing O104:H4 entero-hemorrhagic E. coli and Leishmania sp. and in vitro against Ebola, Marburg and poxviruses and Chlamydiales. An effect is anticipated on other toxins or pathogens that use retrograde trafficking and syntaxin-5. Since Retro-2 targets cell components of the host and not directly the pathogen, no selection of resistant pathogens is expected. These lead compounds need now to be developed as drugs for human use.

Inhibition of Cholera Toxin and Other AB Toxins by Polyphenolic Compounds.

Cholera toxin (CT) is an AB-type protein toxin that contains a catalytic A1 subunit, an A2 linker, and a cell-binding B homopentamer. The CT holotoxin is released into the extracellular environment, but CTA1 attacks a target within the cytosol of a host cell. We recently reported that grape extract confers substantial resistance to CT. Here, we used a cell culture system to identify twelve individual phenolic compounds from grape extract that inhibit CT. Additional studies determined the mechanism of inhibition for a subset of the compounds: two inhibited CT binding to the cell surface and even stripped CT from the plasma membrane of a target cell; two inhibited the enzymatic activity of CTA1; and four blocked cytosolic toxin activity without directly affecting the enzymatic function of CTA1. Individual polyphenolic compounds from grape extract could also generate cellular resistance to diphtheria toxin, exotoxin A, and ricin. We have thus identified individual toxin inhibitors from grape extract and some of their mechanisms of inhibition against CT.

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.

Quantitative profiling of the in vivo enzymatic activity of ricin reveals disparate depurination of different pulmonary cell types.

The plant-derived toxins ricin and abrin, operate by site-specific depurination of ribosomes, which in turn leads to protein synthesis arrest. The clinical manifestation following pulmonary exposure to these toxins is that of a severe lung inflammation and respiratory insufficiency. Deciphering the pathways mediating between the catalytic activity and the developing lung inflammation, requires a quantitative appreciation of the catalytic activity of the toxins, in-vivo. In the present study, we monitored truncated cDNA molecules which are formed by reverse transcription when a depurinated 28S rRNA serves as template. We found that maximal depurination after intranasal exposure of mice to 2LD50 ricin was reached 48h, where nearly 40% of the ribosomes have been depurinated and that depurination can be halted by post-exposure administration of anti-ricin antibodies. We next demonstrated that the effect of ricin intoxication on different cell types populating the lungs differs greatly, and that outstandingly high levels of damage (80% depurination), were observed in particular for pulmonary epithelial cells. Finally, we found that the magnitude of depurination induced by the related plant-derived toxin abrin, was significantly lower in comparison to ricin, and can be attributed mostly to reduced depurination of pulmonary epithelial cells by abrin. This study provides for the first time vital information regarding the scope and timing of the catalytic performance of ricin and abrin in the lungs of intact animals.