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.

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.

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.

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.

An in vitro evaluation of epigallocatechin gallate (eGCG) as a biocompatible inhibitor of ricin toxin.

The catechin, epigallocatechin gallate (eGCG), found in green tea, has inhibitory activity against a number of protein toxins and was investigated in relation to its impact upon ricin toxin (RT) in vitro. The IC(50) for RT was 0.08±0.004 ng/mL whereas the IC(50) for RT+100 µM eGCG was 3.02±0.572 ng/mL, indicating that eGCG mediated a significant (p<0.0001) reduction in ricin toxicity. This experiment was repeated in the human macrophage cell line THP-1 and IC(50) values were obtained for RT (0.54±0.024 ng/mL) and RT+100 µM eGCG (0.68±0.235 ng/mL) again using 100 µM eGCG and was significant (p=0.0013). The documented reduction in ricin toxicity mediated by eGCG was found to be eGCG concentration dependent, with 80 and 100 µg/mL (i.e. 178 and 223 µM respectively) of eGCG mediating a significant (p=0.0472 and 0.0232) reduction in ricin toxicity at 20 and 4 ng/ml of RT in Vero and THP-1 cells (respectively). When viability was measured in THP-1 cells by propidium iodide exclusion (as opposed to the MTT assays used previously) 10 ng/mL and 5 ng/mL of RT was used. The addition of 1000 µM and 100 µM eGCG mediated a significant (p=0.0015 and <0.0001 respectively) reduction in ricin toxicity relative to an identical concentration of ricin with 1 µg eGCG. Further, eGCG (100 µM) was found to reduce the binding of RT B chain to lactose-conjugated Sepharose as well as significantly (p=0.0039) reduce the uptake of RT B chain in Vero cells. This data suggests that eGCG may provide a starting point to refine biocompatible substances that can reduce the lethality of ricin.

Monoclonal antibody, mAb 4C13, an effective detoxicant antibody against ricin poisoning.

Ricin is a glycoprotein produced in castor seeds and consists of two polypeptide chains named Ricin Toxin A Chain (RTA) and Ricin Toxin B Chain (RTB), linked via a disulfide bridge. Due to its high toxicity, ricin is regarded as a high terrorist risk for the public. However, antibodies can play a pivotal role in neutralizing the toxin. In this research, the anti-toxicant effect of mAb 4C13, a monoclonal antibody (mAb) established using detoxicated ricin as the immunized antigen, was evaluated. Compared with mAb 4F2 and mAb 5G6, the effective mechanism of mAb 4C13 was analyzed by experiments relating to its cytotoxicity, epitope on ricin, binding kinetics with the toxin, its blockage on the protein synthesis inhibition induced by ricin and the intracelluar tracing of its complex with ricin. Our result indicated that mAb 4C13 could recognize and bind to RTA, RTB and exert its high affinity to the holotoxin. Both cytotoxicity and animal toxicity of ricin were well blocked by pre-incubating the toxin with mAb 4C13. By intravenous injection, mAb 4C13 could rescue the mouse intraperitoneally (ip) injected with a lethal dose of ricin (20µg/kg) even at 6h after the intoxication and its efficacy was dependent on its dosage. This research indicated that mAb 4C13 could be an excellent candidate for therapeutic antibodies. Its potent antitoxic efficiency was related to its recognition on the specific epitope with very high affinity and its blockage of protein synthesis inhibition in cytoplasm followed by cellular internalization with ricin.

Novel class of potential therapeutics that target ricin retrograde translocation.

Ricin toxin, an A-B toxin from Ricinus communis, induces cell death through the inhibition of protein synthesis. The toxin binds to the cell surface via its B chain (RTB) followed by its retrograde trafficking through intracellular compartments to the ER where the A chain (RTA) is transported across the membrane and into the cytosol. Ricin A chain is transported across the ER membrane utilizing cellular proteins involved in the disposal of aberrant ER proteins by a process referred to as retrograde translocation. Given the current lack of therapeutics against ricin intoxication, we developed a high-content screen using an enzymatically attenuated RTA chimera engineered with a carboxy-terminal enhanced green fluorescent protein (RTA(E177Q)egfp) to identify compounds that target RTA retrograde translocation. Stabilizing RTA(E177Q)egfp through the inclusion of proteasome inhibitor produced fluorescent peri-nuclear granules. Quantitative analysis of the fluorescent granules provided the basis to discover compounds from a small chemical library (2080 compounds) with known bioactive properties. Strikingly, the screen found compounds that stabilized RTA molecules within the cell and several compounds limited the ability of wild type RTA to suppress protein synthesis. Collectively, a robust high-content screen was developed to discover novel compounds that stabilize intracellular ricin and limit ricin intoxication.

A retrograde trafficking inhibitor of ricin and Shiga-like toxins inhibits infection of cells by human and monkey polyomaviruses.

UNLABELLED: Polyomaviruses are ubiquitous pathogens that cause severe disease in immunocompromised individuals. JC polyomavirus (JCPyV) is the causative agent of the fatal demyelinating disease progressive multifocal leukoencephalopathy (PML), whereas BK polyomavirus (BKPyV) causes polyomavirus-induced nephropathy and hemorrhagic cystitis. Vaccines or antiviral therapies targeting these viruses do not exist, and treatments focus on reducing the underlying causes of immunosuppression. We demonstrate that retro-2(cycl), an inhibitor of ricin and Shiga-like toxins (SLTs), inhibits infection by JCPyV, BKPyV, and simian virus 40. Retro-2(cycl) inhibits retrograde transport of polyomaviruses to the endoplasmic reticulum, a step necessary for productive infection. Retro-2(cycl) likely inhibits polyomaviruses in a way similar to its ricin and SLT inhibition, suggesting an overlap in the cellular host factors used by bacterial toxins and polyomaviruses. This work establishes retro-2(cycl) as a potential antiviral therapy that broadly inhibits polyomaviruses and possibly other pathogens that use retrograde trafficking. IMPORTANCE: The human polyomaviruses JC polyomavirus (JCPyV) and BK polyomavirus (BKPyV) cause rare but severe diseases in individuals with reduced immune function. During immunosuppression, JCPyV disseminates from the kidney to the central nervous system and destroys oligodendrocytes, resulting in the fatal disease progressive multifocal leukoencephalopathy. Kidney transplant recipients are at increased risk of BKPyV-induced nephropathy, which results in kidney necrosis and loss of the transplanted organ. There are currently no effective therapies for JCPyV and BKPyV. We show that a small molecule named retro-2(cycl) protects cells from infection with JCPyV and BKPyV by inhibiting intracellular viral transport. Retro-2(cycl) treatment reduces viral spreading in already established infections and may therefore be able to control infection in affected patients. Further optimization of retro-2(cycl) may result in the development of an effective antiviral therapy directed toward pathogens that use retrograde trafficking to infect their hosts.

Review of the inhibition of biological activities of food-related selected toxins by natural compounds.

There is a need to develop food-compatible conditions to alter the structures of fungal, bacterial, and plant toxins, thus transforming toxins to nontoxic molecules. The term 'chemical genetics' has been used to describe this approach. This overview attempts to survey and consolidate the widely scattered literature on the inhibition by natural compounds and plant extracts of the biological (toxicological) activity of the following food-related toxins: aflatoxin B1, fumonisins, and ochratoxin A produced by fungi; cholera toxin produced by Vibrio cholerae bacteria; Shiga toxins produced by E. coli bacteria; staphylococcal enterotoxins produced by Staphylococcus aureus bacteria; ricin produced by seeds of the castor plant Ricinus communis; and the glycoalkaloid α-chaconine synthesized in potato tubers and leaves. The reduction of biological activity has been achieved by one or more of the following approaches: inhibition of the release of the toxin into the environment, especially food; an alteration of the structural integrity of the toxin molecules; changes in the optimum microenvironment, especially pH, for toxin activity; and protection against adverse effects of the toxins in cells, animals, and humans (chemoprevention). The results show that food-compatible and safe compounds with anti-toxin properties can be used to reduce the toxic potential of these toxins. Practical applications and research needs are suggested that may further facilitate reducing the toxic burden of the diet. Researchers are challenged to (a) apply the available methods without adversely affecting the nutritional quality, safety, and sensory attributes of animal feed and human food and (b) educate food producers and processors and the public about available approaches to mitigating the undesirable effects of natural toxins that may present in the diet.

High-throughput, cell-based screens to identify small-molecule inhibitors of ricin toxin and related category b ribosome inactivating proteins (RIPs).

Ricin is a member of the ubiquitous ribosome-inactivating protein (RIP) family of toxins. The Centers for Disease Control and Prevention (CDC) classify ricin and related toxins as Category B biothreat agents. There are currently no antidotes or therapeutics to counteract RIPs in humans. The discovery of effective small-molecule inhibitors of RIPs is increasingly possible, however, due to the availability and accessibility of diverse small-molecule chemical libraries coupled with robust robotics and automated screening methodologies. In this article, we describe a cell-based, high-throughput screening strategy and secondary assays that we have successfully used to identify compounds that target ricin toxin's enzymatic activity and intracellular trafficking, as well as stress-activated signaling pathways associated with cell death. The methods described in the protocol are amenable to the other RIPs.

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.

Small-molecule inhibitors of ricin and Shiga toxins.

This review summarizes the successes and continuing challenges associated with the identification of small-molecule inhibitors of ricin and Shiga toxins, members of the RNA N-glycosidase family of toxins that irreversibly inactivate eukaryotic ribosomes through the depurination of a conserved adenosine residue within the sarcin-ricin loop (SRL) of 28S rRNA. Virtual screening of chemical libraries has led to the identification of at least three broad classes of small molecules that bind in or near the toxin's active sites and thereby interfere with RNA N-glycosidase activity. Rational design is being used to improve the specific activity and solubility of a number of these compounds. High-throughput cell-based assays have also led to the identification of small molecules that partially, or in some cases, completely protect cells from ricin- and Shiga-toxin-induced death. A number of these recently identified compounds act on cellular proteins associated with intracellular trafficking or pro-inflammatory/cell death pathways, and one was reported to be sufficient to protect mice in a ricin challenge model.

7-Substituted pterins provide a new direction for ricin A chain inhibitors.

Ricin is a potent toxin found in castor seeds. The A chain, RTA, enzymaticlly depurinates a specific adenosine in ribosomal RNA, inhibiting protein synthesis. Ricin is a known chemical weapons threat having no effective antidote. This makes the discovery of new inhibitors of great importance. We have previously used 6-substituted pterins, such as pteroic acid, as an inhibitor platform with moderate success. We now report the success of 7-carboxy pterin (7CP) as an RTA inhibitor; its binding has been monitored using both kinetic and temperature shift assays and by X-ray crystallography. We also discuss the synthesis of various derivatives of 7CP, and their binding affinity and inhibitory effects, as part of a program to make effective RTA inhibitors.

Inhibition of retrograde transport protects mice from lethal ricin challenge.

Bacterial Shiga-like toxins are virulence factors that constitute a significant public health threat worldwide, and the plant toxin ricin is a potential bioterror weapon. To gain access to their cytosolic target, ribosomal RNA, these toxins follow the retrograde transport route from the plasma membrane to the endoplasmic reticulum, via endosomes and the Golgi apparatus. Here, we used high-throughput screening to identify small molecule inhibitors that protect cells from ricin and Shiga-like toxins. We identified two compounds that selectively block retrograde toxin trafficking at the early endosome-TGN interface, without affecting compartment morphology, endogenous retrograde cargos, or other trafficking steps, demonstrating an unexpected degree of selectivity and lack of toxicity. In mice, one compound clearly protects from lethal nasal exposure to ricin. Our work discovers the first small molecule that shows efficacy against ricin in animal experiments and identifies the retrograde route as a potential therapeutic target.

Vinyldeoxyadenosine in a sarcin-ricin RNA loop and its binding to ricin toxin a-chain.

8-Vinyl-2'-deoxyadenosine (8vdA) is a fluorophore with a quantum yield comparable to that of 2-aminopurine nucleoside. 8vdA was incorporated into a 10-mer stem-tetraloop RNA (8vdA-10) structure for characterization of the properties of the base, 8-vinyladenine (8-vA), with respect to adenine as a substrate or inhibitor for ribosome-inactivating proteins. Ricin toxin A-chain (RTA) and pokeweed antiviral protein (PAP) catalyze the release of adenine from a specific adenosine on a stem-tetraloop (GAGA) sequence at the elongation factor (eEF2) binding site of the 28S subunit of eukaryotic ribosomes, thereby arresting translation. RTA does not catalyze the release of 8-vinyladenine from 8vdA-10. Molecular dynamics simulations implicate a role for Arg180 in oxacarbenium ion destabilization and the lack of catalysis. However, 8vdA-10 is an active site analogue and inhibits RTA with a Ki value of 2.4 microM. Adenine is also released from the second adenosine in the modified tetraloop, demonstrating an alternative mode for the binding of this motif in the RTA active site. The 8vdA analogue defines the specificities of RTA for the two adenylate depurination sites in a RNA substrate with a GAGA tetraloop. The rate of nonenzymatic acid-catalyzed solvolysis of 8-vinyladenine from the stem-loop RNA is described. Unlike RTA, PAP catalyzes the slow release of 8-vinyladenine from 8vdA-10. The isolation of 8-vA and its physicochemical characterization is described.

Circular DNA and DNA/RNA hybrid molecules as scaffolds for ricin inhibitor design.

Ricin Toxin A-chain (RTA) catalyzes the hydrolytic depurination of A4324, the first adenosine of the GAGA tetra-loop portion of 28S eukaryotic ribosomal RNA. Truncated stem-loop versions of the 28S rRNA are RTA substrates. Here, we investigate circular DNA and DNA/RNA hybrid GAGA sequence oligonucleotides as minimal substrates and inhibitor scaffolds for RTA catalysis. Closing the 5'- and 3'-ends of a d(GAGA) tetraloop creates a substrate with 92-fold more activity with RTA (kcat/Km) than that for the d(GAGA) linear form. Circular substrates have catalytic rates (kcat) comparable to and exceeding those of RNA and DNA stem-loop substrates, respectively. RTA inhibition into the nanomolar range has been achieved by introducing an N-benzyl-hydroxypyrrolidine (N-Bn) transition state analogue at the RTA depurination site in a circular GAGA motif. The RNA/DNA hybrid oligonucleotide cyclic GdAGA provides a new scaffold for RTA inhibitor design, and cyclic G(N-Bn)GA is the smallest tight-binding RTA inhibitor (Ki = 70 nM). The design of such molecules that lack the base-paired stem-loop architecture opens new chemical synthetic approaches to RTA inhibition.

Monovalent and polyvalent carbohydrate inhibitors of ricin binding to a model of the cell-surface receptor.

A selection of galactose and lactose analogues was evaluated for their potency in inhibiting the binding of ricin to immobilised asialofetuin, which is a model of the cell-surface receptor for ricin. The aim was to identify compounds that could be used as antagonists of ricin toxicity in vivo, and as more selective, and therefore safer, antitoxins. Although one of these analogues had been identified by molecular modelling in a previous study as a potentially potent inhibitor, it and the other carbohydrates studied were less effective than galactose and lactose themselves (I(50) = 1.39 and 0.74 mM, respectively). In an attempt to increase the potency of carbohydrate-based inhibitors, galactose was coupled to the surface of dendrimers. No synergistic interactions were observed from this multivalent approach. Encouraging results, however, were obtained with a self-assembled lyotropic mesophase gel containing novel synthetic galactose-based surfactants, which was able to sequester ricin from aqueous solution in a 2-phase system.

A novel designed single domain antibody on 3-D structure of ricin A chain remarkably blocked ricin-induced cytotoxicity.

Ricin A chain (RA), an N-glycosidase, is able to fatally disrupt protein synthesis by attacking the Achilles heel of the ribosome RNA (rRNA). As specific immunotoxins, emergence of inhibitors for RA may obtain access to antagonistics against ricin intoxication and contribute to ameliorate the concomitant side effects. Many experimental results showed that the engineered VHs, which possessed solubility, stability, small size and consequently easier to express, purify and manipulate in vitro, were self and long-lived molecules compared to synthetic peptides. In this study, based on the crystal structure of RA, a novel recombinant human single-domain antibody expressing a polypeptide against RA in the CDR3 loop (named rVH(PT)) was obtained using computer-guided molecular design method. Theoretically, rVH(PT) could penetrate deeply into the active cleft of RA and act as a potent antagonist analogue to block the RA-rRNA interaction. Followed results showed that the recombinant VH(PT) was easily expressed of high-yield production and in a partially soluble fusion form in Escherichia coli. In vitro cytotoxicity experiments demonstrated that it possessed remarkable ability to block ricin-induced cytotoxicity. This study highlights the potential of human VH to display biostructure and biofunction of peptides designed on RA functional domain and could be useful in developing new antidotes with potential therapeutic uses to neutralize unintended exposure to ricin.

Monoclonal antibodies to ricin: in vitro inhibition of toxicity and utility as diagnostic reagents.

Monoclonal antibodies (MAbs) against ricin toxin (RT) and its subunits were produced in mice. The MAbs were initially selected based upon the ability to either bind ricin or the individual subunits in a solid-phase enzyme-linked immunosorbent assay (ELISA). Several candidates were selected for further evaluation, including their ability to inhibit ricin intoxication in vitro and their utility as immunodiagnostic reagents. Although their ability to capture antigen when bound to the solid phase was poor, some MAbs demonstrated potential utility as detection reagents in solid-phase immunoassays. Several MAbs were also able to inhibit ricin-mediated eukaryotic cell cytotoxicity in vitro. These MAbs may prove useful for preventing and/or treating ricin intoxication.

A novel neutralizing monoclonal antibody against cell-binding polypeptide of ricin.

One strain of neutralizing monoclonal antibody (MAb) against cell-binding polypeptide of ricin, named 3E1, was generated efficiently. The antibody recognized the linearity epitope of RTB located in a toxin structure domain characterized by Western blotting. The safe period of mice for intraperitoneal injection of 100 microg of antibody was 20 min after intraperitoneal injection of 2 microg of Ricin (10 times LD50). The neutralizing MAb we obtained could be developed into an immunotherapeutic agent to counteract the use of ricin as a terrorist or biological warfare weapon. It might be useful, as well, for antibody-based prophylaxis.