Heinz Faulstich

Amatoxins, phallotoxins, phallolysin, and antamanide: the biologically active components of poisonous Amanita mushrooms.

This review gives a comprehensive account of the molecular toxicology of the bicyclic peptides obtained from the poisonous mushrooms of the genus Amanita. The discussion of the biochemical events will be preceded by a consideration of the chemistry of the toxic peptides. The structural features essential for biological activities of both the amatoxins and the phallotoxins will be discussed, also including the most important analytical data. Similar consideration will be given to antamanide, a cyclic peptide, which counteracts phalloidin. In addition, the phallolysins, three cytolytic proteins from Amanita phalloides will be discussed. The report on the biological activity of the amatoxins will deal with the sensitivity of the different RNA-polymerases towards the toxins and with their action on various cell types. Consideration will also be given to systems in which alpha-amanitin was used and can be used as a molecular tool; in the past, many investigators used the inhibitor in molecular biology, genetics, and even in physiological research. As for the phallotoxins, discussion of the affinity of these toxins for actin is provied. Further discussion attempts to understand the course of intoxication by filling in the gap between the first molecular event, formation of microfilaments, and the various lesions in hepatocytes during the intoxication.

Involvement of ezrin/moesin in de novo actin assembly on phagosomal membranes

The current study focuses on the molecular mechanisms responsible for actin assembly on a defined membrane surface: the phagosome. Mature phagosomes were surrounded by filamentous actin in vivo in two different cell types. Fluorescence microscopy was used to study in vitro actin nucleation/polymerization (assembly) on the surface of phagosomes isolated from J774 mouse macrophages. In order to prevent non‐specific actin polymerization during the assay, fluorescent G‐actin was mixed with thymosin β4. The cytoplasmic side of phagosomes induced de novo assembly and barbed end growth of actin filaments. This activity varied cyclically with the maturation state of phagosomes, both in vivo and in vitro. Peripheral membrane proteins are crucial components of this actin assembly machinery, and we demonstrate a role for ezrin and/or moesin in this process. We propose that this actin assembly process facilitates phagosome/endosome aggregation prior to membrane fusion.

Fluorescent phallotoxins as probes for filamentous actin

ConclusionsPhallotoxins have been conjugated to four different fluorescent labels: fluorescein, tetramethylrhodamine, nitrobenzoxadiazole and coumarine. Although endowed with different substrate affinities, they are all specific for polymeric actin.Useful advantage can and has been taken of the special characteristics of the fluorescent phallotoxin derivatives. Among them:1.Their specific labelling of F-actin, native or chemically fixed, in a simple one-step procedure, making them useful probes for single and multiple fluorescent labelling of cytoskeletal and other preparations.2.Their stabilization of F-actin against depolymerization and benign effects on actomyosin-based movement, important factors inin vitro motility systems. The stabilizing effect of phalloidin leads also to improved structural preservation in immunofluorescence preparations.3.Their small size, allowing ready penetration into cytoskeletal networks, in fixed and living cells.4.Their long-term stability, dry or in organic solvents. From their various useful properties we can expect fluorescent phallotoxins to find further and new applications in studies of actin-based phenomena.

Preparation of tetramethylrhodaminyl-phalloidin and uptake of the toxin into short-term cultured hepatocytes by endocytosis

A fluorescent phallotoxin with high photostability, tetramethylrhodaminyl-phalloidin (Rh-phalloidin), has been prepared. The affinity of this compound to rabbit muscle actin has been determined to be about 6 times lower than that of phalloidin. In freshly isolated hepatocytes the internalized fluorescent toxin stains the cellular actin. In contrary, there is no actin staining visible in cultured hepatocytes. Short-term cultured hepatocytes (5 h of culturing) incorporate the toxin by endocytosis; it is kept sealed in the endocytotic vesicles, which are usually found accumulated at the sites where cells touch after reaggregation.

Therapeutic Potential of Amanitin-Conjugated Anti-Epithelial Cell Adhesion Molecule Monoclonal Antibody Against Pancreatic Carcinoma

BACKGROUND Human epithelial cell adhesion molecule (EpCAM) is overexpressed in many cancers. Anti-EpCAM antibodies have shown promise in preclinical studies, but showed no tumor regression in a recent phase II clinical trial. Therefore, we generated a novel anti-EpCAM antibody-drug conjugate and assessed whether it showed enhanced antitumor effects. METHODS Chemical cross-linking was conducted to covalently conjugate α-amanitin, a toxin known to inhibit DNA transcription, with chiHEA125, a chimerized anti-EpCAM monoclonal antibody, to generate the antibody-drug conjugate α-amanitin-glutarate-chiHEA125 (chiHEA125-Ama). Antiproliferative activity of chiHEA125-Ama was tested in human pancreatic (BxPc-3 and Capan-1), colorectal (Colo205), breast (MCF-7), and bile duct (OZ) cancer cell lines in vitro using [(3)H]-thymidine incorporation assay. Antitumor activity of chiHEA125-Ama was assessed in vivo in immunocompromised mice bearing subcutaneous human BxPc-3 pancreatic carcinoma xenograft tumors (n = 66 mice). Cell proliferation and apoptosis were evaluated in xenograft tumors by immunohistochemistry. All statistical tests were two-sided. RESULTS In all cell lines, chiHEA125-Ama reduced cell proliferation (mean half maximal inhibitory concentration [IC(50)] = 2.5 × 10(-10) to 5.4 × 10(-12) M). A single dose of chiHEA125-Ama inhibited BxPc-3 xenograft tumor growth (chiHEA125 [control, n = 4 mice] vs. chiHEA125-Ama [n = 6 mice], dose of 15 mg/kg with respect to IgG and 50 μg/kg with respect to α-amanitin, mean relative increase in tumor volume on day 16 = 884% vs. -79%, difference = 963%, 95% CI = 582% to 1344%, P = .019). Two higher doses of chiHEA125-Ama (100 μg/kg with respect to α-amanitin), administered 1 week apart (n = 10 mice per group), led to complete tumor regression in nine of 10 (90%) mice compared with chiHEA125, during the observation period of 16 days; increased apoptosis and reduced cell proliferation were observed in mice treated with chiHEA125-Ama. CONCLUSION This preclinical study suggests that anti-EpCAM antibody conjugates with α-amanitin have the potential to be highly effective therapeutic agents for pancreatic carcinomas and various EpCAM-expressing malignancies.

Fifty years of amanitin

Pharmacokinetic studies have provided new insights into humanAmanita poisoning, but it appears to be impossible to treat this intoxication by immunotherapy. New synthetic analogs have revealed structure-activity relationships that were unknown so far. The main toxin, α-amanitin, is in constant use as a tool in molecular biology and in biological research. First experiments have been reported in which amanitin bound to polymers could be internalized into tumor cells via a receptor-mediated endocytosis.

Simultaneous assay for amatoxins and phallotoxins in Amanita phalloides Fr. by high-performance liquid chromatography

A reversed-phase high-performance liquid chromatographic method is described that allows the simultaneous determination of up to eight amatoxins and phallotoxins. The method identifies both neutral toxins (alpha- and gamma-amanitin, phalloidin, phallisin and phalloin) and acidic toxins (beta-amanitin, phallacidin and phallisacin). Toxins were separated, identified and determined by gradient elution with 0.02 M aqueous ammonium acetate-acetonitrile and simultaneous monitoring of the absorbances at 214 and 295 nm. The assay was successfully applied to the analysis of the toxins in a crude extract of Amanita phalloides. The limit of detection for each toxin was 10 ng/ml of extraction medium. The assay was further validated by analysing the toxin content in Galerina marginata, a species containing only amatoxins. This relatively simple method should be suitable for the detection of amatoxins and phallotoxins in almost any species of mushrooms.

Characterization of a transporting system in rat hepatocytes. Studies with competitive and non-competitive inhibitors of phalloidin transport

Primary cultures of rat hepatocytes were used for assaying several drugs not previously known for inhibiting the transport of phalloidin. In order to have 50% inhibition (IC50) of the entrance of a tritiated phallotoxin derivative ([3H]demethylphalloin, 1 microM) from the medium into the cells the following concentrations (microM) of the various inhibitors were determined: cyclolinopeptide (0.5), Nocloprost (5.0), Nileprost (7.0), beta-estradiol (42), Verapamil (70). For comparison, the corresponding IC50 values of some known antagonists of phalloidin toxicity were determined by the same method. Moreover, we studied several natural and synthetic phallotoxins and alpha-amanitin for their ability to displace [3H]demethylphalloin from the transporting system. Lineweaver-Burk plots made it obvious that two groups of inhibitors exist. Competitive inhibitors are, for example, antamanide, beta-estradiol, silybin, Nileprost, taurocholate, and the cyclic somatostatin analog cyclo[Phe-Thr-Lys-Trp-Phe-D-Pro], whereas Verapamil and monensin inhibit phallotoxin uptake in a non-competitive way. Considering the very different chemical features of the competitive inhibitors, we tentatively conclude that the phallotoxin transport system selects compounds not on the basis of their chemical features, but rather their physical properties. The physical properties of a typical substrate are low molecular mass, lipophilic nature, and, possibly the presence of rigid ring structures. Negative charges accelerate the transport of a substrate, while positive charges have the opposite effect. The phalloidin-transporting system may represent part of a hepatic equipment which clears portal blood from, for example, bile acids, lipophilic hormones, or xenobiotics. By chance, the transporting system incorporates phallotoxins into the hepatocytes leading to the death of these cells.

A rapid radioimmunoassay, using a nylon support, for amatoxins from Amanita mushrooms

A fetuin derivative of alpha-amanitin was prepared and used as an antigen in rabbits. The antigen was superior to previous bovine serum albumin derivatives of beta-amanitin by its lower toxicity and high immunogenicity. On the other hand, the antibodies raised with the alpha-amanitin derivative did not show full crossreactivity with the other amatoxins, as did the immunoglobulins induced by protein derivatives of beta-amanitin. The procedure for activating nylon surfaces and coupling proteins onto them was improved with respect to surface charge and homogeneity. A partially purified IgG-fraction derived from the sera of rabbits immunized against amatoxins was covalently attached to the activated nylon surfaces. The covalently coupled immunoglobulins were complexed with a tritiated amatoxin. Then small pieces of the nylon sheet were punched out and incubated with the amatoxin solution to be analyzed. This procedure represents a method for dosing, in one step and without pipetting, the immunoglobulins and the labelled hapten. Determination of amatoxin concentrations was achieved by counting the radioactivity in the incubation fluid. The limit of detection was about 3 ng of amatoxins per ml. The radioimmunoassay was used to measure amatoxin concentrations in serum, urine, duodenal fluid, and gastric juice of patients with Amanita poisoning. Since such assays can be performed in 2-3 hr, the results can be used to determine the therapeutic protocol. The assay was likewise used to determine the concentration of amatoxins in mushroom tissue. For Amanita phalloides, for example, we found that the amatoxin concentration (mg/g dry weight) is 4.5 times higher in the gills than in the bulb.

Use of bimanyl actin derivative (TMB-actin) for studying complexation of β-thymosins: Inhibition of actin polymerization by thymosin β9

By reacting trimethylammoniobromobimane bromide (TMB bromide) with rabbit muscle actin, a fluorescent reporter group was linked to cysteine at position 374. Fluorescence of TMB‐actin decreased significantly on addition of thymosin β4 (Tβ4), a peptide of 43 amino acid residues reported to bind to monomeric actin and to prevent filament formation. Based on this effect, we determined the K d value of the thymosin β4 complex as 0.8 μM, a value that is in agreement with previous determinations. In addition to the main compound thymosin β4, bovine tissue contains a related peptide, thymosin β9 (Tβ9), which has 41 amino acid residues and ca. 75% sequence homology. In the present study we show for the first time that Tβ9, similar to Tβ4, forms a 1:1 complex with monomeric actin, and hereby inhibits actin polymerization. With a K d value of 1.1 μM the affinity of Tβ9 is in the same range as that of Tβ4, suggesting that Tβ9, like Tβ4, contributes to maintaining the pool of monomeric actin in bovine non‐muscle cells. Further proof of the interaction of Tβ9 with actin was provided by native PAGE, where the complex showed the reported higher mobility, as well as by crosslinking experiments. Using different crosslinking reagents, like water‐soluble carbodiimide (EDC), m‐maleimidobenzoyl‐N‐hydroxysuccinimidate (MBS), and disuccinimidylsuberate (DSS), we were able to produce conjugates of 47 kDa. In one of these (from MBS) both actin and Tβ9 could be identified by immunoblotting. When, in the MBS crosslinking experiments, native actin was replaced with (374‐NEM)actin, the 47 kDa band was not seen, indicating that Cys‐374 takes part in the thiol‐specific crosslinking reaction. This suggests that part of the binding site of Tβ9 must be located close to the carboxy‐tenninus.