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New in silico insights into the inhibition of RNAP II by α-amanitin and the protective effect mediated by effective antidotes

Publication . Garcia, Juliana; Carvalho, Alexandra T.P.; Dourado, Daniel F.A.R.; Baptista, Paula; Bastos, Maria de Lourdes; Carvalho, Félix

Poisonous α-amanitin-containing mushrooms are responsible for the major cases of fatalities after mushroom ingestion. α-Amanitin is known to inhibit the RNA polymerase II (RNAP II), although the underlying mechanisms are not fully understood. Benzylpenicillin, ceftazidime and silybin have been the most frequently used drugs in the management of α-amanitin poisoning, mostly based on empirical rationale. The present study provides an in silico insight into the inhibition of RNAP II by α-amanitin and also on the interaction of the antidotes on the active site of this enzyme. Docking and molecular dynamics (MD) simulations combined with molecular mechanics- generalized Born surface area method (MM-GBSA) were carried out to investigate the binding of α-amanitin and three antidotes benzylpenicillin, ceftazidime and silybin to RNAP II. Our results reveal that α-amanitin should affects RNAP II transcription by compromising trigger loop (TL) function. The observed direct interactions between α-amanitin and TL residues Leu1081, Asn1082, Thr1083, His1085 and Gly1088 alters the elongation process and thus contribute to the inhibition of RNAP II. We also present evidences that α-amanitin can interact directly with the bridge helix residues Gly819, Gly820 and Glu822, and indirectly with His816 and Phe815. This destabilizes the bridge helix, possibly causing RNAP II activity loss. We demonstrate that benzylpenicillin, ceftazidime and silybin are able to bind to the same site as α-amanitin, although not replicating the unique α-amanitin binding mode. They establish considerably less intermolecular interactions and the ones existing are essential confine to the bridge helix and adjacent residues. Therefore, the therapeutic effect of these antidotes does not seem to be directly related with binding to RNAP II. RNAP II α-amanitin binding site can be divided into specific zones with different properties provi ding a reliable platform for the structure-based drug design of novel antidotes for α-amatoxin poisoning. An ideal drug candidate should be a competitive RNAP II binder that interacts with Arg726, Ile756, Ala759, Gln760 and Gln767, but not with TL and bridge helix residues.

2014Journal article

Open access

Co-ingestion of amatoxins and isoxazoles-containing mushrooms and successful treatment: A case report

Publication . Garcia, Juliana; Costa, Vera M.; Costa, Ana Elisa; Andrade, Sérgio; Carneiro, Ana Cristina; Conceição, Filipe; Paiva, José Artur; Pinho, P. Guedes de; Baptista, Paula; Bastos, Maria de Lourdes; Carvalho, Félix

Mushroom poisonings occur when ingestion of wild mushrooms containing toxins takes place, placing the consumers at life-threatening risk. In the present case report, an unusual multiple poisoning with isoxazoles- and amatoxins-containing mushrooms in a context of altered mental state and poorly controlled hypertension is presented. A 68-year-old female was presented to São João hospital (Portugal) with complaints of extreme dizziness, hallucinations, vertigo and imbalance, 3 h after consuming a stew of wild mushrooms. The first observations revealed altered mental state and elevated blood pressure. The examination of cooked mushroom fragments allowed a preliminary identification of Amanita pantherina. Gas chromatography-mass spectrometry (GC-MS) showed the presence of muscimol in urine. Moreover, through high-performance liquid chromatography-ultraviolet detection (HPLC-UV) analysis of the gastric juice, the presence of α-amanitin was found, showing that amatoxins-containing mushrooms were also included in the stew. After 4 days of supportive treatment, activated charcoal, silybin and N-acetylcysteine, the patient recovered being discharged 10 days post-ingestion with no organ complications. The prompt and appropriate therapy protocol for life-threatening amatoxins toxicity probably saved the patient's life as oral absorption was decreased and also supportive care was immediately started.

2015Journal article

Open access

Amanita phalloides poisoning: Mechanisms of toxicity and treatment

Publication . Garcia, Juliana; Costa, Vera M.; Carvalho, Alexandra T.P.; Baptista, Paula; Pinho, P. Guedes de; Bastos, Maria de Lourdes; Carvalho, Félix

Amanita phalloides, also known as 'death cap', is one of the most poisonous mushrooms, being involved in the majority of human fatal cases of mushroom poisoning worldwide. This species contains three main groups of toxins: amatoxins, phallotoxins, and virotoxins. From these, amatoxins, especially α-amanitin, are the main responsible for the toxic effects in humans. It is recognized that α-amanitin inhibits RNA polymerase II, causing protein deficit and ultimately cell death, although other mechanisms are thought to be involved. The liver is the main target organ of toxicity, but other organs are also affected, especially the kidneys. Intoxication symptoms usually appear after a latent period and may include gastrointestinal disorders followed by jaundice, seizures, and coma, culminating in death. Therapy consists in supportive measures, gastric decontamination, drug therapy and, ultimately, liver transplantation if clinical condition worsens. The discovery of an effective antidote is still a major unsolved issue. The present paper examines the clinical toxicology of A. phalloides, providing the currently available information on the mechanisms of toxicityinvolved and on the current knowledge on the treatment prescribed against this type of mushrooms. Antidotal perspectives will be raised as to set the pace to new and improved therapy against these mushrooms.

2015Journal article

Open access

A breakthrough on Amanita phalloides poisoning: an effective antidotal effect by polymyxin B

Publication . Garcia, Juliana; Costa, Vera M.; Carvalho, Alexandra T.P.; Silvestre, Ricardo; Duarte, José A.; Dourado, Daniel F.A.R.; Arbo, Marcelo D.; Baltazar, Teresa; Dinis-Oliveira, Ricardo Jorge; Baptista, Paula; Bastos, Maria de Lourdes; Carvalho, Félix

Amanita phalloides is responsible for more than 90 % of mushroom-related fatalities, and no effective antidote is available. α-Amanitin, the main toxin of A. phalloides, inhibits RNA polymerase II (RNAP II), causing hepatic and kidney failure. In silico studies included docking and molecular dynamics simulation coupled to molecular mechanics with generalized Born and surface area method energy decomposition on RNAP II. They were performed with a clinical drug that shares chemical similarities to α-amanitin, polymyxin B. The results show that polymyxin B potentially binds to RNAP II in the same interface of α-amanitin, preventing the toxin from binding to RNAP II. In vivo, the inhibition of the mRNA transcripts elicited by α-amanitin was efficiently reverted by polymyxin B in the kidneys. Moreover, polymyxin B significantly decreased the hepatic and renal α-amanitin-induced injury as seen by the histology and hepatic aminotransferases plasma data. In the survival assay, all animals exposed to α-amanitin died within 5 days, whereas 50 % survived up to 30 days when polymyxin B was administered 4, 8, and 12 h post-α-amanitin. Moreover, a single dose of polymyxin B administered concomitantly with α-amanitin was able to guarantee 100 % survival. Polymyxin B protects RNAP II from inactivation leading to an effective prevention of organ damage and increasing survival in α-amanitin-treated animals. The present use of clinically relevant concentrations of an already human-use-approved drug prompts the use of polymyxin B as an antidote for A. phalloides poisoning in humans.

2015Journal article

Open access

Determination of amatoxins and phallotoxins in Amanita phalloides mushrooms from northeastern Portugal by HPLC-DAD-MS

Publication . Garcia, Juliana; Oliveira, Ana Paula Leandro; Pinho, P. Guedes de; Freitas, Victor; Carvalho, Alexandra T.P.; Baptista, Paula; Pereira, Eric Carvalho; Bastos, Maria de Lourdes; Carvalho, Félix

Amanita phalloides is a toxic mushroom responsible for the majority of deaths occurring after mushrooms ingestion, mainly due to amatoxins. In the present study the contents and distribution of the major amatoxins and phallotoxins in different tissues of A. phalloides from two different sites of Portugal were analyzed by liquid chromatography (LC) coupled to diode array (DAD) and mass spectrometry (MS) detection. The main toxins were separated by LC and its chemical structures confirmed by MS. a-Amanitin contents in caps, stipe and volva tissues were quantified by RP-HPLC. The results show that caps have the highest content of amatoxins, whereas the volva was richest in phallotoxins. Moreover variability in the toxins composition from different geographic sites was also observed. This study provides for the first time the content of toxins in A. phalloides from Portugal.

2015Journal article

Open access