Chemoenzymatic Synthesis of 4,5-Dihydroxyisoleucine Fragment of α-Amanitin.

The ability of α-amanitin to potently inhibit RNA polymerase II (RNAP II) has elicited further research into its use as a novel payload for antibody-drug conjugates. Despite this promise, the de novo synthesis of α-amanitin is still a major challenge as it possesses an unusual bicyclic octapeptide structure that contains several oxidized amino acids, most notably 4,5-dihydroxy-l-isoleucine. Here, we report a concise chemoenzymatic synthesis of this key amino acid residue, which features two regioselective and diastereoselective enzymatic C-H oxidations on l-isoleucine.

Ultrasensitive Aptasensor for α-Amatoxin Detection Based on the DNA Tetrahedral Nanostructure Triggering Rolling Circle Amplification and Signal Amplification.

Poisonous mushrooms containing α-amatoxin can be lethal, making it imperative to develop a rapid and sensitive detection method for α-amatoxin. Utilizing the DNA tetrahedral structure as its foundation, the aptamer allows controlled density and orientation. Consequently, we designed aptamer tetrahedral functionalized magnetic beads that specifically target α-amanitin to release complementary DNA (C-DNA) strands. These strands were then employed as primers to initiate rolling circle amplification (RCA) with fluorescent dyes. The combination of SYBR Green I detection probes facilitated the amplification of the detection signal, enhancing the detection sensitivity of the aptasensor. The calculated detection limit was determined to be 3 ng/mL, a magnitude lower than that of other aptasensors by 2 orders of magnitude. The aptasensor integrates the advantages of high sensitivity and specificity, offering a simple and reliable rapid detection method for α-amanitin analysis.

The occurrence of ansamers in the synthesis of cyclic peptides.

α-Amanitin is a bicyclic octapeptide composed of a macrolactam with a tryptathionine cross-link forming a handle. Previously, the occurrence of isomers of amanitin, termed atropisomers has been postulated. Although the total synthesis of α-amanitin has been accomplished this aspect still remains unsolved. We perform the synthesis of amanitin analogs, accompanied by in-depth spectroscopic, crystallographic and molecular dynamics studies. The data unambiguously confirms the synthesis of two amatoxin-type isomers, for which we propose the term ansamers. The natural structure of the P-ansamer can be ansa-selectively synthesized using an optimized synthetic strategy. We believe that the here described terminology does also have implications for many other peptide structures, e.g. norbornapeptides, lasso peptides, tryptorubins and others, and helps to unambiguously describe conformational isomerism of cyclic peptides.

Utilizing the DNA Aptamer to Determine Lethal α-Amanitin in Mushroom Samples and Urine by Magnetic Bead-ELISA (MELISA).

Amanita poisoning is one of the most deadly types of mushroom poisoning. α-Amanitin is the main lethal toxin in amanita, and the human-lethal dose is about 0.1 mg/kg. Most of the commonly used detection techniques for α-amanitin require expensive instruments. In this study, the α-amanitin aptamer was selected as the research object, and the stem-loop structure of the original aptamer was not damaged by truncating the redundant bases, in order to improve the affinity and specificity of the aptamer. The specificity and affinity of the truncated aptamers were determined using isothermal titration calorimetry (ITC) and gold nanoparticles (AuNPs), and the affinity and specificity of the aptamers decreased after truncation. Therefore, the original aptamer was selected to establish a simple and specific magnetic bead-based enzyme linked immunoassay (MELISA) method for α-amanitin. The detection limit was 0.369 µg/mL, while, in mushroom it was 0.372 µg/mL and in urine 0.337 µg/mL. Recovery studies were performed by spiking urine and mushroom samples with α-amanitin, and these confirmed the desirable accuracy and practical applicability of our method. The α-amanitin and aptamer recognition sites and binding pockets were investigated in an in vitro molecular docking environment, and the main binding bases of both were T3, G4, C5, T6, T7, C67, and A68. This study truncated the α-amanitin aptamer and proposes a method of detecting α-amanitin.

Development of a simple and reliable method for α-amanitin detection in rat plasma and its application to a toxicokinetic study.

RATIONALE: α-Amanitin is a highly toxic peptide widely found in species of poisonous mushrooms. The matrix effect has been a major obstacle for accurate determination of α-amanitin in plasma samples by liquid chromatography/tandem mass spectrometry (LC/MS/MS). In this study, the strategy to eliminate the matrix effect of α-amanitin with a one-step dilution approach after deproteinization was applied. METHODS: Rat plasma samples were processed by protein precipitation with methanol followed by a nine-fold dilution with pure water. The matrix effect value of α-amanitin was 19.7%-22.2% by protein precipitation and then changed to 87.5%-88.7% after dilution. α-Amanitin and the internal standard (roxithromycin) were analyzed on an ACQUITY UPLC® BEH C18 (50 mm × 2.1 mm, 1.7 µm) column within 3.0 min by gradient elution. RESULTS: The linear ranges were 0.90-600 ng/mL with a correlation coefficient r >0.9958. A lower limit of quantification (LLOQ) of 0.90 ng/mL was achieved using only 50 µL of rat plasma. The intra- and inter-day precisions for the analyte ranged from 3.2% to 7.5% and 3.1% to 7.1%, respectively, and the accuracy ranged from -5.3% to -8.0%. CONCLUSIONS: The matrix effect of α-amanitin was reduced by sample dilution after plasma deproteinization. A reliable LC/MS/MS method for the determination of α-amanitin in rat plasma was developed. This method was successfully applied for a toxicokinetic study of rats after intravenous injection of α-amanitin with a subacute toxicity dose at 0.10 mg/kg.

In vivo and in vitro α-amanitin metabolism studies using molecular networking.

Amanitin poisonings are among the most life-threatening mushroom poisonings, and are mainly caused by the genus Amanita. Hepatotoxicity is the hallmark of amanitins, powerful toxins contained in these mushrooms, and can require liver transplant. Among amatoxins, α-amanitin is the most studied. However, the hypothesis of a possible metabolism of amanitins is still controversial in this pathophysiology. Therefore, there is a need of clarification using cutting-edge tools allowing metabolism study. Molecular network has emerged as powerful tool allowing metabolism study through organization and representation of untargeted tandem mass spectrometry (MS/MS) data in a graphical form. The aim of this study is to investigate amanitin metabolism using molecular networking. In vivo (four positive amanitin urine samples) and in vitro (differentiated HepaRG cells supernatant incubated with α-amanitin 2 µM for 24 h) samples were extracted and analyzed by LC-HRMS/MS using a Q Exactive™ Orbitrap mass spectrometer. Using molecular networking on both in vitro and in vivo, we have demonstrated that α-amanitin does not undergo metabolism in human. Thus, we provide solid evidence that a possible production of amanitin metabolites cannot be involved in its toxicity pathways. These findings can help to settle the debate on amanitin metabolism and toxicity.

Enhancing the Pharmacokinetics and Antitumor Activity of an α-Amanitin-Based Small-Molecule Drug Conjugate via Conjugation with an Fc Domain.

Herein we describe the design and biological evaluation of a novel antitumor therapeutic platform that combines the most favorable properties of small-molecule drug conjugates (SMDCs) and antibody drug conjugates (ADCs). Although the small size of SMDCs, compared to ADCs, is an appealing feature for their application in the treatment of solid tumors, SMDCs usually suffer from poor pharmacokinetics, which severely limits their therapeutic efficacy. To overcome this limitation, in this proof-of-concept study we grafted an α-amanitin-based SMDC that targets prostate cancer cells onto an immunoglobulin Fc domain via a two-step "program and arm" chemoenzymatic strategy. We demonstrated the superior pharmacokinetic properties and therapeutic efficacy of the resulting Fc-SMDC over the SMDC in a prostate cancer xenograft mouse model. This approach may provide a general strategy toward effective antitumor therapeutics combining small size with pharmacokinetic properties close to those of an ADC.

Toxin components and toxicological importance of Galerina marginata from Turkey.

Amatoxins, most of which are hepatotoxic, can cause fatal intoxication. While mushrooms in the amatoxin-containing Galerina genus are rare, they can poison humans and animals worldwide. Few studies have profiled the toxicity of Galerina marginata. In addition, many studies indicate that macrofungi can have different characteristics in different regions. In this study, the quantities of toxins present in G. marginata from different provinces in Turkey were analysed using reversed-phase high-performance liquid chromatography with ultraviolet detection (RP-HPLC-UV) and liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS). G. marginata samples were collected from three different regions of Turkey. The taxonomic categorization of mushrooms was based on their micro- and macroscopic characteristics. The presence of toxins α-amanitin (AA), ß-amanitin (BA), γ-amanitin (GA), phalloidin (PHD) and phallacidin (PHC) quantities were measured using RP-HPLC-UV and then were confirmed using LC-ESI-MS/MS. BA levels were higher than AA levels in G. marginata mushrooms collected from all three regions. Moreover, the levels of GA were below the detection limit and no phallotoxins were detected. This is the first study to identify and test the toxicity of G. marginata collected from three different regions of Turkey using RP-HPLC-UV. This is also the first study to confirm the UV absorption of amatoxins in G. marginata using LC-ESI-MS/MS, which is a far more sensitive process. More studies evaluating the toxicity of G. marginata in other geographic regions of the world are needed.

Extensive screening of cyclopeptide toxins in mushrooms by ultra-high-performance liquid chromatography coupled with quadrupole-Orbitrap mass spectrometry.

A non-target screening method of cyclopeptide toxins and their analogues in mushroom was developed, using ultra-high-performance liquid chromatography coupled with quadrupole Orbitrap mass spectrometry (UHPLC-Q-Orbitrap MS) followed by mass spectrometry databases retrieval and software tools analysis for the candidate analogues. Three cyclopeptide toxins in the toxic mushroom Amanita rimosa were firstly screened without standard, and two of them were unknown analogues which were tentatively identified by the accurate masses, isotopic patterns and characteristic fragments. A validated quantitative method was performed to rapidly quantify three major cyclopeptide toxins in the Amanita rimosa sample including α-manitin, ß-amanitin and phalloidin, and their contents were detected to be 4.52 mg/kg, 2.37 mg/kg and 2.53 mg/kg, respectively. The developed method has good selectivity and sensitivity for rapid and comprehensive screening the cyclopeptide toxins and their analogues in mushrooms at trace levels. Successful non-target screening of trace cyclopeptide toxin analogues will guarantee the food safety in mushrooms consumption.

Total Synthesis of α- and ß-Amanitin.

α-Amanitin and related amatoxins have been studied for more than six decades mostly by isolation from death cap mushrooms. The total synthesis, however, remained challenging due to unique structural features. α-Amanitin is a potent inhibitor of RNA polymerase II. Interrupting the basic transcription processes of eukaryotes leads to apoptosis of the cell. This unique mechanism makes the toxin an ideal payload for antibody-drug conjugates (ADCs). Only microgram quantities of toxins, when delivered selectively to tumor sites through conjugation to antibodies, are sufficient to eliminate malignant tumor cells of almost every origin. By solving the stereoselective access to dihydroxyisoleucine, a photochemical synthesis of the tryptathion precursor, solid-phase peptide synthesis, and macrolactamization we obtained a scalable synthetic route towards synthetic α-amanitin. This makes α-amanitin and derivatives now accessible for the development of new ADCs.

A Convergent Total Synthesis of the Death Cap Toxin α-Amanitin.

The toxic bicyclic octapeptide α-amanitin is mostly found in different species of the mushroom genus Amanita, with the death cap (Amanita phalloides) as one of the most prominent members. Due to its high selective inhibition of RNA polymerase II, which is directly linked to its high toxicity, particularly to hepatocytes, α-amanitin received an increased attention as a toxin-component of antibody-drug conjugates (ADC) in cancer research. Furthermore, the isolation of α-amanitin from mushrooms as the sole source severely restricts compound supply as well as further investigations, as structure-activity relationship (SAR) studies. Based on a straightforward access to the non-proteinogenic amino acid dihydroxyisoleucine, we herein present a robust total synthesis of α-amanitin providing options for production at larger scale as well as future structural diversifications.

Novel Method for Preparation of Site-Specific, Stoichiometric-Controlled Dual Warhead Conjugate of FGF2 via Dimerization Employing Sortase A-Mediated Ligation.

Targeted therapies are rapidly evolving modalities of cancer treatment. The largest group of currently developed biopharmaceuticals is antibody-drug conjugates (ADCs). Here, we developed a new modular strategy for the generation of cytotoxic bioconjugates, containing a homodimer of targeting protein and two highly potent anticancer drugs with distinct mechanisms of action. Instead of antibody, we applied human fibroblast growth factor 2 (FGF2) as a targeting protein. We produced a conjugate of FGF2 with either monomethyl auristatin E (MMAE) or α-amanitin (αAMTN) as a cytotoxic agent and subsequently applied a sortase A-mediated ligation to obtain a dimeric conjugate containing both MMAE and αAMTN. The developed method ensures site-specific conjugation and a controlled drug-to-protein ratio. We validated our approach by demonstrating that dimeric dual warhead conjugate exhibits higher cytotoxic potency against fibroblast growth factor receptor-positive cell lines than single-warhead conjugates. Our modular technology can be applied to other targeting proteins or drugs and thus can be used for preparation of different bioconjugates.

FGF2 Dual Warhead Conjugate with Monomethyl Auristatin E and α-Amanitin Displays a Cytotoxic Effect towards Cancer Cells Overproducing FGF Receptor 1.

In the rapidly developing field of targeted cancer therapy there is growing interest towards therapeutics combining two or more compounds to achieve synergistic action and minimize the chance of cancer resistance to treatment. We developed a fibroblast growth factor 2 (FGF2)-conjugate bearing two cytotoxic drugs with independent mode of action: α-amanitin and monomethyl auristatin E. Drugs are covalently attached to the targeting protein in a site-specific manner via maleimide-thiol conjugation and Cu(I)-catalyzed alkyne-azide cycloaddition. The dual warhead conjugate binds to FGF receptor 1 (FGFR1) and utilizes receptor-mediated endocytosis for selective internalization into cancer cells with FGFR1. The developed conjugate displays high cytotoxicity towards all tested FGFR1-positive cell lines. Most importantly, the improved cytotoxic effect of both drugs is observed for lung cancer cell line NCI-H446. The single drug-FGF2 conjugates have no impact on the viability of NCI-H446 cells, whereas the dual warhead-FGF2 conjugate selectively and efficiently kills these FGFR1 positive cancer cells. Due to the diversified mode of action the dual warhead-FGF2 conjugate may overcome the potential acquired resistance of FGFR1-overproducing cancer cells towards single cytotoxic drugs.

Chemical Synthesis of Bioactive Naturally Derived Cyclic Peptides Containing Ene-Like Rigidifying Motifs.

The development of synthetic methods to prepare conformationally constrained peptides and peptide-polyketide hybrids remain an important chemical challenge. It is known that structural rigidity correlates with the specificity, bioactivity, and stability of these peptide systems, thus rigid systems are particularly attractive leads for development of potent biopharmaceuticals. Herein we provide an overview of recent developments in the syntheses of naturally derived constrained peptides and peptide-polyketide hybrids, with a particular emphasis on those systems containing an ene-like bond.

Synthesis of the Death-Cap Mushroom Toxin α-Amanitin.

α-Amanitin is an extremely toxic bicyclic octapeptide isolated from the death-cap mushroom, Amanita phalloides. As a potent inhibitor of RNA polymerase II, α-amanitin is toxic to eukaryotic cells. Recent interest in α-amanitin arises from its promise as a payload for antibody-drug conjugates. For over 60 years, A. phalloides has been the only source of α-amanitin. Here we report a synthesis of α-amanitin, which surmounts the key challenges for installing the 6-hydroxy-tryptathionine sulfoxide bridge, enantioselective synthesis of (2 S,3 R,4 R)-4,5-dihydroxy-isoleucine, and diastereoselective sulfoxidation.

Cryo-EM structure of a mammalian RNA polymerase II elongation complex inhibited by α-amanitin.

RNA polymerase II (Pol II) is the central enzyme that transcribes eukaryotic protein-coding genes to produce mRNA. The mushroom toxin α-amanitin binds Pol II and inhibits transcription at the step of RNA chain elongation. Pol II from yeast binds α-amanitin with micromolar affinity, whereas metazoan Pol II enzymes exhibit nanomolar affinities. Here, we present the high-resolution cryo-EM structure of α-amanitin bound to and inhibited by its natural target, the mammalian Pol II elongation complex. The structure revealed that the toxin is located in a pocket previously identified in yeast Pol II but forms additional contacts with metazoan-specific residues, which explains why its affinity to mammalian Pol II is ∼3000 times higher than for yeast Pol II. Our work provides the structural basis for the inhibition of mammalian Pol II by the natural toxin α-amanitin and highlights that cryo-EM is well suited to studying interactions of a small molecule with its macromolecular target.

A cost-effective LC-MS/MS method for identification and quantification of α-amanitin in rat plasma: Application to toxicokinetic study.

α-Amanitin is the main lethal component of amanita mushrooms, and data on its toxicokinetics are few. The aim of this study was to develop a sensitive and cost-effective method to identify α-amanitin and investigate its toxicokinetic parameters using liquid chromatography-triple quadrupole tandem mass spectrometry. The colchicine was used as the internal standard (IS). The compounds were extracted from plasma samples by protein precipitation with acetonitrile (containing 1% formic acid). The analysis was performed through multiple reactions monitoring. The molecular ions and fragment ions of α-amanitin could be used as characteristic ions to perform qualitative analysis of α-amanitin. The assay was successfully validated by selectivity, linearity, matrix effect, precision and accuracy, recovery and stability according to the U.S. Food and Drug Administration Guidance, and applied to study the toxicokinetic profile of α-amanitin in rats after a single intraperitoneal administration.

The capacity and effectiveness of diosmectite and charcoal in trapping the compounds causing the most frequent intoxications in acute medicine: A comparative study.

The aim of the study was to compare the adsorption ability of two adsorbent materials, namely diosmectite and activated charcoal towards selected model compounds that are most commonly involved in acute intoxication. Eleven model compounds were selected: acetylsalicylic acid, α-amanitin, amlodipine, digoxin, phenobarbital, ibuprofen, imipramine, carbamazepine, oxazepam, promethazine, and theophylline. Of the tested compounds, promethazine and imipramine were the most effectively adsorbed to diosmectite. Their adsorption to diosmectite (0.356±0.029mg promethazine/mg diosmectite and 0.354±0.019mg imipramine/mg diosmectite, respectively) was significantly higher than their adsorption to activated charcoal. The effect of temperature and pH on the adsorption efficiencies was also evaluated. In the case of experiments with mixture of both adsorbents, they mostly behaved in a solution independently or in a slightly antagonistic way. Using various methods such as N2 adsorption and thermogravimetric analysis, the structure and texture of diosmectite and activated charcoal were attained.

Deconvolution method for specific and nonspecific binding of ligand to multiprotein complex by native mass spectrometry.

In native mass spectrometry, it has been difficult to discriminate between specific bindings of a ligand to a multiprotein complex target from the nonspecific interactions. Here, we present a deconvolution model that consists of two levels of data reduction. At the first level, the apparent association binding constants are extracted from the measured intensities of the target/ligand complexes by varying ligand concentration. At the second level, two functional forms representing the specific and nonspecific binding events are fit to the apparent binding constants obtained from the first level of modeling. Using this approach, we found that a power-law distribution described nonspecific binding of α-amanitin to yeast RNA polymerase II. Moreover, treating the concentration of the multiprotein complex as a fitting parameter reduced the impact of inaccuracies in this experimental measurement on the apparent association constants. This model improves upon current methods for separating specific and nonspecific binding to large, multiprotein complexes in native mass spectrometry, by modeling nonspecific binding with a power-law function.

Quantification of alpha-amanitin in biological samples by HPLC using simultaneous UV- diode array and electrochemical detection.

α-Amanitin is a natural bicyclic octapeptide, from the family of amatoxins, present in the deadly mushroom species Amanita phalloides. The toxicological and clinical interests raised by this toxin, require highly sensitive, accurate and reproducible quantification methods for pharmacokinetic studies. In the present work, a high-performance liquid chromatographic (HPLC) method with in-line connected diode-array (DAD) and electrochemical (EC) detection was developed and validated to quantify α-amanitin in biological samples (namely liver and kidney). Sample pre-treatment consisted of a simple and unique deproteinization step with 5% perchloric acid followed by centrifugation at 16,000×g, 4°C, for 20min. The high recovery found for α-amanitin (≥96.8%) makes this procedure suitable for extracting α-amanitin from liver and kidney homogenates. The resulting supernatant was collected and injected into the HPLC. Mobile phase was composed by 20% methanol in 50mM citric acid, and 0.46mM octanessulfonic acid, adjusted to pH 5.5. The chromatographic runs took less than 22min and no significant endogenous interferences were observed at the α-amanitin retention time. Calibration curves were linear with regression coefficients higher than 0.994. The overall inter- and intra-assay precision did not exceed 15.3%. The present method has low interferences with simple and fast processing steps, being a suitable procedure to support in vivo toxicokinetic studies involving α-amanitin. In fact, the validated method was successfully applied to quantify α-amanitin in biological samples following intraperitoneal α-amanitin administration to rats. Moreover, human plasma was also used as matrix and the purposed method was adequate for detection of α-amanitin in that matrix. The results clearly indicate that the proposed method is suitable to investigate the pharmacokinetic and tissue distribution of α-amanitin. Additionally, the method will be very useful in the development of novel and potent antidotes against amatoxins poisoning and to improve the knowledge of α-amanitin toxicity.