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.

Lateral flow immunoassay (LFIA) for the detection of lethal amatoxins from mushrooms.

The mushroom poison that causes the most deaths is the class of toxins known as amatoxins. Current methods to sensitively and selectively detect these toxins are limited by the need for expensive equipment, or they lack accuracy due to cross-reactivity with other chemicals found in mushrooms. In this work, we report the development of a competition-based lateral flow immunoassay (LFIA) for the rapid, portable, selective, and sensitive detection of amatoxins. Our assay clearly indicates the presence of 10 ng/mL of α-AMA or γ-AMA and the method including extraction and detection can be completed in approximately 10 minutes. The test can be easily read by eye and has a presumed shelf-life of at least 1 year. From testing 110 wild mushrooms, the LFIA identified 6 out of 6 species that were known to contain amatoxins. Other poisonous mushrooms known not to contain amatoxins tested negative by LFIA. This LFIA can be used to quickly identify amatoxin-containing mushrooms.

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.

Amanitins and their development as a payload for antibody-drug conjugates.

Amanitin-based ADCs represent a new class of ADCs using a novel mode of action. This payload introduces a novel mode of action into oncology therapy, the inhibition of RNA Polymerase II. The high potency of the toxin leads to highly efficacious ADCs. The development of the technology around this toxin will be described. These developments support the clinical development of amanitin-based ADCs by using a toxin with a new mode of action and with a favorable therapeutic index. HDP-101 is an Amanitin based ADC directed against BCMA and will be advancing to the clinical phase in 2019.

Peptides of pHLIP family for targeted intracellular and extracellular delivery of cargo molecules to tumors.

The pH (low) insertion peptides (pHLIPs) target acidity at the surfaces of cancer cells and show utility in a wide range of applications, including tumor imaging and intracellular delivery of therapeutic agents. Here we report pHLIP constructs that significantly improve the targeted delivery of agents into tumor cells. The investigated constructs include pHLIP bundles (conjugates consisting of two or four pHLIP peptides linked by polyethylene glycol) and Var3 pHLIPs containing either the nonstandard amino acid, γ-carboxyglutamic acid, or a glycine-leucine-leucine motif. The performance of the constructs in vitro and in vivo was compared with previous pHLIP variants. A wide range of experiments was performed on nine constructs including (i) biophysical measurements using steady-state and kinetic fluorescence, circular dichroism, and oriented circular dichroism to study the pH-dependent insertion of pHLIP variants across the membrane lipid bilayer; (ii) cell viability assays to gauge the pH-dependent potency of peptide-toxin constructs by assessing the intracellular delivery of the polar, cell-impermeable cargo molecule amanitin at physiological and low pH (pH 7.4 and 6.0, respectively); and (iii) tumor targeting and biodistribution measurements using fluorophore-peptide conjugates in a breast cancer mouse model. The main principles of the design of pHLIP variants for a range of medical applications are discussed.

Synthesis of a Cytotoxic Amanitin for Biorthogonal Conjugation.

Alpha-amanitin is an exceedingly toxic, naturally occurring, bicyclic octapeptide that inhibits RNA polymerase and results in cellular and organismal death. Here we report the straightforward synthesis of an amanitin analogue that exhibited near-native toxicity. A pendant alkyne was readily installed to enable copper-catalyzed alkyne-azide cycloaddition (CuAAC) to azido-rhodamine and two azide-bearing versions of the RGD peptide. The fluorescent toxin analogue entered cells and provoked morphological changes consistent with cell death. The latter two conjugates are as toxic as the parent alkyne precursor, which demonstrates that conjugation does not diminish toxicity. In addition, we showed that toxicity depends on a single diastereomer of the unnatural amino acid, dihydroxyisoleucine (DHIle), at position 3. The convenient synthesis of a heptapeptide precursor now provides access to bioactive amanitin analogues that may be readily conjugated to biomolecules of interest.

Cytotoxic constituents of Amanita subjunquillea.

As part of our systematic study of Korean toxic mushrooms, we have investigated the constituents of Amanita subjunquillea. The column chromatographic separation of the MeOH extract of A. subjunquillea led to the isolation of four ergosterols, two cerebrosides and four cyclopeptides. Their structures were determined by spectroscopic methods to be (22E,24R)-5alpha,8alpha-epidioxyergosta-6,9,22-triene-3beta-ol (1), (22E,24R)-5alpha,8alpha-epidioxyergosta-6,22-dien-3beta-ol (2), (22E,24R)-5alpha,6alpha-epoxyergosta-8,22-diene-3beta,7beta-diol (3), (24S)-ergost-7-en-3beta-ol (4), 8,9-dihydrosoyacerebroside I (5), soyacerebroside I (6), beta-amanitin (7), phalloin (8), alpha-amanitin (9), and phalloidin (10). The compounds 1-6 and 8 were isolated for the first time from this mushroom. The isolated compounds were evaluated for the cytotoxicity against A549, SK-OV-3, SK-MEL-2 and HCT15 cells. Compound 9 exhibited significant cytotoxic activity against A549, SK-OV-3, SK-MEL-2 and HCT15 with ED(50) values of 1.47, 0.26, 1.57 and 1.32 microM, respectively.

Tryptathionine bridges in peptide synthesis.

The tryptathionine linkage is a crosslink formed between tryptophan and cysteine. This feature is characteristic of the bicyclic peptides: the phallotoxins and the amatoxins. These peptides both bind to protein folds of their respective targets (F-actin and RNA pol II, respectively) with extremely high affinities. Studies on these peptides have shown that the tryptathionine crosslink is essential for this binding affinity. Tryptathionines have been investigated for many years and several syntheses exist for their formation. In this review, we report on the various methodologies employed in tryptathionine synthesis, and discuss some of the advantages and disadvantages associated with each of them.

Transcriptional blockade induces p53-dependent apoptosis associated with translocation of p53 to mitochondria.

The tumor suppressor p53 functions as a transcriptional activator to induce cell cycle arrest and apoptosis in response to DNA damage. Although p53 was also shown to mediate apoptosis in a manner independent of its transactivation activity, the mechanism and conditions that trigger such cell death have remained largely unknown. We have now shown that inhibition of RNA polymerase II-mediated transcription by alpha-amanitin or RNA interference induced p53-dependent apoptosis. Inhibition of pol II-mediated transcription resulted in down-regulation of p21Cip1, which was caused by both transcriptional suppression and protein degradation, despite eliciting p53 accumulation, allowing the cells to progress into S phase and then to undergo apoptosis. This cell death did not require the transcription of p53 target genes and was preceded by translocation of the accumulated p53 to mitochondria. Our data thus suggested that blockade of pol II-mediated transcription induced p53 accumulation in mitochondria and was the critical factor for eliciting p53-dependent but transcription-independent apoptosis.

Elucidation of the structure of constrained bicyclopeptides in solution by two-dimensional cross-relaxation spectroscopy: amatoxin analogues.

The evaluation of peptide structures in solution is made feasible by the combined use of two-dimensional NMR in the laboratory (NOESY) and rotating frames (ROESY), and by the use of molecular dynamics calculations. The present paper describes how both the NMR method and molecular dynamics calculations were applied to very rigid synthetic bicycle peptides that are analogues of natural amatoxins. The NMR theory, which allows the estimate of interatomic distances between interacting nuclei, is briefly discussed. The experimental data were compared with those of known solid-state structures. Three amatoxin analogues have been examined. Of these, one is biologically active (S-deoxo gamma[R] OH-Ile3-amaninamide) and its structure in the solid state has recently been worked out. The second and third analogues (S-dexo-Ile3-Ala5-amaninamide and S-deoxo-D-Ile3-amaninamide, respectively) are inactive and their solid-state structures are unknown. The data presented confirm the authors previous hypothesis that lack of biological activity of S-deoxo-Ile3-Ala5-amaninamide is due to the masking of the tryptophan ring by the methyl group of L-Ala and not to massive conformational changes of the analogue.

Fifty years of amanitin.

Pharmacokinetic studies have provided new insights into human Amanita 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, alpha-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.