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.

Amanitins: The Most Poisonous Molecules of the Fungal World.

Among the toxic metabolites of the fungal world, those that, due to their strong biological effect, can seriously (even fatally) damage the life processes of humans (and certain groups of animals) stand out. Amatoxin-containing mushrooms and the poisonings caused by them stand out from the higher fungi, the mushrooms. There are already historical data and records about such poisonings, but scientific research on the responsible molecules began in the middle of the last century. The goals of this review work are as follows: presentation of the cosmopolitan mushroom species that produce amanitins (which are known from certain genera of four mushroom families), an overview of the chemical structure and specific properties of amanitins, a summary of the analytical methods applicable to them, a presentation of the "medical history" of poisonings, and a summary of the therapeutic methods used so far. The main responsible molecules (the amanitins) are bicyclic octapeptides, whose structure is characterized by an outer loop and an inner loop (bridge). It follows from the unusual properties of amanitins, especially their extreme stability (against heat, the acidic pH of the medium, and their resistance to human, and animal, digestive enzymes), that they are absorbed almost without hindrance and quickly transported to our vital organs. Adding to the problems is that accidental consumption causes no noticeable symptoms for a few hours (or even 24-36 h) after consumption, but the toxins already damage the metabolism of the target organs and the synthesis of nucleic acid and proteins. The biochemical catastrophe of the cells causes irreversible structural changes, which lead to necrotic damage (in the liver and kidneys) and death. The scientific topicality of the review is due to the recent publication of new data on the probable antidote molecule (ICR: indocyanine green) against amanitins. Further research can provide a new foundation for the therapeutic treatment of poisonings, and the toxicological situation, which currently still poses a deadly threat, could even be tamed into a controllable problem. We also draw attention to the review conclusions, as well as the mycological and social tasks related to amanitin poisonings (prevention of poisonings).

Synthesis and evaluation of "Ama-Flash", a photocaged amatoxin prodrug for light-activated RNA Pol II inhibition and cell death.

Amanitin is used extensively as a research tool to inhibit RNA Pol II thereby implicating its role in mRNA transcription. Recently, amanitin has gained traction as a toxic payload for targeted therapy. Here we report the first-ever photocaged amanitin analog, that is non-toxic and can be pre-loaded into cells. Light provides a means to inhibit RNA Pol II and provoke cell death on-demand.

HDP-101, an Anti-BCMA Antibody-Drug Conjugate, Safely Delivers Amanitin to Induce Cell Death in Proliferating and Resting Multiple Myeloma Cells.

Despite major treatment advances in recent years, patients with multiple myeloma inevitably relapse. The RNA polymerase II complex has been identified as a promising therapeutic target in both proliferating and dormant cancer cells. Alpha-amanitin, a toxin so far without clinical application due to high liver toxicity, specifically inhibits this complex. Here, we describe the development of HDP-101, an anti-B-cell maturation antigen (BCMA) antibody conjugated with an amanitin derivative. HDP-101 displayed high efficacy against both proliferating and resting myeloma cells in vitro, sparing BCMA-negative cells. In subcutaneous and disseminated murine xenograft models, HDP-101 induced tumor regression at low doses, including durable complete remissions after a single intravenous dose. In cynomolgus monkeys, HDP-101 was well tolerated with a promising therapeutic index. In conclusion, HDP-101 safely and selectively delivers amanitin to myeloma cells and provides a novel therapeutic approach to overcome drug resistance in this disease.

The cyclopeptide -amatoxin induced hepatic injury via the mitochondrial apoptotic pathway associated with oxidative stress.

In order to explore the role of apoptosis in alpha-amatoxin (α-AMA) induced liver injury and probable upstream activation signals, we established animal and cellular models, respectively, for this pathophysiological condition. To this end, we evaluated the survival rate and serum biochemical parameters in BALB/c mice exposed to α-AMA at different time periods, along with the levels of oxidative and antioxidant enzymes in the liver tissue of these mice and proteins involved in apoptosis-related pathways. Our results reveal that α-AMA-induced apoptosis occurs primarily through the mitochondrial apoptotic pathway and is associated with oxidative damage. Further, in order to verify the key nodes and important upstream activators in this apoptotic pathway, we estimated the levels of p53 protein and downstream mitochondrial apoptotic pathway-related proteins in L-02 cells, all of which were found to change significantly. We also found that the levels of total and mitochondrial reactive oxygen species (ROS) in L-02 cells increased with time. Collectively, our findings suggest that α-AMA affects many cellular processes, including the expression of p53 independent of transcription and the expression of Bax and Bcl-2, thereby activating the subsequent caspase cascade pathways. In addition, we identified ROS to be an upstream signaling molecule involved in the α-AMA-induced apoptosis of mouse liver cells and L-02 cells.

Differences in blastomere totipotency in 2-cell mouse embryos are a maternal trait mediated by asymmetric mRNA distribution.

It is widely held that the first two blastomeres of mammalian embryos are equally totipotent and that this totipotency belongs to the group of regulative properties. However, this interpretation neglects an important aspect: evidence only came from successful monozygotic twins which can speak only for those pairs of half-embryos that are able to regulate in the first place. Are the frequently occurring incomplete pairs simply an artefact, or do they represent a real difference, be it in the imperfect blastomere's ability to regulate growth or in the distribution of any compound X that constrains regulation? Using the model system of mouse embryos bisected at the 2-cell stage after fertilization, we present evidence that the interblastomere differences evade regulation by external factors and are already latent in oocytes. Specifically, an interblastomere imbalance of epiblast production persists under the most diverse culture conditions and applies to the same extent in parthenogenetic counterparts. As a result, cases in which twin blastocysts continued to develop in only one member account for 65 and 57% of zygotic and parthenogenetic pairs, respectively. The interblastomere imbalance is related to the subcellular distribution of gene products, as documented for the epiblast-related gene Cops3, using mRNA FISH in super-resolution mode confocal microscopy. Blastomere patterns of Cops3 mRNA distribution are α-amanitin-resistant. Thus, the imbalance originates not from de novo transcription, but from influences which are effective before fertilisation. These data expose previously unrecognized limits of regulative capacities of 2-cell stage blastomeres and point to aspects of cytoplasmic organization of the mouse oocyte that segregate unequally to blastomeres during cleavage.

The hnRNP-Htt axis regulates necrotic cell death induced by transcriptional repression through impaired RNA splicing.

In this study, we identify signaling network of necrotic cell death induced by transcriptional repression (TRIAD) by α-amanitin (AMA), the selective RNA polymerase II inhibitor, as a model of neurodegenerative cell death. We performed genetic screen of a knockdown (KD) fly library by measuring the ratio of transformation from pupa to larva (PL ratio) under TRIAD, and selected the cell death-promoting genes. Systems biology analysis of the positive genes mapped on protein-protein interaction databases predicted the signaling network of TRIAD and the core pathway including heterogeneous nuclear ribonucleoproteins (hnRNPs) and huntingtin (Htt). RNA sequencing revealed that AMA impaired transcription and RNA splicing of Htt, which is known as an endoplasmic reticulum (ER)-stabilizing molecule. The impairment in RNA splicing and PL ratio was rescued by overexpresion of hnRNP that had been also affected by transcriptional repression. Fly genetics with suppressor or expresser of Htt and hnRNP worsened or ameliorated the decreased PL ratio by AMA, respectively. Collectively, these results suggested involvement of RNA splicing and a regulatory role of the hnRNP-Htt axis in the process of the transcriptional repression-induced necrosis.

[Determination of main peptide toxins from Amanita pallidorosea with HPLC and their antifungal action on Blastomyces albicans].

OBJECTIVE: To detect peptide toxins in Amanita pallidorasea and to study the antifungal activities of peptide toxins against Blastomyces albicans. METHODS: We separated and identified peptide toxins and determined its contents in the fruiting body, pileus and the mixture of stipe and volva from A. pallidorasea by HPLC and ESI-MS methods. Meanwhile, we detected antifungal activities of the crude toxin and the separated peptide toxins against Blastomyces albicans JLC31680 and JLC31681 by the paper disk method. RESULTS: We totally got three peptide toxins: alpha-amanitin (alpha-AMA), beta-amanitin (beta-AMA) and phalloidin (PHD). The contents of alpha-AMA, beta-AMA and PHD were 30.3 mg/g, 6.99 mg/g and 9.95 mg/g in fruiting body, and 45.0 mg/g, 11.1 mg/g and 11.3 mg/g in pileus. The contents of alpha-AMA and PHD were 11.7 mg/g and 7.98 mg/g in the mixture of stipe and volva , but the beta-AMA was not detected in this part. The inhibition ratio of the crude toxin and alpha-AMA, beta-AMA and PHD to B. albicans JLC31680 were 11.96%, 32.52%, 23.29% (P<0.01) and 15.46% (P<0.05). The inhibition ratio of the crude toxin and beta-AMA to B. albicans JLC31681 was 10.16% and 11.10% (P < 0.01), while that of alpha-AMA's was 6.89% (P < 0.05). CONCLUSIONS: A. pallidorasea is a new resource of peptide toxins with antifungal activity.

Tumor therapy with Amanita phalloides (death cap): stabilization of B-cell chronic lymphatic leukemia.

BACKGROUND: Molecular events that cause tumor formation upregulate a number of HOX genes, called switch genes, coding for RNA polymerase II transcription factors. Thus, in tumor cells, RNA polymerase II is more active than in other somatic cells. Amanita phalloides contains amanitin, inhibiting RNA polymerase II. Partial inhibition with amanitin influences tumor cell--but not normal cell--activity. OBJECTIVES: To widen the treatment spectrum, homeopathic dilutions of Amanita phalloides, containing amanitin, were given to a patient with leukemia. Monitoring the leukemic cell count, different doses of amanitin were given. RESULTS: The former duplication time of leukemic cells was 21 months. Within a period of 21 months, the cell count is stabilized to around 10(5)/µL. No leukemia-associated symptoms, liver damage, or continuous erythrocyte deprivation occur. CONCLUSIONS: This new principle of tumor therapy shows high potential to provide a gentle medical treatment.

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.

Transcription of hepatitis delta virus RNA by RNA polymerase II.

Previous studies have indicated that the replication of the RNA genome of hepatitis delta virus (HDV) involves redirection of RNA polymerase II (Pol II), a host enzyme that normally uses DNA as a template. However, there has been some controversy about whether in one part of this HDV RNA transcription, a polymerase other than Pol II is involved. The present study applied a recently described cell system (293-HDV) of tetracycline-inducible HDV RNA replication to provide new data regarding the involvement of host polymerases in HDV transcription. The data generated with a nuclear run-on assay demonstrated that synthesis not only of genomic RNA but also of its complement, the antigenome, could be inhibited by low concentrations of amanitin specific for Pol II transcription. Subsequent studies used immunoprecipitation and rate-zonal sedimentation of nuclear extracts together with double immunostaining of 293-HDV cells, in order to examine the associations between Pol II and HDV RNAs, as well as the small delta antigen, an HDV-encoded protein known to be essential for replication. Findings include evidence that HDV replication is somehow able to direct the available delta antigen to sites in the nucleoplasm, almost exclusively colocalized with Pol II in what others have described as transcription factories.

Dynamical modeling of syncytial mitotic cycles in Drosophila embryos.

Immediately following fertilization, the fruit fly embryo undergoes 13 rapid, synchronous, syncytial nuclear division cycles driven by maternal genes and proteins. During these mitotic cycles, there are barely detectable oscillations in the total level of B-type cyclins. In this paper, we propose a dynamical model for the molecular events underlying these early nuclear division cycles in Drosophila. The model distinguishes nuclear and cytoplasmic compartments of the embryo and permits exploration of a variety of rules for protein transport between the compartments. Numerical simulations reproduce the main features of wild-type mitotic cycles: patterns of protein accumulation and degradation, lengthening of later cycles, and arrest in interphase 14. The model is consistent with mutations that introduce subtle changes in the number of mitotic cycles before interphase arrest. Bifurcation analysis of the differential equations reveals the dependence of mitotic oscillations on cycle number, and how this dependence is altered by mutations. The model can be used to predict the phenotypes of novel mutations and effective ranges of the unmeasured rate constants and transport coefficients in the proposed mechanism.

Cotranscriptional recruitment of the dosage compensation complex to X-linked target genes.

Dosage compensation is a process required to balance the expression of X-linked genes between males and females. In Drosophila this is achieved by targeting the dosage compensation complex or the male-specific lethal (MSL) complex to the male X chromosome. In order to study the mechanism of targeting, we have studied two X-chromosomal genes, mof and CG3016, using chromatin immunoprecipitation as well as immuno-FISH analysis on transgenic flies. We show that MSL complex recruitment requires the genes to be in a transcriptionally active state. MSL complex recruitment is reversible because blocking transcription severely reduces MSL binding to its target genes. Furthermore, targeting cues are found toward the 3' end of the gene and depend on the passage of the transcription machinery through the gene, whereby the type of promoter and the direction of transcription are dispensable. We propose a model of dynamic MSL complex binding to active genes based on exposed DNA target elements.

Accumulation and dynamics of proteins of the MCM family during mouse oogenesis and the first embryonic cell cycle.

We describe the localization of three proteins of the minichromosome maintenance (MCM) family, Mcm2, -6 and -7 in mouse ovarian oocytes. We showed that Mcm proteins are stored in two forms: soluble and insoluble. Soluble Mcm2, -6 and -7 were uniformly distributed in the nuclei of ovarian oocytes. Insoluble Mcm2 and Mcm7 (but not Mcm6) were detected in the nuclei of resting, growing and fully-grown transcribing oocytes. In transcriptionally inactive fully-grown oocytes, Mcm2 underwent redistribution and Mcm7 disappeared. A similar effect was observed when transcription in growing oocytes was inhibited with alpha-amanitin. We postulate that in mouse oogenesis, the insoluble Mcm proteins are engaged in processes related to regulation of transcription and/or chromatin organization. In oocytes preparing for meiotic maturation, aggregates of the insoluble form of Mcm2 fragmented, dispersed and ultimately disappeared from the nuclei. Numerous Mcm2-positive deposits were observed in the cytoplasm of maturing oocytes. In the one-cell embryo, insoluble Mcm2 appeared in the G1 nucleus, persisted in the S phase and was undetectable in the G2 nucleus. Such behavior of Mcm2 supports its involvement in chromatin licensing in the first embryonic cell cycle.

Genes associated with opening and senescence of Mirabilis jalapa flowers.

A modest ethylene climacteric accompanies flower senescence in Mirabilis jalapa L., and exogenous ethylene accelerates the process. However, inhibitors of ethylene action and synthesis have little effect on the life-span of these ephemeral flowers. Treatment with alpha-amanitin, an inhibitor of DNA-dependent RNA synthesis, substantially delays the onset of senescence. This effect falls linearly between 7 h and 8 h after the start of flower opening. Subtractive hybridization was used to isolate transcripts that were up- and down-regulated during this critical period. Eighty-two up-regulated and 65 down-regulated transcripts were isolated. The genes identified encode homologues of a range of transcription factors, and of proteins involved in protein turnover and degradation. Real-time quantitative RT-PCR was used to examine expression patterns of these genes during flower opening and senescence. Genes that were identified as being down-regulated during senescence showed a common pattern of very high expression during floral opening. These genes included a homologue of CCA1, a 'clock' gene identified in Arabidopsis thaliana and an aspartyl protease. Up-regulated genes commonly showed a pattern of increase during the critical period (4-9 h after opening), and some showed very strong up-regulation. For example, the abundance of transcripts encoding a RING zinc finger protein increased >40 000 fold during the critical period.

Nonapoptotic cell death caused by the inhibition of RNA polymerase disrupts organelle distribution.

It is controversial whether the mode of cell death induced by CAG repeat diseases is apoptotic. One technical problem that affects this issue is that the very methods used for DNA injection may induce artificial apoptosis. A recent study demonstrated that the functions of RNA polymerase II are disrupted in spinocerebellar ataxia type 1 (SCA 1) pathology, one of the CAG repeat diseases, and that alpha-amanitin can inhibit the activity of RNA polymerase. To examine the cell death mechanisms involved in CAG repeat diseases, we treated cultured rat neurons with alpha-amanitin to avoid the artifacts caused by DNA transfection. Mature and immature rat neurons were treated with alpha-amanitin for 4-6 days and the effects of the treatment on the elongation of neurites, the distribution or morphology of organelles, and the nature of cell death were assessed by immunocytochemistry and quantitative analysis. Neurons exhibited a disruption of neurite elongation and eventually died by day 15 of the treatment. However, apoptosis was not detected. When the neurons survived well, but showed altered neurites, Golgi complexes and lysosomes exhibited changes in their normal intracellular distribution or morphology, but the endoplasmic reticulum and mitochondria did not. The distribution of phosphorylated Trk receptors was also disrupted in the neurites of treated neurons. The signal intensity of the dynein intermediate chain was markedly decreased in the treated neurons. Thus, organelle transport systems, particularly a minus-end-directed microtubule-dependent pathway, would be disrupted by the inhibition of RNA polymerase, and this change is likely to be an early event involved in SCA 1 pathology.

Plasmodium falciparum var gene expression is developmentally controlled at the level of RNA polymerase II-mediated transcription initiation.

The Plasmodium falciparum var gene family codes for a major virulence factor in this most lethal of human malaria parasites. A single var protein variant type is expressed on each infected red blood cell, with antigenic variation allowing progeny parasites to escape host immune detection. The control of mutually exclusive var gene expression in the parasite relies on in situ epigenetic changes. Whether control of expression occurs at transcription initiation or post transcription, however, remains to be established. Recent evidence supports existence of a unique var transcription site at the nuclear periphery containing the dominantly expressed var gene, although silent var genes can colocalize to the same region. We demonstrate here that exclusive var gene expression is controlled at the level of transcription initiation during ring stages and that var genes are transcribed by RNA polymerase II. This represents another example where P. falciparum differs from the paradigm for antigenic variation, Trypanosoma brucei.

Nuclear repositioning of the VSG promoter during developmental silencing in Trypanosoma brucei.

Interphase nuclear repositioning of chromosomes has been implicated in the epigenetic regulation of RNA polymerase (pol) II transcription. However, little is known about the nuclear position-dependent regulation of RNA pol I-transcribed loci. Trypanosoma brucei is an excellent model system to address this question because its two main surface protein genes, procyclin and variant surface glycoprotein (VSG), are transcribed by pol I and undergo distinct transcriptional activation or downregulation events during developmental differentiation. Although the monoallelically expressed VSG locus is exclusively localized to an extranucleolar body in the bloodstream form, in this study, we report that nonmutually exclusive procyclin genes are located at the nucleolar periphery. Interestingly, ribosomal DNA loci and pol I transcription activity are restricted to similar perinucleolar positions. Upon developmental transcriptional downregulation, however, the active VSG promoter selectively undergoes a rapid and dramatic repositioning to the nuclear envelope. Subsequently, the VSG promoter region was subjected to chromatin condensation. We propose a model whereby the VSG expression site pol I promoter is selectively targeted by temporal nuclear repositioning during developmental silencing.

RNA polymerase II transcription is required for human papillomavirus type 16 E7- and hydroxyurea-induced centriole overduplication.

Aberrant centrosome numbers are detected in virtually all human cancers where they can contribute to chromosomal instability by promoting mitotic spindle abnormalities. Despite their widespread occurrence, the molecular mechanisms that underlie centrosome amplification are only beginning to emerge. Here, we present evidence for a novel regulatory circuit involved in centrosome overduplication that centers on RNA polymerase II (pol II). We found that human papillomavirus type 16 E7 (HPV-16 E7)- and hydroxyurea (HU)-induced centriole overduplication are abrogated by alpha-amanitin, a potent and specific RNA pol II inhibitor. In contrast, normal centriole duplication proceeded undisturbed in alpha-amanitin-treated cells. Centriole overduplication was significantly reduced by siRNA-mediated knock down of CREB-binding protein (CBP), a transcriptional co-activator. We identified cyclin A2 as a key transcriptional target of RNA pol II during HU-induced centriole overduplication. Collectively, our results show that ongoing RNA pol II transcription is required for centriole overduplication whereas it may be dispensable for normal centriole duplication. Given that many chemotherapeutic agents function through inhibition of transcription, our results may help to develop strategies to target centrosome-mediated chromosomal instability for cancer therapy and prevention.