Wikipedia:WikiProject Chemicals/Chembox validation/VerifiedDataSandbox and Α-Amanitin: Difference between pages

{{short description|Chemical compound}}

{{lowercase title}}

| Verifiedfields = changed

| verifiedrevid = 477318766

| Name=α-Amanitin

| ImageFile=Alpha-amanitin structure.png

| ImageSize=

| ImageFile1=Alpha-amanitin-from-xtal-1k83-3D-sticks-skeletal.png

| ImageFile2=

| IUPACName=

| OtherNames=(cyclic <small>L</small>-asparaginyl-4-hydroxy-<small>L</small>-proly-(''R'')-4,5-dihydroxy-<small>L</small>-isoleucyl-6-hydroxy-2-mercapto-<small>L</small>-tryptophylglycyl-<small>L</small>-isoleucylglycyl-<small>L</small>-cysteinyl) cyclic (4 → 8)-sulfide(''R'')-''S''-oxide.

|Section1={{Chembox Identifiers

| CASNo_Ref = {{cascite|correct|CAS}}

| CASNo=23109-05-9

| UNII_Ref = {{fdacite|correct|FDA}}

| UNII = E04K0QZ999

| PubChem=2100

| ChemSpiderID=16735655

| ChEBI_Ref = {{ebicite|changed|EBI}}

| ChEBI = 37415

| StdInChI_Ref = {{stdinchicite|correct|chemspider}}

|Section2={{Chembox Properties

| Formula=C₃₉H₅₄N₁₀O₁₄S

| MolarMass=918.97 g/mol

| Appearance=

| Density=

| MeltingPt=

| BoilingPt=

| Solubility= Good

|Section3={{Chembox Hazards

| HPhrases = {{H-phrases|300|310|330|373}}

| PPhrases = {{P-phrases|260|262|264|270|271|280|284|301+310|302+350|304+340|310|314|320|321|322|330|361|363|403+233|405|501}}

| GHSPictograms = {{GHS skull and crossbones}}

| MainHazards = Highly toxic}}

'''α-Amanitin''' ('''''alpha''-Amanitin''') is a [[cyclic peptide]] of eight [[amino acid]]s. It is possibly the most deadly of all the [[amatoxin]]s, [[toxin]]s found in several species of the [[mushroom]] genus ''[[Amanita]]'', one being the [[Amanita phalloides|death cap]] (''Amanita phalloides'') as well as the [[destroying angel]], a complex of similar species, principally ''A. virosa'' and ''A. bisporigera''. It is also found in the mushrooms ''[[Galerina marginata]]'', ''[[Lepiota subincarnata]]'' and ''[[Conocybe filaris]]''. The oral {{LD50}} of amanitin is 100&nbsp;μg/kg for rats.

Unlike most [[cyclic peptides]], amatoxins (and [[phallotoxin]]s) are synthesized on [[ribosomes]]. The genes encoding the proprotein for α-amanitin belong to the same family as those that encode for phallacidin (a [[phallotoxin]]).<ref>{{cite journal | vauthors = Hallen HE, Luo H, Scott-Craig JS, Walton JD | title = Gene family encoding the major toxins of lethal Amanita mushrooms | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 104 | issue = 48 | pages = 19097–101 | date = November 2007 | pmid = 18025465 | pmc = 2141914 | doi = 10.1073/pnas.0707340104 | bibcode = 2007PNAS..10419097H | doi-access = free }}</ref>

== Scientific use ==

α-Amanitin is a selective inhibitor of [[RNA polymerase II]] and [[RNA polymerase III|III]] but not [[RNA polymerase I|I]].<ref>{{Cite web|url=https://www.adcreview.com/the-review/cytotoxic-agents/what-is-alpha-amanitin/|title=What is Alpha-Amanitin? | author = ADC Review Team | department = Editorial|date=2019-03-23|website=ADC Review|language=en-US|access-date=2020-04-17}}</ref><ref>{{cite journal | vauthors = Meinecke B, Meinecke-Tillmann S | title = Effects of alpha-amanitin on nuclear maturation of porcine oocytes in vitro | journal = Journal of Reproduction and Fertility | volume = 98 | issue = 1 | pages = 195–201 | date = May 1993 | pmid = 8345464 | doi = 10.1530/jrf.0.0980195 | doi-access = free }}</ref> This mechanism makes it a deadly toxin.{{citation needed|date=February 2023}}

α-Amanitin can also be used to determine which types of RNA polymerase are present. This is done by testing the sensitivity of the polymerase in the presence of α-amanitin. RNA polymerase I is insensitive, RNA polymerase II is highly sensitive (inhibited at 1μg/ml), RNA polymerase III is moderately sensitive (inhibited at 10μg/ml), and [[RNA polymerase IV]] is slightly sensitive (inhibited at 50μg/ml).{{Citation needed|date=March 2012}}<ref>{{cite journal | vauthors = Gao Z, Herrera-Carrillo E, Berkhout B | title = RNA Polymerase II Activity of Type 3 Pol III Promoters | journal = Molecular Therapy: Nucleic Acids | volume = 12 | pages = 135–145 | date = September 2018 | pmid = 30195753 | pmc = 6023835 | doi = 10.1016/j.omtn.2018.05.001 }}</ref><ref>{{Cite book|url=https://books.google.com/books?id=c-tTDwAAQBAJ&q=RNA+polymerase+II+highly+sensitive+(inhibited+at+1%CE%BCg/ml),+RNA+polymerase+III+is+moderately+sensitive+(inhibited+at+10%CE%BCg/ml)&pg=SA3-PA77|title=Gene Control |last=Latchman |first=David | name-list-style = vanc |date=2018-03-29|publisher=Garland Science|isbn=9781136844201|language=en}}</ref>

== Chemical structure ==

α-amanitin is a highly modified bicyclic octapeptide consisting of an outer and an inner loop. The outer loop is formed by [[peptide bond]]s between a carboxyl terminus of an amino acid to the subsequent amino terminus of the next residue. The inner loop is closed by a tryptathionine linkage between 6-hydroxy-[[tryptophan]] and [[cysteine]]. In addition, α-amanitin is decorated with modified [[amino acid]] side chains (2''S'',3''R'',4''R'')-4,5-dihydroxy-[[isoleucine]], ''trans''-4-hydroxy-[[proline]], which gives its high affinity for [[RNA polymerase II]] and [[RNA polymerase III|III]].<ref>{{cite journal | vauthors = Meinecke B, Meinecke-Tillmann S | title = Effects of alpha-amanitin on nuclear maturation of porcine oocytes in vitro | journal = Journal of Reproduction and Fertility | volume = 98 | issue = 1 | pages = 195–201 | date = May 1993 | pmid = 8345464 | doi = 10.1530/jrf.0.0980195 | url = http://www.reproduction-online.org/content/98/1/195 | doi-access = free }}</ref>

== Detection techniques ==

Early methods to detect alpha-amanitin included [[thin-layer chromatography]] (TLC). In most solvent systems used in TLC, alpha-amanitin and beta-amanitin would travel at different rates, thus allowing individual identification of each toxin. Another early method was the [[Meixner test#Meixner test for amatoxins|Meixner test]] (also known as the Wieland test), which would detect amatoxins, but also yielded false positives for some compounds, such as [[psilocin]].<ref>{{cite journal | vauthors = Beuhler M, Lee DC, Gerkin R | title = The Meixner test in the detection of alpha-amanitin and false-positive reactions caused by psilocin and 5-substituted tryptamines | journal = Annals of Emergency Medicine | volume = 44 | issue = 2 | pages = 114–20 | date = August 2004 | pmid = 15278082 | doi = 10.1016/j.annemergmed.2004.03.017 }}</ref> [[Capillary zone electrophoresis]] was also developed, but was not adequately sensitive for clinical samples, but sufficient for mushroom extracts.<ref>{{cite journal | vauthors = Brüggemann O, Meder M, Freitag R | title = Analysis of amatoxins alpha-amanitin and beta-amanitin in toadstool extracts and body fluids by capillary zone electrophoresis with photodiode array detection | journal = Journal of Chromatography A | volume = 744 | issue = 1–2 | pages = 167–76 | date = September 1996 | pmid = 8843665 | doi = 10.1016/0021-9673(96)00173-2 | series = 8th International Symposium on High Performance Capillary Electrophoresis Part I }}</ref>

More recently, the use of [[high-performance liquid chromatography]] (HPLC) has become the preferred method, which allows for better resolution, reproducibility, and higher sensitivity.<ref>{{Cite book|last=Walton | first = Jonathan | name-list-style = vanc |title=The cyclic peptide toxins of Amanita and other poisonous mushrooms| date = 9 May 2018 |isbn=978-3-319-76822-9|location=Cham, Switzerland|oclc=1035556400}}</ref> A range of detectors can be paired with HPLC, such as UV or [[mass spectrometry]].

As early as the 1980s, antibody-based assays ([[immunoassay]]s) were developed for amanitin (but more often recognize amatoxins as the antibodies cross-react with some of the [[Congener (chemistry)|congeners]]). The earliest immunoassays were [[radioimmunoassay]]s and then enzyme linked immunosorbent assays ([[ELISA]]s). More, recently, in 2020, a monoclonal antibody-based [[Lateral flow test|lateral flow]] immunoassay (similar to a pregnancy test) has been developed that can quickly and selectively detect amatoxins in mushrooms<ref>{{cite journal | vauthors = Bever CS, Adams CA, Hnasko RM, Cheng LW, Stanker LH | title = Lateral flow immunoassay (LFIA) for the detection of lethal amatoxins from mushrooms | journal = PLOS ONE | volume = 15 | issue = 4 | pages = e0231781 | date = 2020-04-17 | pmid = 32302363 | pmc = 7164595 | doi = 10.1371/journal.pone.0231781 | bibcode = 2020PLoSO..1531781B | doi-access = free }}</ref> and in urine samples.<ref>{{cite journal | vauthors = Bever CS, Swanson KD, Hamelin EI, Filigenzi M, Poppenga RH, Kaae J, Cheng LW, Stanker LH | display-authors = 6 | title = Rapid, Sensitive, and Accurate Point-of-Care Detection of Lethal Amatoxins in Urine | journal = Toxins | volume = 12 | issue = 2 | pages = 123 | date = February 2020 | pmid = 32075251 | doi = 10.3390/toxins12020123 | pmc = 7076753 | doi-access = free }}</ref>

== Total synthesis ==

Matinkhoo et al. devised strategies to surmount three synthetic hurdles to give α-amanitin in 2018.<ref name=":0">{{cite journal | vauthors = Matinkhoo K, Pryyma A, Todorovic M, Patrick BO, Perrin DM | title = Synthesis of the Death-Cap Mushroom Toxin α-Amanitin | journal = Journal of the American Chemical Society | volume = 140 | issue = 21 | pages = 6513–6517 | date = May 2018 | pmid = 29561592 | doi = 10.1021/jacs.7b12698 }}</ref> First, [[enantioselective synthesis]] of [[Peptide synthesis|solid phase peptide synthesis]]-compatible (2''S'',3''R'',4''R'')-4,5-dihydroxyisoleucine was afforded in 11 steps from 2-(benzyloxy)acetaldehyde. Two key stereochemistry-defining steps include Brown crotylation at (''3R'',''4R'')-positions, and asymmetric [[Strecker amino acid synthesis]] at the (''2S'')-[[Alpha and beta carbon|α carbon]].<ref>{{cite journal | vauthors = Mohapatra DK, Das PP, Pattanayak MR, Yadav JS | title = Iodine-catalyzed highly diastereoselective synthesis of trans-2,6-disubstituted-3,4-dihydropyrans: application to concise construction of C28-C37 bicyclic core of (+)-sorangicin A | journal = Chemistry: A European Journal | volume = 16 | issue = 7 | pages = 2072–8 | date = February 2010 | pmid = 20099288 | doi = 10.1002/chem.200902999 }}</ref> Secondly, chemoselective inner ring closure by fluorocyclization between 6-hydroxytrytophan and cysteine was achieved by intra-annular Savige-Fontana reaction. This requires a solid phase peptide synthesis-compatible, and methyliminodiacetic acid (MIDA), a boron protecting group, orthogonal amino acid in 5 steps.<ref name=":0" /> As a final step, enantioselective [[Redox|oxidation]] at the tryptathionine linkage was achieved using a bulky organic [[oxidizing agent]] and an optimized solvent system to afford the desired bio-reactive (''R'')-[[enantiomer]] [[sulfoxide]], completing the [[total synthesis]].{{citation needed|date=February 2023}}

== Symptoms of poisoning ==

α-Amanitin has an unusually strong and specific attraction to the enzyme RNA polymerase II. Upon ingestion and uptake by liver cells, it binds to the RNA polymerase II enzyme, effectively causing [[cytolysis]] of [[hepatocyte]]s (liver cells).<ref>{{cite journal | vauthors = Michelot D, Labia R | s2cid = 23872903 | title = alpha-Amanitin: a possible suicide substrate-like toxin involving the sulphoxide moiety of the bridged cyclopeptide | journal = Drug Metabolism and Drug Interactions | volume = 6 | issue = 3–4 | pages = 265–74 | year = 1988 | pmid = 3078291 | doi = 10.1515/dmdi.1988.6.3-4.265 }}</ref> Few effects are reported within 10 hours; it is not unusual for significant effects to take as long as 24 hours after ingestion to appear, with this delay in symptoms making α-amanitin poisoning even more difficult to diagnose and all the more dangerous. By then, it is far past the time in which [[stomach pumping]] would yield an efficient result. [[Diarrhea]] and [[cramps]] are the first symptoms, but those pass, giving a false sign of remission. Typically, on the 4th to 5th day, the toxin starts to have severe effects on the [[liver]] and [[kidneys]], leading to total system failure in both. Death usually takes place around a week from ingestion.<ref>{{cite journal | vauthors = Mas A | title = Mushrooms, amatoxins and the liver | journal = Journal of Hepatology | volume = 42 | issue = 2 | pages = 166–9 | date = February 2005 | pmid = 15664239 | doi = 10.1016/j.jhep.2004.12.003 }}</ref>

Around 15% of those poisoned will die within 10 days, progressing through a comatose stage to [[kidney failure]], [[liver failure]], [[Hepatic encephalopathy|hepatic coma]], [[respiratory failure]] and death. Those who recover are at risk of permanent liver damage.<ref name ="benjamin95">{{cite book |vauthors =Benjamin DR |chapter=Amatoxin syndrome |pages=198–214 |title=Mushrooms: poisons and panaceas — a handbook for naturalists, mycologists and physicians |url=https://archive.org/details/mushroomspoisons0000benj |url-access=registration |publisher=New York: WH Freeman and Company |year=1995}}</ref> Diagnosis is difficult, and is established by observation of the clinical symptoms as well as the presence of α-amanitin in the [[urine]]. Urine screening is generally most useful within 48 hours of ingestion. Treatment is mainly supportive ([[gastric lavage]], [[activated carbon]], [[Fluid replacement|fluid resuscitation]]) but includes various drugs to counter the amatoxins, including intravenous [[penicillin]] and [[cephalosporin]] derivatives, and, in cases of greater ingestion, can extend to an [[orthotopic liver transplant]]. The most reliable method to treat amanitin poisoning is through [[gastric lavage]] immediately after ingestion; however, the onset of symptoms is generally too late for this to be an option. Chemically modified [[silibinin]], silibinin dihydrogen disuccinate disodium (trade name Legalon SIL) a solution for [[Intravenous therapy|IV administration]], is used in treatment of severe intoxications with [[hepatotoxic]] substances such as [[paracetamol]] and [[amanitins]].<ref name="Mitchell2009">{{ClinicalTrialsGov|NCT00915681|Intravenous Milk Thistle (Silibinin-Legalon) for Hepatic Failure Induced by Amatoxin/Amanita Mushroom Poisoning}}</ref>

== Mode of inhibitory action ==

[[Image:Alpha-Amanitin–RNA polymerase II complex 1K83.png|thumb|α-Amanitin (red) bound to RNA polymerase II from ''[[Saccharomyces cerevisiae]]'' (brewer's yeast). From {{PDB|1K83}}.<ref name=Bushnell/>]]

Based on a 2002 crystal structure analysis, α-amanitin interacts with the bridge helix in [[RNA polymerase II]] (pol II).<ref name="Bushnell">{{cite journal | vauthors = Bushnell DA, Cramer P, Kornberg RD | title = Structural basis of transcription: alpha-amanitin-RNA polymerase II cocrystal at 2.8 A resolution | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 99 | issue = 3 | pages = 1218–22 | date = February 2002 | pmid = 11805306 | pmc = 122170 | doi = 10.1073/pnas.251664698 | bibcode = 2002PNAS...99.1218B | doi-access = free }}</ref> This interaction interferes with the translocation of RNA and DNA needed to empty the site for the next round of RNA synthesis. The addition of α-amanitin can reduce the rate of pol II translocating on DNA from several thousand to a few nucleotides per minute,<ref>{{cite journal | vauthors = Chafin DR, Guo H, Price DH | title = Action of alpha-amanitin during pyrophosphorolysis and elongation by RNA polymerase II | journal = The Journal of Biological Chemistry | volume = 270 | issue = 32 | pages = 19114–9 | date = August 1995 | pmid = 7642577 | doi = 10.1074/jbc.270.32.19114 | doi-access = free }}</ref><ref>{{cite journal | vauthors = Rudd MD, Luse DS | title = Amanitin greatly reduces the rate of transcription by RNA polymerase II ternary complexes but fails to inhibit some transcript cleavage modes | journal = The Journal of Biological Chemistry | volume = 271 | issue = 35 | pages = 21549–58 | date = August 1996 | pmid = 8702941 | doi = 10.1074/jbc.271.35.21549 | doi-access = free }}</ref> but has little effect on the affinity of pol II for nucleoside triphosphate,<ref>{{cite journal | vauthors = Cochet-Meilhac M, Chambon P | title = Animal DNA-dependent RNA polymerases. 11. Mechanism of the inhibition of RNA polymerases B by amatoxins | journal = Biochimica et Biophysica Acta (BBA) - Nucleic Acids and Protein Synthesis | volume = 353 | issue = 2 | pages = 160–84 | date = June 1974 | pmid = 4601749 | doi = 10.1016/0005-2787(74)90182-8 }}</ref> and a phosphodiester bond can still be formed.<ref>{{cite journal | vauthors = Vaisius AC, Wieland T | title = Formation of a single phosphodiester bond by RNA polymerase B from calf thymus is not inhibited by alpha-amanitin | journal = Biochemistry | volume = 21 | issue = 13 | pages = 3097–101 | date = June 1982 | pmid = 7104312 | doi = 10.1021/bi00256a010 }}</ref><ref>{{cite journal | vauthors = Gu W, Powell W, Mote J, Reines D | title = Nascent RNA cleavage by arrested RNA polymerase II does not require upstream translocation of the elongation complex on DNA | journal = The Journal of Biological Chemistry | volume = 268 | issue = 34 | pages = 25604–16 | date = December 1993 | doi = 10.1016/S0021-9258(19)74433-0 | pmid = 7503982 | pmc = 3373964 | doi-access = free }}</ref> The bridge helix has evolved to be flexible and its movement is required for translocation of the polymerase along the DNA backbone. Binding of α-amanitin puts a constraint on its mobility, hence slowing down the translocation of the polymerase and the rate of synthesis of the RNA molecule.{{citation needed|date=February 2023}}

== Use in antibody-drug conjugates ==

A new [[antibody-drug conjugate]] (ADC) technology based on α-amanitin has shown activity in therapy-resistant tumor cells, e.g. cells expressing multi-drug resistant transporters, tumor-initiating cells and non-dividing cells at picomolar concentrations.<ref name="adc-ama">{{cite web|url=http://adcreview.com/adc-university/adcs-101/cytotoxic-agents/α-amanitin/ |title=Alpha Amanitin|website=ADC Review / Journal of Antibody-drug Conjugates |issn=2327-0152 |access-date=26 May 2017}}</ref>

The unique [[mode of action]] of α-amanitin seems to make the amanitin-based ADCs a suitable toxic payload.<ref>{{cite web | author = ADC Review Editorial Team |url=http://adcreview.com/adc-university/adcs-101/antibody-drug-conjugates-adcs/ |title=What are antibody-drug conjugates? |website=ADC Review / Journal of Antibody-drug Conjugates |issn=2327-0152 |access-date=26 May 2017}}</ref> Amanitin has a water-soluble structure, resulting in ADCs with low tendency for aggregation.<ref>{{cite journal | vauthors = Moldenhauer G, Salnikov AV, Lüttgau S, Herr I, Anderl J, Faulstich H | title = Therapeutic potential of amanitin-conjugated anti-epithelial cell adhesion molecule monoclonal antibody against pancreatic carcinoma. | journal = Journal of the National Cancer Institute | date = April 2012 | volume = 104 | issue = 8 | pages = 622–34 | doi = 10.1093/jnci/djs140 | pmid = 22457476 | doi-access = free }}</ref><ref>{{cite conference | vauthors = Hechler T, Kulke M, Müller C, Pahl A, Anderl J | author-link2 = Matthew Kulke | title = Amanitin-based antibody-drug conjugates targeting the prostate-specific membrane antigen PSMA. Poster #664 | conference = AACR Annual Meeting | date = 2014 | doi = 10.1158/1538-7445.AM2014-664 }}</ref>

== See also ==

* [[Mushroom poisoning]]

* [[Antibody-drug conjugate]]

== References ==

{{reflist}}

== External links ==

* [http://www.heidelberg-pharma.com/ Heidelberg Pharma, GmbH]

* [http://americanmushrooms.com/toxicms.htm Poisonous American Mushrooms]

{{Poisonous Amanitas}}

{{Toxins}}

{{DEFAULTSORT:Amanitin, alpha-}}

[[Category:Cyclic peptides]]

[[Category:Amatoxins]]

[[Category:Hepatotoxins]]

[[Category:Tryptamines]]

[[Category:Enzyme inhibitors]]

[[Category:RNA polymerase inhibitors]]

[[Category:Sulfoxides]]

[[Category:Experimental cancer drugs]]