Fig 1.0 Structure of α-Amanitin
Fig 1.0 Structure of α-Amanitin

Systematic name: (cyclic(L)-asparaginyl-4-hydroxy-L-proly-(R)-4,5-dihydroxy-L-isoleucyl-6-hydroxy-2-mercapto-L-tryptophylglycyl-L-isoleucylglycyl-L-cysteinyl) cyclic (4 → 8)-sulfide(R)-S-oxide.
Molecular Formula: C39H54N10O14S
Molecular weight: 918.97 g/mol
Average mass: 918.970 Da
Monoisotopic mass: 918.354187 Da
CAS Registry: 23109-05-9
Solubility: Soluble in water (1.0 mg/ml), ethanol (5 mM), DMSO, DMF, methanol, and acetonitrile.
Storage: Store at 4° C
Melting Point: 254-255 °C (lit.)
Boiling Point: 1622.18 °C at 760 mmHg (Predicted)
Density: 1.57 g/mL (Predicted)

α-(alpha)-Amanitin is a bicyclic octapeptide which belongs to a large group of protoplasmic mushroom toxins known as amatoxins. Among the mushroom species is the green death cap mushroom (Amanita phalloides) as well as  Amanita verna, Amanita virosa, Amanita bisporigera, Amanita ocreata, Amanita tenuifolia, Galerina and Conocybe filaris. These mushroom species produce α-amanitin in amounts sufficient to poison an adult person with liver damage and fatal outcome (LD50, p.o. humans; 0.1mg/kg). α-Amanitin kills cells by inhibiting RNA polymerase II (Pol II) and shutting down gene transcription.[1]

α-Amanitin is synthesized as a proprotein, on ribosomes, 34 to 35 amino acids in length and then cleaved at specific proline residues by an enzyme belonging to the prolyl oligopeptidase (POP) subfamily. The toxin shows remarkable binding affinity for eukaryotic RNA polymerase II, slightly binds to RNA polymerase III, and shows no activity on RNA polymerase I. The drug has been used to determine which types of RNA polymerase are present in a given sample. The toxin works by binding to the bridging helix of RNA polymerase II inhibiting the translocation of RNA and DNA needed to empty the site for the next round of synthesis, thereby slowing the rate of transcription by over 1,000 fold.

Use in medicine
Heidelberg Pharmam, GmbH, based in Ladenburg, Germany, a company providing pre-clinical drug discovery and development services,  has developed a new ADC technology based on α-Amanitin.

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The trial drug has shown the outstanding activity of amanitin-based ADCs in therapy-resistant tumor cells, e.g. cells expressing multi-drug resistant transporters, tumor-initiating cells and non-dividing cells at picomolar concentrations.

α-Amanitin seems to be a suitable toxic payload for use in an Antibody-drug Conjugate or ADC  because of the unique mode of action and the molecular characteristics of the toxin.[2]

Clinical trials
The tolerability and therapeutic window of amanitin-based ADCs has been determined in a variety of rodent and non-human primate models. Furthermore, amanitin has a water-soluble structure, resulting in Antibody-drug Conjugates with low tendency for aggregation, even using higher drug to antibody ratios (DAR).

In preclinical mouse models of prostate cancer, α-amanitin conjugated to an antibody against prostate-specific membrane antigen (PSMA; FOLH1; GCPII) showed high antitumoral activity and caused complete remission at single i.v. doses of 150 μg/kg of toxin, with no more than marginal weight loss in treated animals.[3]

α-Amanitin is highly active in drug-resistant cells, independent of the status of expression of multi-drug resistant transporters because of its hydrophilic structure. Inhibition of RNA polymerase II amanitin-binding not only leads to apoptosis of dividing cells, but also of slowly growing cells – which is often observed in prostate cancer.[3]

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