Toxic Mechanisms of α-Amanitin and Its Potential to Fight Cancer

Xueke Bai

†

, Peixi Jiang

†

and Yuyou Wu

†

Department of Chemistry, The University of Manchester, Manchester, U.K.

Department of Applied Chemistry, Central South University, Changsha, Hunan, China

Faculty of Arts and Science, University of Toronto, Toronto, ON, Canada

†

These authors contributed equally

Keywords: α-Amanitin, Cancer Treatment, Selective RNA Inhibition.

Abstract: In fighting one of the major health problems in the world, the common approach to cancer treatment is the

combination of chemotherapy and radiation therapy yet from which the cytotoxic effects on normal tissues

and the drug tolerance gained through remain a huge obstacle ahead; thus, new approaches are in immediate

demand. In the past decades, toxins such as α-Amanitin have been studied in treating colorectal cancer, breast

cancer and pancreatic tumor as a therapeutic agent mostly by conjugating to moieties with targeting property

and reduced toxicity. The Amanitin toxin blocks RNA polymerase II activity and is the most specific and

most potent inhibitor of the eukaryotic transcription, hepatotoxicity being the main syndrome. For the

selective inhibition of RNAPII and induced RNPII activity in cancer cells, using this transcriptional arrest to

fight cancer cells appears to be a novel approach with broad applications. Considering the liver toxicity of α-

Amanitin, conjugation of α-Amanitin to antibodies or small molecules minimizes its toxicity and increases

the efficacy of treatment with relatively accurate targeting properties. In addition, further enhancements such

as photocaged α-Amanitin analog, conjugation with pH low insert peptide (pHLIP) and Fc Domain provide

access to more controlled drug release and ideal pharmacokinetics.

1 INTRODUCTION

Cancer is a major health problem across the world;

from the statistics of the National Health Center, 28.4

million of new cancer cases are expected by 2040

Sung. Cancer is a disease caused by certain genes

changes; TP53 is such a well-known tumor

suppressor gene and is frequently inactivated by a

deletion in a majority of human tumors. Cancer cells

grow uncontrollably and invade other parts of the

body, instead of dying through a process known as

programmed cell death as normal cells do.

Additionally, cancer relapse after chemotherapy is a

frequent biological phenomenon, along with survival

of the subpopulations drug-tolerant colonies (DTCs).

While common cytotoxic therapies are detrimental to

normal tissues, cancer cells can also develop

resistance to chemotherapy. Consequently, new

approaches are urgently required for successful

http://orcid.org/0000-0003-0058-4432

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administration in humans where the inhibitory effect

is strongly selective thus reducing adverse effects on

normal tissues. Recent studies have found the

enhanced antitumor activity of monoclonal

antibodies by conjugation to cytotoxic agents

(Shuptrine, 2012). Combining the highly-selective

property of antibody and the cytotoxic molecules

(payloads) with a linker providing covalent binding,

Antibody-Drug Conjugates (ADC) make it possible

for this specific route where the toxin is delivered to

kill target cells avoiding normal tissues; several of

these them are currently being evaluated in clinical

trials against cancer (Strassz, 2020). Except for

ADCs, Small Molecule-Drug Conjugates (SMDC)

have also been designed to overcome limitations in

ADCs. Following specific approaches to targets, the

conjugates readily enter tumor cells and release active

payloads for further inhibition. So far ADCs are

based on only a few toxic compounds, which are

Bai, X., Jiang, P. and Wu, Y.

Toxic Mechanisms of -Amanitin and Its Potential to Fight Cancer.

DOI: 10.5220/0011378000003443

In Proceedings of the 4th International Conference on Biomedical Engineering and Bioinformatics (ICBEB 2022), pages 1095-1103

ISBN: 978-989-758-595-1

1095

largely limited to microtubule- or DNA-targeting

toxins that mainly impact proliferating cells and have

limited efficacy in diseases with a low proliferative

fraction such as indolent lymphomas or multiple

myeloma (Strassz, 2020). This limitation further

urges the studies of new compounds with alternative

toxic mechanisms and the ability to selectively inhibit

cancer cells.

Amatoxins can be found in several species of the

mushroom genus Amanita, one being the famous

death cap (Amanita Phalloides), and also in the

mushrooms Galerina marginate and Conocybe filaris.

Among the amatoxin family, α-Amanitin is possibly

the most fatal one. The toxin is notorious for its

specific and non-covalent binding to RNA

polymerase II, thereby decreasing mRNA levels and

protein synthesis, which is the primary toxic

mechanism for liver necrosis or apoptosis (Arima,

2005, Leu and George, 2007, Ljungman, 1999).

Among the twelve subunits in the human RNA

polymerase II complex, POLR2A encodes the largest

subunit that is indispensable for the polymerase

activity in mRNA synthesis. In addition, genomic

deletion of the tumor suppressor gene TP53

frequently comes with encompassment of

neighboring essential gene POLR2A, rendering

cancer cells with hemizygous TP53 deletion

vulnerable to further suppression of POLR2A

expression, which can be inhibited by α-Amanitin

(Liu, 2015). Molecular events that cause tumor

formation involve a number of Homeobox (Hox)

genes, proteins of which can bind to regulatory DNA

at the level of transcription of target gene DNA to

messenger RNA by RNA polymerase II (Boube,

2014). Comparing with other toxins employed in

ADCs development such as microtubule inhibitors

and DNA cross-linkers, α-Amanitin ADCs have an

effect on slowly dividing tumor cells (Hechler, 2014).

Additionally, considering the number of intracellular

targets of ADCs, RNAPII are fewer than targets of

other developments, which leads to a lower

concentration of α-Amanitin having complete

inhibition (Rudd and Luse, 1996). Thus, the selective

inhibition of RNAPII plays a crucial role in the

inhibitory effect against cancer.

This review discusses the recent research on α-

Amanitin as an RNAPII inhibitor with a focus on its

high selectivity and controlled drug pathway that

allows new innovative and effective therapeutics

against cancer. The chemical properties of α-

Amanitin and its activity in cancer cells are also

discussed in this review. As the non-stop mRNA

synthesis and gene expression are essential in the

endless growth of cancer cells, and studies have

demonstrated the mechanism underlying relapse is

based on transcriptional regulation, α-Amanitin

presents itself as a novel therapeutic agent against

human cancer (Kume, 2016). Although applications

of free α-Amanitin are limited in use due to its liver

toxicity, α-Amanitin-based conjugates were

evaluated with reduced toxicity for its potential to

suppress DTC formation and reduce cancer cells

resistance. The conjugates appeared optimistic in

vivo. While the large size of ADCs impairs the

penetration and SMDCs have better tumor-

penetrating properties, the pharmacokinetics of

SMDCs limit their anti-tumor activity. Consequently,

more enhancements of these approaches are

discussed to overcome the limitations; photocaged

Amanitin analogs are introduced as a novel method

to control drug release (Matinkhoo, 2021);

conjugation with an Fc Domain enhances the

pharmacokinetics and anti-tumor activity of α-

Amanitin-based-SMDCs (Gallo, 2021).

2 PROPERTIES OF AMANITINS

α-Amanitin is a highly toxic bicyclic octapeptide with

eight amino acids and is produced by the hepatotoxic

mushroom genus Amanita with its relatives β-, γ-,

and ε-Amanitin. The Amanitins are special peptides

with the amino acid chains branched and the branches

giving rise to an inner loop. The structural derivatives

of α-Amanitin show the importance of bridge helix

interaction for inhibitory activity (Fig.1a) (Wang,

2011, ZANOTTI, 1987). α-Amanitin is possibly

considered to be the deadliest among all the

amatoxins by far. The mushroom species which

called Amanita Phalloides ("death cap") or Amanita

verna (“destroying angel”) is famous for its red cap

and lethal toxin Amanitin. It has been estimated that

Amanita Phalloides is responsible for 90% of the

mushroom-ingested fatalities worldwide with

hepatocellular failure being the main syndrome (α-

Amanitin poisoning may require liver transplantation

when the toxicity is severe). Chemical and physical

properties of α-Amanitin were shown in Table 1.

Amatoxins can be absorbed rapidly after ingestion.

The ingested amounts as low as 0.1 mg/kg are

sufficient to be lethal (Lewis and Seeff 2020). The

oral LD₅₀ of Amanitin is 0.4-0.8 mg/kg in mice and

it takes nearly 2–8 days to cause death (Saravanapriya

and Devi 2021).

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Table 1: Chemical and physical properties of α-Amanitin.

Pro

ert

Name Pro

ert

Value

irical Formula C

Average Molar Mass 918.97 g/mol

dro

en Bond Donor Count 13

Hydrogen Bond Acceptor Count 15

Rotatable Bond Count 7

Melting Point 254-255 ºC

Water Solubility

ൎ1-10 g/L

(a) The bicyclic octapeptide amatoxins

(b) Interaction of α-amanitin with RNAPII

Figure 1: Chemical structure of α-Amanitin with residues

of with RNAPII (Bushnell, 2002).

3 MOLECULAR TOXICITY

MECHANISMS OF ALPHA-

AMANITIN

Amatoxins can block nuclear RNA polymerases via

the target organs (intestinal mucosa, liver and

kidneys) to inhibit synthesis of proteins. Liver is the

privileged target of α-Amanitin (Amatoxins can be

absorbed by the gastrointestinal tract and then travel

within the enterohepatic circulation and reach the

hepatocytes, where the inhibition of mRNA

production and protein synthesis occurred, causing

cell necrosis, inducing apoptosis and glutathione

depletion), internalizing the toxin through the

organic-anion transporter 1B3 (OATP1B3)

(Letschert, 2006), and thus receiving a massive

amount of Amanitin after rapid gastrointestinal

absorption.

3.1 Pro-apoptosis Activity

Inside the cell, α-Amanitin induces the stress signals

(ex. RNA polymerase inhibition) which lead to an

induction of the p53 protein, allowing the formation

of complexes with the two anti-apoptotic proteins

which called B-cell lymphoma-extra-large (Bcl-XL)

and B-cell lymphoma-2 (Bcl-2) and triggering

apoptosis by the mitochondrial release of cytochrome

c in the cytosol (Arima 2005, Leu and George 2007,

Ljungman 1999). F.A. Derheimer et al. presented two

possible mechanisms of tumor suppressor gene p53

induction by α-Amanitin (Derheimer 2007). The

study suggested that the export of p53 from the

nucleus is dependent on the export of mRNA so that

when the synthesis or export of mRNA is blocked,

p53 accumulates in the nucleus by default. Secondly,

inhibition of transcription elongation leads to the

phosphorylation of the Ser-15 site of p53. Blockage

of transcription is sufficient for the nuclear

accumulation of p53 even though it's unclear that

which mechanism takes place in the cells. A report

pointed out that α-Amanitin induced significant

changes in the mitochondrial proteome, associated

with the destruction of membrane potential (Wang

2018). Hence, Amanitin-induced apoptosis has been

considered to play a vital role in the pathophysiology

of these intoxications (Figure 2) (Magdalan, 2010).

3.2 Enhancement of Oxidate Stress

Some mechanisms have been suggested the

formation of reactive oxygen species (ROS) leading

to oxidative stress-related damages. The production

of oxidative stress has been seen as an essential factor

in the development of that severe hepatotoxicity. In

fact, some studies have shown that the accumulation

of α-Amanitin leads to the increase of superoxide

dismutase (SOD) and glutathione (GSH) peroxidase

activities, malondialdehyde products, and lipid

peroxidation, which is related to the inhibition of

catalase activity (Dündar, 2017, Rodrigues, 2020,

Toxic Mechanisms of -Amanitin and Its Potential to Fight Cancer

1097

Steurer, 2018, Zheleva, 2013, Zheleva, 2007) (Figure

2). Recent researches have indicated that α-Amanitin

induces the production of GSH and tGSH, confirming

the hypothesis of the involvement of oxidative stress

in the pathophysiology (Rodrigues 2020, Steurer

2018). A scientist found that α-Amanitin could form

phenoxyl-free radicals that might be involved in the

increased production of reactive oxygen species

(Figure 2) (Zheleva, 2013). With the case of

Amanitin intoxication, induction of the NF-κB

(nuclear factor-kappa B) pathway has been observed

to have a certain protective effect without

establishing a link with the levels of production of

SOD, GSH, or catalase (Garcia, 2015, Morgan and

Liu, 2011).

3.3 Inhibition of Protein Synthesis

Furthermore, the cytotoxicity α-Amanitin will result

in the inhibition of RNA polymerase, especially RNA

polymerase II (it is more sensitive to this mushroom

toxin than other polymerases in eukaryotic cells).

Since RNA polymerase II is responsible for mRNA

synthesis in the cell, α-Amanitin is a potent and

selective inhibitor of mRNA synthesis (Kume, 2016).

The main toxicity mechanism of Amanitin is

attributed to non-covalent nuclear inhibition of RNA

polymerase type II (RNAP II), which reduces mRNA

levels and protein synthesis (WIELAND 1983). By

complexing with the intracellular RNA polymerase II

enzyme, amatoxins inhibit the formation of mRNA

and then restrain the protein synthesis, leading to cell

necrosis rapidly. This enzyme inhibition has been

proved to be the cause of RNAP II ubiquitination and

its degradation by proteasomes, which is related to

the increased intracellular ATP concentration

(Rodrigues, 2020, Steurer, 2018) (Figure 2). Up to

now, there are still many unknown areas about the

toxic effects of Amanitin at the cellular level that

need further exploration. The treatment mainly

remains symptomatic.

Figure 2: Main toxicity mechanisms of Amanitin within

hepatocytes (modified from (Le Daré, 2021)

4 INTRACELLULAR

ACTIVITIES OF

ALPHA-AMANITIN

4.1 Inhibition of Tumor Growth

through Suppression of POLR2A

While there is no denying that most cancers come

along with the deletion of the tumor suppressor gene

p53, no effective p53-based therapy has been

successfully applied in clinical treatment due to its

complexity in signalling. However, 104 (53%) out of

195 colorectal cancer cells (CRC) cases bear the

hemizygous loss in the 17p13 region, resulting in

concomitant deletion of TP53 and POLR2A(Liu,

2015). The gene POLR2A encodes the largest subunit

of RNA polymerase II and is indispensable in cell

proliferation. No homozygous deletion was observed

in cancer cells, in accordance with the fact that

POLR2A is essential for cancer cell survival. Studies

demonstrated that POLR2A expression tightly

correlates with its gene copy numbers, resulting in

significantly lower levels of POLR2A loss

(hemizygous deletion) cells(Liu, 2015). However, by

comparing POLR2Aneutral cells and POLR2Aloss

cells, the similar proliferation rates indicate this

hemizygous loss is sufficient to main proliferation in

HCT116 cells. The half-maximum inhibitory

concentration (IC50) for the POLR2Aneutral cells

was 10-fold greater than the POLR2Aloss cells; when

examined for drug sensitivity to different

chemotherapy drugs of these two cell types, POLR2A

inhibited by α-Amanitin notably increased the cell-

killing effects yet no significant enhancements were

observed in normal cells. POLR2Aloss cells were

more sensitive to POLR2A inhibition by α-Amanitin

while re-expression of POLR2A rescued resistance to

α-Amanitin, where this loss happens to be concurrent

with p53 deletion in major cancer cases. High

concentrations of α-Amanitin caused complete deaths

while with low doses, the inhibition had significantly

higher levels of cell-killing effect on the POLR2A

loss cells than the neutral ones(Liu 2015). Taken

together, this research implied using α-Amanitin as a

potential therapeutic method against CRC.

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4.2 Inhibition of RNAPII by Amanitin

via TAF15 Mrna

Κume et al. investigated the impact of RNA

polymerase II inhibition on DTC survival(Kume,

2016). Among four compounds that affect different

steps including chromatin formation, transcription, or

protein synthesis, α-Amanitin showed remarkable

suppression of colony formation in the shortest

exposure period. Moreover, colony formation was

also clearly suppressed by the other less-specific

RNAP inhibitor AMD, suggesting the selective

inhibition of RNAPII plays a crucial role in

suppressing colony formation. Their study also

demonstrated that TAF15 is especially responsible

for DTC formation as TAF15 gene products are

upregulated in DTC-forming cells. Recent studies

had suggested TAF15 binds the C-terminal domain of

RNAPII more avidly than other RNA-binding

proteins in the TET family and acts as a coactivator

of RNAPII(Kwon, 2013). Both TAF15 mRNA and

protein levels were decreased after treatment,

suggesting RNAPII activity towards TAF15 mRNA

was inhibited by α-Amanitin. Furthermore, the

subsequent colony formation assay showed that

TAF15 knockdown suppressed the emergence of

both DTCs with or without α-Amanitin treatment,

meaning TAF15 depletion inhibited DTC formation.

Additionally, TAF15-knockdown and α-Amanitin-

treated cells induced similar morphological changes,

suggesting a TAF15 depletion by α-Amanitin

treatment. Both TAF15 mRNA and protein levels

were decreased in response to α-Amanitin, consistent

with similar morphological changes. These results

suggested TAF15 is a mediator of RNA polymerase

II-dependent DTC formation and a crucial target of

α-Amanitin. The RNAPII-dependent inhibition of

early-phase mRNA synthesis is sufficient to produce

a nearly complete suppression of colony formation,

and the practicality of α-Amanitin treatment for

disease caused by DTCs, such as Peritonitis

Carcinomatosa by chemotherapy, via inhibition of

TAF15.

5 THERAPEUTIC USE OF

ALPHA-AMANITIN

α--Amanitin has a low cellular uptake due to its polar

structure and low permeation except in liver cells

where OATP1B3 internalizes the toxin(Bodero

2018). Free Amanitin permeation mediated by

transporter protein results in apoptosis and necrosis

of hepatocytes(Bodero, 2018, Letschert, 2006). The

strong inhibition effect caused by Amanitin aroused

the interest of scientists to target cancer cells while

preventing liver toxicity. Numerous studies have

shown promising cell-killing effects after direct α-

Amanitin treatment in tumor cells. In order to

enhance the selectivity of Amanitin treatment,

scientists have developed conjugates targeting tumor

cells and novel drug release methods.

5.1 Antibody-drug Conjugates

Approach

Amanitin antibody-drug conjugates (ADC) were

found to successfully increase α-Amanitin activity in

target cells while not being a substrate of OATP1B3

to avoid systematic toxicity. The antibody is

responsible for targeting specific antigens and

bringing the toxin α-Amanitin into the cells.

Moreover, epithelial cell adhesion molecule

(EpCAM) is a target antigen highly and frequently

expressed on carcinoma cells and its metastasis

(Gires 2020). α-Amanitin that was conjugated with

chiHEA125, a chimerized anti-EpCAM antibody,

reduced cell proliferation in human pancreatic

(BxPc-3 and Capan-1), colorectal (Colo205), breast

(MCF-7), and bile duct (OZ) cancer cell lines (IC50

= 2.5×10−10 to 5.4×10−12 M)(Moldenhauer 2012).

The antiproliferative effect of chiHEA125-ama was

up to 10,000 -fold higher than α-Amanitin alone.

Anti-tumor effects of chiHEA125-Amanitin

conjugates were tested in an experimental human

BxPc-3 pancreatic cancer model, induced by

injecting BxPc-3 cancer cells subcutaneously into the

right flank of female mice. A single dose (50 μg/kg

with respect to α-Amanitin) suppressed BxPc-3

xenograft tumor growth, while two higher doses (100

μg/kg with respect to α-Amanitin), administered one

week apart, inhibited tumor recurrence for 3-4 weeks.

Besides, the treatment was well-tolerated in tumor-

bearing mice and no substantial difference was

observed compared with the control mice treated with

unconjugated chiHEA125 (Moldenhauer, 2012).

Drug resistance is one of the major concerns of

existing chemo- and targeted therapies of colorectal

cancer cells (CRC)(Van der Jeught, 2018). Escaping

mechanisms including enhanced DNA repair and

drug metabolism result in worse clinical outcomes.

Research showed that α-Amanitin-HEA125

selectively killed human CRCs with a hemizygous

deletion of POLR2A(Liu 2015). Those cells were

more sensitized to chemotherapy drugs owing to the

strong inhibition of POLR2A by α-Amanitin. It was

observed that a very low dose of α-Amanitin-

Toxic Mechanisms of -Amanitin and Its Potential to Fight Cancer

1099

HEA125 (10μg/kg) was sufficient to inhibit tumor

growth in mice bearing POLR2Aloss HCT116

tumors, reducing the effective doses of α-Amanitin

by at least 10,000-fold (IC50 = 0.01 ng/ml).

Increasing specificity and effects on chemo-resistant

cancer cells by conjugating with antibody HEA125

implied a potential therapeutic way to EpCAM

expressed human cancers and other Amanitin ADCs

might be synthesized in future studies to target other

cancers. Although α-Amanitin ADC has shown

positive outcomes in various animal studies, this

approach is relatively high cost and may result in

unfavorable pharmacokinetics and immune response

(Bodero, 2018).

Antibody-targeting Amanitin conjugates

(ATACs) have a broader application than other toxin

compounds as payloads because of their unique

interaction with RNAPII. Comparing with other

intracellular targets like microtubule(Nasiri 2018),

RNAPII maintains a much lower number of 100 to

1000, rendering low concentration able to achieve

optimistic cell-killing effects. Slow growth of tumor

is common in prostate cancers, and another advantage

of α-Amanitin as a payload is the active inhibition on

dormant cells. Despite the fact that Prostate-specific

membrane antigen (PSMA) are expressed in normal

tissues as well, the expression levels are much higher

in prostate carcinomas, indicating a potential high

specificity for antibody therapy(Osborne 2013).

Scientists conjugated α-Amanitin to the anti-PSMA

mAb 3F11(Hechler, 2014). To test the active

inhibition of ATACs on non-proliferating cells,

growth arrest of LNCaP cells is made by addition of

Interleukin 6 (IL-6). Inhibition effects were observed

both on rapidly dividing and growth arrested LNCaP

cells. The viability curve of dormant cells is

comparable to dividing ones under increasing

concentration of Amanitin ADC, indicating the

proliferation-independent inhibition on tumor by

ATACs (Hechler, 2014).

However, the large size and high affinity to the

target of ADCs restrict their ability of penetration,

especially in solid tumors. Furthermore, their long

circulatory half-life might lead to immunogenicity

and unspecific toxicities. Thus, alternative

approaches may still catch scientists’ attention in the

future.

5.2 Small Molecule-drug Conjugates

Approach

Small molecule-drug conjugates (SMDCs) are an

alternative approach where a specific cell-membrane-

receptor ligand assists drug delivery to the target site

and internalization by the receptor. Tripeptide

arginine-glycine-aspartate (RGD) and the related

sequence isoaspartate-glycine-arginine (isoDGR)

were two ligands that have been conjugated to α-

Amanitin targeting the αVβ integrin receptor family

(Bodero 2018). αVβ integrin receptors are strongly

expressed on blood vessels in human cancers such as

breast cancer, glioblastoma, pancreatic tumor,

prostate carcinoma (Desgrosellier and Cheresh

2010). The conjugates demonstrated great binding

affinity to the receptor like the free ligands. However,

the toxicity and inhibitory effect of the integrin

ligand-α-Amanitin conjugates were either worse or

slightly better than free α-Amanitin. Similar results

were achieved in other studies involving small

molecule-amanitin conjugates (Moshnikova, 2013,

Zhao, 2015). In addition, SMDCs have a shorter half-

life compared to ADCs due to their smaller size,

limiting their distribution to tumor cells and

therapeutic effect. In order to prolong circulatory

half-life, the immunoglobulin Fc domain was

conjugated to α-Amanitin-based SMDCs (Gallo,

2021). Apparently, the interaction between the Fc

domain and neonatal Fc receptors (FcRN) results in

prolonged exposure to drugs that contain Fc peptides

(Wang, 2011, Wu, 2012). Both SMDCs (IC50 =

0.863 nM)) and Fc-SMDCs (IC50 = 15.2 nM) had

higher cytotoxicity in vitro compared to

unconjugated α-Amanitin (IC50 = 476 nM). In vivo

pharmacokinetics and biodistribution study were

conducted to evaluate half-live, tumor, and organ

accumulation of SMDCs and Fc-SMDCs. Fc-SMDCs

showed a dramatic decrease in their clearance, thus

extending their half-lives from 44 min to

approximately 7.2 days (Gallo, 2021).

Table.2: Evaluation of inhibitory effect of α-Amanitin and α-Amanitin conjugates in various cell lines.

Compound (name) IC

(cell lines) References

ChiHEA125-Ama 2 × 10

−12

M (Colorectal,

Colo205), 8.7 × 10

−11

M (Bile

duct, OZ), 2.1 × 10

−11

(Pancreatic, Capan-1), 2.5 × 10

−10

M (Pancreatic, BxPC-3), 5.4 × 10

−12

M (Breast, MCF-7)

(Moldenhauer 2012)

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1100

Ama-HEA125 0.1 μg/ml (POLR2A

loss

HCT116) (Liu 2015)

HDP 30.2284 8.63 × 10

-10

(

LNCaP

)

(

Gallo 2021

)

HDP 30.2972 1.52 × 10

-8

M (LNCaP) (Gallo 2021)

α-Amanitin ~4.76 × 10

-8

(

Gallo 2021

)

To increase the cytotoxicity effect, α-Amanitin

was combined with another cytotoxic drug displaying

an independent mode of action. Dual conjugation of

α-Amanitin and monomethyl auristatin E (MMAE)

with fibroblast growth factor 2 (FGF2) tend to

increase the toxicity of both drugs and maintain the

selectivity (Świderska, 2018). Fluorescence

microscopy was performed to track the

internalization process and found that FGF2 dual

conjugate internalization was strongly correlated with

FGF receptors level on the cell surface. None of the

tested conjugates displayed severe toxicity towards

receptor-negative cells, suggesting its selectivity to

FGF receptor expressed cells. After binding to the

high affinity FGFRs on the cancer cell surface, dual

FGF2 conjugate is internalizing by endocytosis.

Processing through the endosome–lysosome pathway

leads to release of MMAE and α-Amanitin inside the

cell and next respectively inhibit tubulin

polymerization and DNA transcription. α-Amanitin

inhibited DNA transcription and MMAE stopped

microtubule polymerization simultaneously, leading

to cell apoptosis. The dual conjugate had a greater

cytotoxic effect than any of single-drug FGF2

conjugates, which can be explained as a result of the

combined cytotoxic action of α-Amanitin and

MMAE. With the highest concentration, α-

Amanitin/MMAE-FGF2 conjugate reduced 95% of

cell viability. No or minimal immune response was

triggered since the ligand sequence was fully from

Homo sapiens (Świderska, 2018). The positive result

of combining two cytotoxic agents allows more

possibilities in the future against different cancer cells

with different intracellular impair.

5.3 Drug Delivery and Release

On the basis of cell targeting models including ADCs

and SMDCs, controlled drug release further increases

selectivity. Stimuli-responsive prodrugs and carriers

allowed on-demand drug delivery and release

(Dunkel and Ilaš, 2021). Photocaged α-Amanitin

analog, Ama-Flash has been synthesized by

Matinkhoo and coworkers (Matinkhoo 2021).

Nitroveratryl (Nv) ether was attached to modified α-

Amanitin as a photo-masking group which is

extensively used in photo-pharmacology studies. Nv-

protected α-Amanitin was inactive against RNAP II

and didn't result in cell death even at a high dose of

100 μM. α-Amanitin analog was released through a

25 min irradiation with λ = 366 nm and CHO

(Chinese hamster ovary) cell viability was measured

after 48 and 72 hours, with α-Amanitin as a control.

The IC50 value was not significantly different from

free α-Amanitin, suggesting that irradiation may not

affect toxicity. Furthermore, irradiated wavelengths

are minimal cytotoxic and won't be absorbed by

tryptathionine in α-Amanitin. The toxicity of

byproduct 4,5-dimethoxy-2-nitrobenzyl (DMNB)

was not tested. With light-activated α-Amanitin

analog, it creates the possibility to preload the drug

into cells and trigger RNAP II inhibition and

ubiquitination when needed. Amanitin conjugated

with pH low insert peptide (pHLIP) is another drug

delivery method based on pH-dependent

conformational change of pHLIP which leads to its

insertion into the membrane (Moshnikova, 2013).

Acidic environment protonates Asp/Glu residues and

increases hydrophobicity of pHLIP. Studies showed

that the antiproliferative effect was 4-5 times higher

at pH 6 compared to pH 7.4, creating the selectivity

due to the negative transmembrane pH gradient of

cancer cells. Cytotoxic effect was achieved on four

different human cancer cell lines at concentration of

0.25–1 μM. Taken together, the new techniques of

improving drug delivery and release such as ADCs,

SMDCs, as well as photo-pharmacology methods,

give more flexibility to cancer chemotherapy.

6 CONCLUSIONS

In this review, the toxic mechanisms of α-Amanitin,

a fetal chemical present in the mushroom species

Amanita Phalloides were discussed. Its ability to

inhibit mRNA polymerase II and induce p53 protein

makes it a new possibility against cancer. We

reviewed multiple animal studies in which α-

Amanitin conjugates were formed with either

antibodies or small molecules and showed strong

selectivity and toxic effect. Novel drug delivery

methods provide new insight into future cancer

chemotherapy. More in vivo and in vitro animal

studies with larger sample sizes are necessary in order

Toxic Mechanisms of -Amanitin and Its Potential to Fight Cancer

1101

to confirm the cytotoxicity effect as some cytotoxic

mechanisms are still unclear. There are risks

associated with animal-to-human extrapolation due to

differences in metabolism and size. A first-in-human

phase 1/2a study started enrolling participates in early

2021 (Strassz, 2020). and this study aims to

determine the maximum tolerated dose and assess the

anti-tumor activity of HDP101, an ADC targeting

BCMA (B cell maturation antigen) carrying a

synthetic version of Amanitin as a payload. Future

research focusing on the mechanisms of α-Amanitin

anti-cancer effects and related clinical trials may be

required to promote the understanding of α-Amanitin

as a potential therapeutic way for cancer treatment.

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