Chelation
as a Tool of Therapy
M1 Hiroki Umeda
2022/09/01
1

Outline
1. Introduction

2. Recent Attempts
i. Application to cancer
ii. Application to Alzheimer’s disease

3. Perspective & Summary2

Outline
1. Introduction

2. Recent Attempts
i. Application to cancer
ii. Application to Alzheimer’s disease

3. Perspective & Summary3

1. Introduction

What is Chelation ?
Chelation：
Formation of two or more coordination bonds
by a multidentate ligand to one central atom

In Organic Chemistry：e.g., Crown Ether・solubilize inorganic salts in nonpolar solvents
・create an unsolvated high reactive counter anion
・Transport anions as phase-transfer catalysts

https://www.chem-station.com/blog/2002/02/crown_ether.html

In Biochemistry：・Heme complexes are found in various proteins.
e.g., hemoglobin, P450, cytochrome

https://ja.wikipedia.org/wiki/%E3%83%98%E3%83%A04

Conventional Chelating Strategy

1. Introduction

Conventional Application：
To trap metal cations to form stable complexes,
inactivating metal toxicity and helping excretion.

→ Chelation has been used for metal overload“Chelators” are for removal of metal ions
Axel S., et al. Chem. Soc. Rev., 2020, 49, 3726-3747

5

FDA approved Chelation
Deferoxamine (DFO)：

1st Fe3+ chelator approved by FDA
For iron overload

1. Introduction

D-penicillamine (DPA)：

1st Cu2+ chelator approved by FDA
For copper overloade.g., Wilson’s disease

Chelation strategies may be applied to cancer and Alzheimer‘s disease treatments !
Caution：Some of these attempts have targeted Fe and Zn, but I will focus on Cu Today.
Axel S., et al. Chem. Soc. Rev., 2020, 49, 3726-3747

6

Outline
1. Introduction

2. Recent Attempts
i. Application to cancer
ii. Application to Alzheimer’s disease

3. Perspective & Summary

7

The role of copper in Cancer cell2-i. Application to cancer

Elevated Cu concentrations have been reported in the tumors or serum withmany type of cancers：breast, lung, gastrointestinal, oral, thyroid, gall bladder, gynecologic, prostate→ Cuproplasia = Cu dependent cell growth and proliferation• Directly activation of proangiogenic factors: VEGF, FGF2, TNF, IL-1

→ Angiogenesis• Arosterically activation of MEK1/2 stimulating RAF-MEK-ERK signaling→ Proliferation• Arosterically activation of ULK1/2→ Autophagy• Cofactor of cytocrom c oxidase→ ATP synthesis→ Cu chelator can inhibit cuproplasia
Ge, E.J., et al. Nat Rev Cancer. 2022. 22, 102–113.

Cu chelators for cancer treatment

2-i. Application to cancer

Drug reporposing/repositioning
・From type II diabetes drug

・From Wilson’s disease drug

Tetrathiomolybate is expected to also inhibit cancer
metastasis by inhibiting ATOX-ATP7A-LOX pathways
Axel S., et al. Chem. Soc. Rev., 2020, 49, 3726-3747
Ge, E.J., et al. Nat Rev Cancer. 2022. 22, 102–113.

Another Metal-targeting therapy

2-i. Application to cancer

Chelator (for removal)：
To disrupt the deleterious interaction of metal
ions with biomolecules and then inactivate themor help their excretion

Ionophores (for redistribution)：
To transfer (across a membrane) the removed metal ions to the biologicalcompartment, where they become beneficial

Increased Cu+ incytosol cause
Cuproptosis

Axel S., et al. Chem. Soc. Rev., 2020, 49, 3726-3747

10

2-i. Application to cancer

Ionophores: Example
Glyoxal-bis(4-methylthiosemicarbazone) (GTSM)：
・Charge neutral
・Lipophilic

Axel S., et al. Chem. Soc. Rev., 2020, 49, 3726-3747
Michael A., et al. ACS Chemical Biology 2013 8 (7), 1621-1631

Prochelators
Prochelator: Like prodrug, to improve selectivity
Disulfiram (DSF)
・FDA-approved aldehyde dehydrogenase (ALDH) inhibitor
for the treatment of alcoholism,
and it has been used for over 60 years.
・Activated by intracellular thiol cleavage of the disulphide bond→Diethyldithiocarbamate (DDTC or DTC): Ionophore・(Zn2+ and) Cu2+ complexes of DTC：
act as ionophore
Inhibition of turnover by targeting NPL4
promote the generation of ROS via redox cycling→ push cancer cells towards various apoptosis pathways
Skrott, Z., et al. Nature. 2017. 552, 194–199.
Axel S., et al. Chem. Soc. Rev., 2020, 49, 3726-3747

2-i. Application to cancer

Metal-Carrying Prochelator

2-i. Application to cancer

metal-carrying prochelator (= prodrug of metal complex)

・co-carry a metal ion and chelator within a single small-molecule compound・synthesize active metal complexes in situ
by intramolecular chelation reactions in specific environments
Triger Event

inert

activeAdvantages① Co-delivery of metal ion and chelator within 1 compound② High metal selectivity due to metal carrying③ Higher target selectivity (than general prodrugs) due to greater structural changes
Zeqian H., et al. Angew. Shem. Int. Ed. 2022, 61, e202203500

2-i. Application to cancer

Metal-Carrying Prochelator
Mechanism and Design

DTC

Phenylborate ester：・H2O2 dependent cleavage and release of DTC※ H2O2 conc. : 100uM (tumor) ≫ 20nM (normal)

2,2’-dipicolylamine (DPA) :・Weaker than DTC※logK：18.35(DPA-Cu) < 25.0 (Cu(DTC)2)
・Stronger than other molecule in physiological environment
Zeqian H., et al. Angew. Shem. Int. Ed. 2022, 61, e202203500

2-i. Application to cancer

Metal-Carrying Prochelator
Properties：DPA moiety
1:1 complex

Stability

Selectivity

Cu(DTC)2 generation

Zeqian H., et al. Angew. Shem. Int. Ed. 2022, 61, e202203500

Metal-Carrying Prochelator
Properties：DPBD (w/ Phenylborate ester)

DPBD

Zeqian H., et al. Angew. Shem. Int. Ed. 2022, 61, e202203500

2-i. Application to cancer

Metal carrying prochelator
cellular / in vivo test

1O 2

Zeqian H., et al. Angew. Shem. Int. Ed. 2022, 61, e202203500

2-i. Application to cancer

Outline
1. Introduction
i. Concept
ii. Conventional Application

2. Recent Attempts
i. Application to cancer
ii. Application to Alzheimer’s disease

3. Perspective & Summary

18

The role of copper in AD

2-ii. Application to AD

Dyshomeostasis of copper in Alzheimer’s diseasee.g, 0.4mM accunmulation of Cu in Aβ plaque・Disfunction of physiologically essential enzyme
・ROS (reactive oxygen species) production through free or Aβ bound Copper・Promotion of Aβ aggregation and stabilization of Aβ oligomer
・Promotion of tau phospholylation

Cu chelator can Alleviate the pathology of Alzheimer's disease
Charlène Esmieu, et al. Inorganic Chemistry. 2019 58 (20), 13509-13527.
Kehinde D. Fasae, et al. J. Trace. Elem. Med. Bio. 2021 67, 126779.

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History of chelators toward AD

2-i. Application to cancer

Clioquinol (CQ)：
・The 1st metal ligand (Ionophore)
explored in neurodegenerative diseases
・Moderate affinity for Cu and Zn✓ It permeated the BBB, facilitated clearance of Aβ deposits
and improved cognitive function
× Adverse effects such as neurotoxicity were observed in a Tier II study (failure).
× Toxic di-iodo impurities were found in the manufacturing process.
PBT2：・Compound with the same 8-hydroxyquinoline backbone✓ It did not show any serious side effects✓ It restored cognitive function in AD mice.
× It did not significantly reduce Aβ plaques (clinical trial failure)Charlène Esmieu, et al. Inorganic Chemistry. 2019 58 (20), 13509-13527.
Kehinde D. Fasae, et al. J. Trace. Elem. Med. Bio. 2021 67, 126779.

Valentina Oliveri. Coord. Chem. Rev. 2020. 422,213474.

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Other Examples

2-i. Application to cancer

・Hydroxyquinoline(HQ) Derivatives (L5-L7)
higher affinity than 8-HQ parent compound
due to tetradentate binding motif.

・Aminoquinoline Derivatives (L8, L9)high affinity to Cu(II)・Tetraazamacrocycles (L10-L17)high selectivity for Cu(II) over Zn(II), low MW・Aminophenol Derivatives (L18-L20)

multifunctional agents- phenolic moiety：ROS scavenger- carbohydrate：solubility, low toxicity, BBB penetrationhigh selectivity for Cu(II) over Zn(II)・Aminopyridine Ligands (L22)
L21 cannot compete with Aβ.

・Phosphine Derivatives (L23)

first Cu(I) ligand able to retrieve both Cu(I/II) from Aβ・Bis(thiosemicarbazonato) Derivatives (L24, L25)
IonophoreCharlène Esmieu, et al. Inorganic Chemistry. 2019 58 (20), 13509-13527.

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“2-in-1” bifunctional chelator

2-i. Application to cancer

Incorporate structural elements
known to interact with Aβ species→ improve selectivity(Another way to increase selectivity than prochelators)

Aβ interaction scaffold(A) Stilbene-like (L33-L44)
including resveratrol derivatives (RESV-)(B) IMPY-like (L45-L47)(C) PiB(ThT)-like (L48-L53)

Charlène Esmieu, et al. Inorganic
Chemistry. 2019 58 (20), 13509-13527.
Masha G. Savelieff, et al. Chemical
Reviews 2019 119 (2), 1221-1322

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New Type Chelator：Design

2-ii. Application to Alzheimer’s disease

Stilbene-like“2-in-1” bifunctional chelator① Accommodation of the coordination geometries

S and N donor atoms in thiophen-2-ylmethanamine: for Cu(I) and Cc(II) (respectively∵HSAB rule)
N,N-dimethylaniline (DMA) moiety: for Aβ amyloid② Redox Potential

N,N-dimethyl-p-phenylenediamine (DMPD) moiety in L1：{E1/2: 0.11 V vs. Ag/Ag(I) in H2O}
Cu(II)–Aβ：{E0: approximately 0.083 V vs. Ag/Ag(I) in H2O}
→ critical in L1 capacity as a reducing agent③ Promotion of copper-O2 chemistry

Thiophene moiety：weaker σ-bonding ligand→ potential for O2 binding at the metal center in Cu(I/II)–AβMarinary Y. C., Richard W. V. J. Am. Soc. Mass. Spectrom. 2002. 13, 813-825.
Jiyeon Han, et al.PNAS. 2020. 117 (10), 5160-5167.

23

New Type Chelator：Mechanism

2-ii. Application to Alzheimer’s disease

MW: +96

MW: +16
Jiyeon Han, et al.PNAS. 2020. 117 (10), 5160-5167.

24

New type chelator：Mechanism

Jiyeon Han, et al.PNAS. 2020. 117 (10), 5160-5167.

2-ii. Application to Alzheimer’s disease25

New type chelator：Selectivity
Metal ion selectivity

Jiyeon Han, et al.PNAS. 2020. 117 (10), 5160-5167.

※Zn: non-redox

2-ii. Application to Alzheimer’s disease
Protein selectivity※Ubiquitin: non-amyloidogenic

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New type chelator：Efficiency※ L2,L3: L1-like non-reactive compounds
Jiyeon Han, et al.PNAS. 2020. 117 (10), 5160-5167.

2-ii. Application to Alzheimer’s disease27

Outline
1. Introduction
i. Concept
ii. Conventional Application

2. Recent Attempts
i. Application to cancer
ii. Application to Alzheimer’s disease

3. Perspectives & Summary28

Challenges

3. Perspectives & Summary• Not much relevant data due to short history of the field・Even the advantages of chelation is controversial.
・The role of metals in pathology is also ambiguous.

• Variability of the experimental condition or usage of term
• Many criteria to be met for clinical use
chelation ability, redox inertness, BBB or membrane permeability, solubility
good affinity, selectivity, kinetic property, stability property, low toxicity• Negative impression

・ EDTA toxicity
・ FDA waring in 2010
・ Chelation therapies as alternative medicine

Anne Robert, et al. Accounts of Chemical Research. 2015 48 (5), 1332-1339.
Charlène Esmieu, et al. Inorganic Chemistry. 2019 58 (20), 13509-13527.
Kehinde D. Fasae, et al. J. Trace. Elem. Med. Bio. 2021 67, 126779.

29

Summary

3. Perspectives & Summary• Recent findings on chelation-based therapy in cancer andAlzheimer's disease (focusing on Cu ions) were outlined.
• .Although there are still some issues to be solved,
this treatment method is expected to be a new modality.
• There may be room for consideration of a copper-triggered
switch mechanism ?

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Appendix
31安定度定数 K (logK)

32

DPBD synthesis

33

N-dealkylation のところ↑ひいてたのはこれ

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Cuproptosis
旧説：・アポトーシスの誘導
・カスパーゼ非依存性細胞死・活性酸素種（ROS）誘導・ユビキチン-プロテアソーム系阻害

Li, SR., et al. Sig Transduct Target Ther. 2022. 7, 158.

excess copper promotes the aggregation oflipoylated proteins and destabilization of
Fe-S cluster proteins that results inproteotoxic stress and ultimately cell death

Schematic illustration of cuproptosis
mechanism. The effect of intercellular copper
concentration on cell activity shows that cells
maintain highly bioactive only within a suitable
range of extremely narrow copper ion
concentrations (a). Elesclomol, a copper
ionophore, shuttles copper into the cells. FDX1
encodes a reductase to reduce Cu2+ to Cu+ andis a direct target of elesclomol. DLAT, a protein
target of lipoylation, involves with mediating
carbon entry to the TCA cycle. DLAT lipoylation
was promoted by FDX1, and Cu+ enhanced
lipoylated protein aggregation and iron-sulfur
cluster protein reduction, which triggered
proteotoxic stress and cell death (b).

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自閉症スペクトラムに対するキレーション• Main results• We excluded nine studies because they were non‐randomised trials or werewithdrawn before enrolment. We included one study, which was conducted in twophases. During the first phase of the study, 77 children with ASD were randomly
assigned to receive seven days of glutathione lotion or placebo lotion, followed bythree days of oral dimercaptosuccinic acid (DMSA). Forty‐nine children who were
found to be high excreters of heavy metals during phase one continued on to phase
two to receive three days of oral DMSA or placebo followed by 11 days off, with thecycle repeated up to six times. The second phase thus assessed the effectiveness ofmultiple doses of oral DMSA compared with placebo in children who were highexcreters of heavy metals and who received a three‐day course of oral DMSA. Overall,no evidence suggests that multiple rounds of oral DMSA had an effect on ASD
symptoms.
• Authors' conclusions• This review included data from only one study, which had methodological limitations.
As such, no clinical trial evidence was found to suggest that pharmaceuticalchelation is an effective intervention for ASD. Given prior reports of serious adverse
events, such as hypocalcaemia, renal impairment and reported death, the risks ofusing chelation for ASD currently outweigh proven benefits. Before further trials are
conducted, evidence that supports a causal link between heavy metals and autism
and methods that ensure the safety of participants are needed.

The argument for why chelation therapy may
be helpful for persons with ASD stems fromthe use of mercury in vaccines (Holmes,2010). Mercury, as well as other metals, hasbeen shown to be toxic to humans in highlevels. Thimerosal, which contains mercury,
was used in vaccines given to young children.
Thus, the argument goes that ASD could be
due in part to the presence of heavy metals
in the body.
↑
Given the small amount of Thimerosal invaccines, the fact that Thimerosal was
eliminated from vaccines several years ago,
and the continuing increasing incidence ofautism, the believability of this premise isquite weak.

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Cardiovascular Disease に対するキレーション• Background• EDTA is an intravenous chelating agent with high affinity to divalent cations (lead, cadmium, and calcium) that may bebeneficial in the treatment of cardiovascular disease (CVD). Although a large randomized clinical trial showed benefit,
smaller studies were inconsistent. We conducted a systematic review of published studies to examine the effect of
repeated EDTA on clinical outcomes in adults with CVD.
• Methods and Results• We searched 3 databases (MEDLINE, Embase, and Cochrane) from database inception to October 2021 to identify all
studies involving EDTA treatment in patients with CVD. Predetermined outcomes included mortality, disease severity,
plasma biomarkers of disease chronicity, and quality of life. Twenty‐four studies (4 randomized clinical trials, 15
prospective before/after studies, and 5 retrospective case series) assessed the use of repeated EDTA chelation treatment
in patients with preexistent CVD. Of these, 17 studies (1 randomized clinical trial) found improvement in their respective
outcomes following EDTA treatment. The largest improvements were observed in studies with high prevalence ofparticipants with diabetes and/or severe occlusive arterial disease. A meta‐analysis conducted with 4 studies reporting
ankle‐brachial index indicated an improvement of 0.08 (95% CI, 0.06–0.09) from baseline.
• Conclusions• Overall, 17 studies suggested improved outcomes, 5 reported no statistically significant effect of treatment, and 2
reported no qualitative benefit. Repeated EDTA for CVD treatment may provide more benefit to patients with diabetes
and severe peripheral arterial disease. Differences across infusion regimens, including dosage, solution components, andnumber of infusions, limit comparisons across studies. Additional research is necessary to confirm these findings and toevaluate the potential mediating role of metals.

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Web MD のキレーションに関する記事
Autism：×
The use of chelation therapy to treat this condition is based on the idea that autism is caused by mercury in childhood vaccines. Studies have
proven this idea to be false. But some health care providers also believe that removing metals from the body can improve autism symptoms.
The American Academy of Pediatrics (AAP) says there’s no evidence that chelation is an effective treatment for autism, and it may be dangerous.
One child with the condition died after having this treatment. The AAP doesn't recommend using chelation therapy for autism, except in aclinical trial.
Alzheimer‘s Disease：△
In patients who have this, abnormal proteins called tau and beta amyloid build up in the brain and damage it. To date, no treatment can stop orreverse this disease.
Some researchers think that a buildup of metals like copper, iron, and zinc might also play a role in Alzheimer's disease. If this is true, chelation
therapy might have a place in treating it. So far, there's no evidence that it works.
Heart Disease：△ (糖尿病は〇？)

You get this when fatty deposits called plaques form in your arteries. These substances cause your blood vessels to narrow. They also make themless flexible, so less blood can flow through them. Artery plaques contain calcium. The chelating drug disodium EDTA binds to this mineral. The
idea is that chelation therapy clears it out of the blood vessels. It removes plaques, too.
In 2002, the National Institutes of Health did a big study on chelation therapy, called TACT. It found that this treatment somewhat reduced therisk of heart attacks, strokes, and other heart problems. But it only worked in people with diabetes. The study didn't find enough proof that ittreats heart disease. And so far, the FDA hasn't approved this treatment for the condition. A new study called TACT2 may yield more information.
About TACT：
https://www.nccih.nih.gov/health/questions-and-answers-the-nih-trials-of-edta-chelationtherapy-for-coronary-heart-disease

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GCT prodrug
A prodrug approach is presented to direct copperdependent cytotoxicity to prostate cancer cells. The
prochelator GGTDTC requires activation by γ-glutamyltransferase (GGT) to release the metal chelator
diethyldithiocarbamate from a linker that masks its thiol
reactivity and metal binding properties. In vitro studies
demonstrated successful masking of copper binding aswell as clean liberation of the chelator by GGT. GGTDTC wasstable to non-specific degradation when incubated with aseries of prostate cancer and normal cell lines, withselective release of diethyldithiocarbamate only occurringin cells with measurable GGT activity. The antiproliferative
efficacy of the prochelator correlated with cellular GGTactivity, with 24 h inhibitory concentrations ranging from
800 nm in prostate cancer lines 22Rv1 and LNCaP to over
15 μm in normal prostate PWR-1E cells. These findings
underscore a new strategy to leverage the amplified
copper metabolism of prostate cancer by conditional
activation of a metal-binding pharmacophore.

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他のIonophore for がん40

他のproIonophore for がん
Mechanism of action of HQs and theirproionophores GluHQs and GalHQs. CuHQs diffuse passively through the cell
membrane into the cytosol and release Cu
to their target or increase the intracellularpool of Cu (A). GluHQs and GalHQs cannot
passively cross the cell membrane owing to
the hydrophilicity. The targeting moiety of
GluHQs is recognized by GLUT transportersthat are overexpressed in cancer cells (B).
Upon the internalization, GluHQs arehydrolyzed by the cytosolic β-glucosidase(B). The nontoxic GalHQs are administeredsystemically and hydrolyzed only in thepresence of the exogenous β-galactosidasethat is delivered to cancer cells exploiting
an antigen–antibody interaction (C). In thelatter cases, the released HQs function as
Cu ionophores and release Cu to theirtarget or increase the intracellular pool ofCu (for clarity, the release of Cu is not
represented but is analogous to themechanism represented in A).

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Cu homeostasis
Fig. 1. Cu homeostasis in mammalian cells.
Cu2+ is reduced to Cu+ by reductases on
the cell membrane. Cu is transported into
the cytoplasm by Ctr1. The imported
Cu+ rapidly associates with othermolecules such as metallothioneins, GSH,
and metallochaperones, which distribute
Cu+ to different cellular locations. Atox1
transports Cu to the ATPases, ATP7A, andATP7B in the Golgi complex, whereas
Cox17 and Sco proteins are involved in the
trafficking of Cu to the mitochondria forincorporation into cytochrome c oxidase(COX). The Cu chaperone for Cu/Znsuperoxide dismutase (CCS) transfers Cu to
Cu/Zn superoxide dismutase (Cu/Zn
SOD/SOD1) that is involved in
the detoxification of superoxide radicals.
For clarity, only the oxidation state of Cu isrepresented in the figure, however Cu isalways bound to biologically-relevantmolecules.
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Metal shuttle
Metal shuttles do not significantly affect the
intracellular metal concentration but deliver the
metal to a precise target, or the metal complex
itself is the active compound

https://www.sciencedirect.com/science/article/pii/S001085
4520303349?via%3Dihub

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CQ の役割
CQ (or PBT2) sequester metal ions that are in equilibrium with amyloid-beta (Aβ) complexes
and promote plaque dissociation.
CQ (or PBT2) can form ternary complexes with the metal ion and Aβ.
These complexes are internalized by neurons, where the metals and ligands dissociate andare addressed to different pathways.
The metal ions may activate phosphoinositol 3-kinase (PI3K).
This leads to downstream activation of Akt, c-Jun-N-terminal kinase (JNK), extracellularsignal-regulated kinase (ERK), and consequently matrix metalloproteinases (MMPs).
MMPs promote Aβ clearance, whereas Akt pathway inhibits the α- and β-isoforms ofglycogen synthase kinase 3 (GSK3), a serine/threonine kinase.
GSK3β is considered a central target in AD as it is associated with the hyperphosphorylationof tau protein.
GSK3β is inhibited by phosphorylation at the regulatory site ser-9.
Overall, CQ (or PBT2) reduces tau phosphorylation and mediate Aβ clearance trough theactivation of the above-described pathways.

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Cu-AβからのROS発生

45