The Iron Metabolism and Chelation Program - CCIA

Des RichardsonDes Richardson

Aims of the Iron Metabolism and Chelation Program

(1) To develop new therapeutic agents to treat cancer and a variety of other diseases.

(2) To understand how cancer and normal cells metabolise iron – this knowledge is critical for achieving goal #1

Techniques Used• Wide Range of Techniques Used: Chemistry and Biology

• Synthetic and Inorganic Chemistry - Collaboration with P. Bernhardt (U.Q.)

• X-ray crystallography and molecular modeling

• Cell Biology

• Molecular Biology – Simple to Complex (Gene Knockout Studies in Mice to Determine Molecular Function )

• Animal Studies- Analysing Effects of Compounds in Vivo in Animal Models

Projects Underway in the Iron Program

1. Development of novel anti-metabolites: iron chelators/metal complexes with selective anti-tumour activity

2. Examining the role of iron in the cell cycle – p21 and p53

3. Examination of the function of the melanoma tumour antigen, melanotransferrin- generation of melanotransferrin KO mouse.

4. Examination of the cytotoxic effects of anthracyclines via their avid interaction with cellular iron

5. The effects of nitric oxide and carbon monoxide on Fe metabolism. NO has a high affinity for Fe and plays a role in the anti-tumour effects of macrophages (a role for CO ??)

6. Development of iron chelators for the treatment of Friedreich’s Ataxia and -Thalassaemia and understanding the function of frataxin in intracellular Fe trafficking.

Development of New Chelators

• Design of novel chelators is required for commercial interest and clinical application

• Provisional Patent – Hybrid Iron Chelators

New NT series analogues

OH

N

HN

S

NH2

OH

NN

S

NH2

CH3

OH

N

HN

S

HN

CH3

OH

N

HN

S

N

CH3

CH3

OH

N

HN

S

HN

CH2

CH3

OH

N

HN

S

HN

CH2

HC

CH2

OH

N

HN

S

HN

OH

N

HN

S

NH2

NT N2mT N4mT N44mT

N4eT N4aT N4pT

ST

Thiosemicarbazones - ‘NT’ series

OH

N

HN

O

N

Hydrazones

311OH

N

HN

O

N

311m - NNHOH

N

HN

O

N

N44pHOH

N

HN

O

NoctH

effect of increasingly lipophilic substituents at terminal N4

Some PIH analogues markedly inhibit tumor cell growth

ID50 mol/L2-Hydroxy-1-

Pyridoxal SalicylaldehydeNaphthaldehydeHydrazone (100 series) (200 series) (300 series)Benzoyl (01) 35 5 3p-Hydroxybenzoyl (02) >80 36 4p-Methylbenzoyl (03) 28 - -p-Nitrobenzoyl (04) >80 49 -p-Aminobenzoyl (05) >80 76 8p-t-Butylbenzoyl (06) 7 1 -p-Methoxybenzoyl (07) 52 8 2m-Chlorobenzoyl (08) 24 20 1m-Florobenzoyl (09) 17 2 1m-Bromobenzoyl (10) 41 - 2Isonicotinoyl (11) 75 21 1Acetyl (12) >80 >80 72-Pyridyl (13) 7 - -2-Furoyl (14) >80 - -2-Thiophenecarboxyl (15) 30 8 1

ID50 mol/L

DFO 22Richardson D, Tran E, Ponka P Blood (1995)

N

OH

N NH

O

H3C

CH2

HO

OH

N NH

O

OH

N NH

O

Pyridoxal - 100 series

Salicylaldehyde - 200 series

2-Hydroxy-1-naphthaldehyde - 300 series

Selective anti-proliferative activity

311

311m

NT

N4mT

N44mT

N2mT

DFO

IC50 (M)

>25

>25

>25

>25

>25

22

0.3

0.3

0.5

>12.5

0.5

1.5

SK-N-MC neuroepithelioma MRC-5 fibroblasts

>25

>25

EPR of ribonucleotide reductase activity

Control cells

311 (25M)

Triapine (25M)

OH

N

HN

O

N

N

N

HN

S

NH2

NH2

Mechanisms of activity - effect on cell cycle control molecules

Fe chelation decreases nuclear p21 protein

CO

N

DF

O

311

Cis-pla

tin

Act D

MCF-7

CO

N

DF

O

311

Cis-pla

tin

Act D

MRC-5

p53

p21

β-actin

p53

p21

β-actin

Le and Richardson (2003) Carcinogenesis 24:1045-1058

NUCLEAR LYSATES

Decrease in nuclear p21 expression not due to cytoplasmic localization

p21

CON DFO 311 Act D

p53

MCF-7

Le and Richardson (2003) Carcinogenesis 24:1045-1058

Gene Array Analysisp53 GEarray Q series

GENES

% r

ela

tive

to

th

e c

on

tro

l

0

200

400

600

800

1000

CONDFO311ActD

The increase in NDRG1 mRNA is Fe dependent

NDRG1

β-actin

CO

N

CO

N +

FA

C

DF

O

DF

O +

ME

M

DF

O +

FA

C

311

311

+ M

EM

311

+ F

AC

MCF-7 Breast Cancer Cells

TfR1

In Vivo Experimental Design

M109 lung carcinoma animal model

Tumor weight body weight Blood cell count, RBC, WBC, Platelet, Hb

Outcome assessment

Tumor H&E staining and TUNEL assay

Tumor implantation

4

Drugs injection

9

Stop injection

Harvesting

11Day 0

The Role of Membrane-Bound Melanotransferrin (MTf) in Iron

Uptake by Cells

IntercellularAdhesion

Scavenging Extracellular Fe

MetalloproteaseActivity

Zn

MTf

TfR

?

?

?

POSSIBLE FUNCTIONS OF MELANOTRANSFERRIN

Iron uptake as a function of time at 37oCand 4ºC for CHO cells transfected with or without MTf.

Time (min)

0 60 120 180 240

Inte

rnal

ized

Iro

n (

pm

ol/1

06 cel

ls)

0

3

6

9

12-MTF 40C+MTf 40C-MTf 370C+MTf 370C

PI-PLC: Label: 125I-MoAb

125I-MoAb 59Fe 67Ga59Fe 67Ga

Cel

lula

r U

pta

ke (

% C

on

tro

l)

0

20

40

60

80

100

120

64Cu

64Cu

65Zn 65Zn

Effect of PI-PLC on Metal Ion Uptake by Melanoma Cells

E P E P

MTf 2 MTf 1

1 2 3 4 5 6 1 2 3 4 5 6 6b 7 8 9 10 11 12 13 14 15 16 16b

MTf2 transcript ? Short MTf1 transcript (1.6 kb), exons 1 to 6 + exon 6bLong MTf1 transcript

(2.3 kb) exons 1 to 16

Long MTf1 transcript (2.6 kb) exons 1 to 16 + exon 16b

The relative positions of the originally identified melanotransferrin 1 (MTf1) gene compared to the newly identified MTf2 gene.

Total MTf Knockout Vector

5’ arm 3’ arm

PGKneoTargeting Vector

PGKneo

5’ Probe 3’ Probe

Homologous Recombination

WT

Targeted

Deletion Arm

= Nhe I Restriction Enzyme site

= Eco47II Restriction Site

= Hind III Restriction Site

Expected Restriction Enzyme Fragments From WT and Targeted Gene

5’ ProbeEco47 II /Hind III double digest

WT = 15 kbTargeted = 11.4

3’ Probe

Nhe I digest WT = 12.6 kb

Targeted = 8.3

Examination of MTf Knockout Mouse

• Histopathology/Blood Counts/Clinical Chemistry/X-ray

• Morphology/Morphometry

• Behaviour

• Conditional Knockout