The Iron Metabolism and Chelation Program - CCIA Des Richardson.
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Transcript of The Iron Metabolism and Chelation Program - CCIA Des Richardson.
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