1. The Iron Metabolism and Chelation Program - CCIA Des Richardson

2. 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

3. 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

4. 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.

5. Development of New Chelators Design of novel chelators is required for commercial interest and clinical application Provisional Patent – Hybrid Iron Chelators

6. New NT series analogues NTN2mTN4mTN44mT N4eT N4aTN4pT ST Thiosemicarbazones - ‘NT’ series Hydrazones 311311m - NNHN44pHNoctH  effect of increasingly lipophilic substituents at terminal N4

7. Some PIH analogues markedly inhibit tumor cell growth Richardson D, Tran E, Ponka P Blood (1995) Pyridoxal - 100 series Salicylaldehyde - 200 series 2-Hydroxy-1-naphthaldehyde - 300 series

8. Selective anti-proliferative activity

9. EPR of ribonucleotide reductase activity Control cells 311 (25  M) Triapine (25  M)

10. Mechanisms of activity - effect on cell cycle control molecules

12. Fe chelation decreases nuclear p21 protein CON DFO 311 Cis-platin Act D MCF-7 CON DFO 311 Cis-platin Act D MRC-5 p53 p21 β-actin p53 p21 β-actin Le and Richardson (2003) Carcinogenesis 24:1045-1058 NUCLEAR LYSATES

13. Decrease in nuclear p21 expression not due to cytoplasmic localization p21 CONDFO311Act D p53 MCF-7 Le and Richardson (2003) Carcinogenesis 24:1045-1058

14. Gene Array Analysis

15. The increase in NDRG1 mRNA is Fe dependent NDRG1 β-actin CON CON + FAC DFO DFO + MEM DFO + FAC 311 311 + MEM 311 + FAC MCF-7 Breast Cancer Cells TfR1

16. 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 11 Day 0

17. The Role of Membrane-Bound Melanotransferrin (MTf) in Iron Uptake by Cells

18. Intercellular Adhesion Scavenging Extracellular Fe Metalloprotease Activity Zn MTf TfR ? ? ? POSSIBLE FUNCTIONS OF MELANOTRANSFERRIN

19. Iron uptake as a function of time at 37 o C and 4ºC for CHO cells transfected with or without MTf. Time (min) 060120180240 Internalized Iron (pmol/10 6 cells) 0 3 6 9 12 -MTF 4 0 C +MTf 4 0 C -MTf 37 0 C +MTf 37 0 C

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

21. 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 6b Long 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.

23. Total MTf Knockout Vector 5’ arm 3’ arm PGKneo Targeting 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’ Probe Eco47 II /Hind III double digest WT = 15 kb Targeted = 11.4 3’ Probe Nhe I digest WT = 12.6 kb Targeted = 8.3

24. Examination of MTf Knockout Mouse Histopathology/Blood Counts/Clinical Chemistry/X-ray Morphology/Morphometry Behaviour Conditional Knockout