1 / 24

The Iron Metabolism and Chelation Program - CCIA

The Iron Metabolism and Chelation Program - CCIA. Des 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

genna
Télécharger la présentation

The Iron Metabolism and Chelation Program - CCIA

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  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 Thiosemicarbazones - ‘NT’ series ST NT N2mT N4mT N44mT  effect of increasingly lipophilic substituents at terminal N4 N4aT N4pT N4eT Hydrazones 311 311m - NNH N44pH NoctH

  7. m ID mol/L 50 2-Hydroxy-1- Pyridoxal Salicylaldehyde Naphthaldehyde Hydrazone (100 series) (200 series) (300 series) Benzoyl (01) 35 5 3 p- Hydroxybenzoyl (02) >80 36 4 p- Methylbenzoyl (03) 28 - - p- Nitrobenzoyl (04) >80 49 - p- Aminobenzoyl (05) >80 76 8 p-t- Butylbenzoyl (06) 7 1 - p- Methoxybenzoyl (07) 52 8 2 m- Chlorobenzoyl (08) 24 20 1 m- Florobenzoyl (09) 17 2 1 m- Bromobenzoyl (10) 41 - 2 Isonicotinoyl (11) 75 21 1 Acetyl (12) >80 >80 7 2-Pyridyl (13) 7 - - 2-Furoyl (14) >80 - - 2-Thiophenecarboxyl (15) 30 8 1 m ID mol/L 50 DFO 22 Some PIH analogues markedly inhibit tumor cell growth Pyridoxal - 100 series Salicylaldehyde - 200 series 2-Hydroxy-1-naphthaldehyde - 300 series Richardson D, Tran E, Ponka P Blood (1995)

  8. Selective anti-proliferative activity

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

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

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

  12. Decrease in nuclear p21 expression not due to cytoplasmic localization CON DFO 311 Act D p53 p21 MCF-7 Le and Richardson (2003) Carcinogenesis 24:1045-1058

  13. Gene Array Analysis

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

  15. In Vivo Experimental Design M109 lung carcinoma animal model 11 Day 0 4 9 Drugs injection Stop injection Tumor implantation Harvesting Outcome assessment Tumor weight Tumor H&E staining and TUNEL assay body weight Blood cell count, RBC, WBC, Platelet, Hb

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

  17. POSSIBLE FUNCTIONS OF MELANOTRANSFERRIN ScavengingExtracellularFe Intercellular Adhesion ? ? TfR Zn ? MTf Metalloprotease Activity

  18. 12 0 -MTF 4 C 0 +MTf 4 C 0 -MTf 37 C 0 +MTf 37 C 9 6 Internalized Iron (pmol/106cells) 3 0 0 60 120 180 240 Time (min) Iron uptake as a function of time at 37oC and 4ºC for CHO cells transfected with or without MTf.

  19. Effect of PI-PLC on Metal Ion Uptake by Melanoma Cells 1 2 0 1 0 0 8 0 Cellular Uptake (% Control) 6 0 4 0 2 0 0 _ _ _ _ _ + + + P I - P L C : + + 1 2 5 5 9 6 5 1 2 5 5 9 6 7 6 4 6 7 6 5 6 4 L a b e l : F e Z n I - M o A b F e G a C u G a Z n I - M o A b C u

  20. The relative positions of the originally identified melanotransferrin 1 (MTf1) gene compared to the newly identified MTf2 gene. MTf 1 MTf 2 1 2 3 4 5 6 1 2 3 4 5 6 6b 7 8 9 10 11 12 13 14 15 16 16b E P E P Short MTf1 transcript (1.6 kb), exons 1 to 6 + exon 6b MTf2 transcript ? Long MTf1 transcript (2.3 kb) exons 1 to 16 Long MTf1 transcript (2.6 kb) exons 1 to 16 + exon 16b

  21. Total MTf Knockout Vector 3’ Probe 5’ Probe Deletion Arm WT Homologous Recombination 3’ arm 5’ arm Targeting Vector PGKneo Targeted PGKneo = Nhe I Restriction Enzyme site = Eco47II Restriction Site = Hind III Restriction Site Expected Restriction Enzyme Fragments From WT and Targeted Gene 3’ Probe 5’ Probe Nhe I digest WT = 12.6 kb Targeted = 8.3 Eco47 II /Hind III double digest WT = 15 kb Targeted = 11.4

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

More Related