The Iron Metabolism and Chelation Program - CCIA

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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 - PowerPoint PPT Presentation

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  • 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 cancer and normal cells metabolise iron this knowledge is critical for achieving goal #1

  • Techniques UsedWide 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 Friedreichs Ataxia and -Thalassaemia and understanding the function of frataxin in intracellular Fe trafficking.

  • Development of New ChelatorsDesign of novel chelators is required for commercial interest and clinical application

    Provisional Patent Hybrid Iron Chelators

  • New NT series analoguesNTN2mTN4mTN44mTN4eTN4aTN4pTSTThiosemicarbazones - NT seriesHydrazones311311m - NNHN44pHNoctH effect of increasingly lipophilic substituents at terminal N4

  • Some PIH analogues markedly inhibit tumor cell growthRichardson D, Tran E, Ponka P Blood (1995)

    Pyridoxal - 100 seriesSalicylaldehyde - 200 series2-Hydroxy-1-naphthaldehyde - 300 series

  • Selective anti-proliferative activity

    efflux

    3.50693183770.1820638981

    7.41471903890.6185408672

    43.64671204811.0110996768

    41.27784325060.9172270926

    3.17903572130.2462487945

    12.93220284590.6142202956

    4.41681565450.3103442422

    28.98766324721.3863013841

    43.26727938140.9459199834

    34.85951462782.0327302508

    36.06188774611.8365922503

    37.26110391180.9013484931

    27.70191009140.1538082955

    35.69730951351.2223406338

    Fe uptake 22.2.00

    % Cellular Iron Released

    effluc TC

    2.731846317510.744623312964.969932998719.74343674864.41064018251.06669882491.06669882490.74804382540.74804382540.27096307170.27096307171.879076851.879076850.68985204990.6898520499

    3.839762840421.83607994571.830945954931.510971413869.99795298161.45610905611.45610905611.59483454661.59483454660.83750398930.83750398930.84334338580.84334338580.27517510430.2751751043

    6.962486854441.371010879378.468884731245.940835788276.43375906721.23248493921.23248493921.19167495611.19167495610.36256068640.36256068640.34061423560.34061423560.4365638650.436563865

    10.649926782654.875444758180.378032606753.907218528481.03815488653.73662454633.73662454631.28040970681.28040970680.64698383140.64698383141.06219697981.06219697980.99268792080.9926879208

    Control

    DFO

    311

    NT

    N44mT

    Time (h)

    % Cellular Iron Released

    uptake

    1004.2

    92.62165206144.28

    6.66700876320.94

    9.46381226840.56

    95.88901370359

    90.14222784212.8818577655

    90.98.1

    41.953.3712474269

    4.970.288267196

    19.642.6852675951

    18.742.0933098294

    25.352.1496329757

    25.358.5636126411

    38.82372683451.2023389055

    Internalized Iron Uptake (% control)

    uptake TC

    10090.50275466648.280279882989.04327844575.44336658426.28289349796.282893497913.608381911413.60838191145.15827503555.15827503550.30598498460.30598498461.25697177491.2569717749

    10084.74642645835.36161963982.3944966243.78245486985.66824721975.66824721976.92268937756.92268937753.21668697663.21668697660.59273445570.59273445570.86497459860.8649745986

    10075.91294755854.13544646375.11757666542.69832989465.95443972935.95443972932.58650513082.58650513089.03465428319.03465428310.12984510570.12984510570.29040154550.2904015455

    10047.63132696811.631420494560.62028416260.67914372042.30621680382.30621680381.44351145051.44351145051.86579133091.86579133090.0554884710.0554884710.48459841780.4845984178

    Control

    DFO

    311

    NT

    N44mT

    Time (h)

    Internalized Iron Uptake (% control)

    MTT

    0.1101.9404639287110.862125264497.2354058355104.9141939268128.920653707898.370699989292.25754223394.317077289494.8043811677100.6973960731112.896045037497.6901608582101.7433031097

    0.293.693476925772.002881808590.138965084989.486296821396.745117676582.651088282997.63388544990.439568596990.97111.30670876694.3869100455109.072633638698.7695575086

    0.3940.434940399633.705492360392.5655.891796777697.327809896755.5859507208102.855019961384.295716274584.113738050798.412571076281.5103.7673831976102.6235184192

    102.038605285522.4613.773181107478.2631.963311159986.812036236226.499995097871.358426093773.682039329984.780730623990.225328154793.7112.957045914693.5352921497

    92.226196435413.13688471857.928909812762.579814247216.674.976100514413.409640079948.363349937651.508125656967.946427408193.256533121696.188644201987.832807529996.7665844068

    96.410.63993131436.161.68.481.13078708977.696607573318.404687351328.33085988739.044121359250.921654156896.681.696716567184.6423594758

    88.69.96.653.6780892639786.16561205448.17100853679.620264427222.838642790626.887150449211.458283012175.316085860529.643362877466.9312856893

    56.67.84115572925.308965429351.55152782225.475779897764.03241225478.02972009288.68409473517.40481483217.891502760210.155699067640.44939064599.958504008961.8961895217

    DFO

    311

    NNH

    ST

    NT

    N2mT

    N4mT

    N44mT

    N4eT

    N4aT

    N4pT

    NOctH

    N44pH

    FeNT

    Chelator Concentration (mM)

    Cellular Proliferation (% control)

    MTTMRC51

    0.1101.9404639287104.9141939268128.920653707898.370699989292.2575422330.192.6174496644100.0583089522107.125243570694.403198175896.7415732506

    0.293.693476925789.486296821396.745117676582.651088282997.6338854490.289.115844762297.1966868704108.8498564152103.283268587994.0952103656

    0.3940.434940399655.891796777697.327809896755.5859507208102.85501996130.3995.8856142399101.5539348621107.10403499196.251645117898.3389849717

    102.038605285522.4631.963311159986.812036236226.499995097871.358426093790.235714148183.7467172454104.9878563539103.7669430193101.6428110784106.2921032183

    92.226196435413.136884718516.674.976100514413.409640079948.363349937691.198054774690.2830463963107.6426712987110.3259988661100.0240211224103.0557669922

    96.410.63993131438.481.13078708977.696607573318.404687351386.289956934766.2386927342106.4242803477111.8752133616100.763901805389.3963823523

    88.69.9786.16561205448.17100853679.620264427283.039164584660.1692442369101.8314546437105.6021913722105.932619046185.6532706677

    56.67.841155729212.564.03241225478.02972009288.68409473580.679212909775.576305806898.8238078623111.649294796497.695573033371.0267890763

    72.363922414675.576305806885.0602317064111.306727347692.081543233569.8859743024

    MRC-5Fibroblasts

    SK-N-MCNeuroepithelioma

    DFO (S)

    311 (S)

    NT (S)

    N2mT (S)

    N4mT (S)

    N44mT (S)

    DFO (F)

    311 (F)

    NT (F)

    N2mT (F)

    N4mT (F)

    N44mT (F)

    Chelator Concentration (mM)

    Cellular Proliferation (% control)

    NTpapMRC52

    0.1101.9404639287110.8621252644104.9141939268128.920653707898.370699989292.2575422330.192.617449664493.048745313100.0583089522107.125243570694.403198175896.7415732506

    0.293.693476925772.002881808589.486296821396.745117676582.651088282997.6338854490.289.115844762286.126334006397.1966868704108.8498564152103.283268587994.0952103656

    0.3940.434940399633.705492360355.891796777697.327809896755.5859507208102.85501996130.3995.885614239993.5102394001101.5539348621107.10403499196.251645117898.3389849717

    102.038605285522.4613.773181107431.963311159986.812036236226.499995097871.358426093790.235714148183.746717245488.2030573983104.9878563539103.7669430193101.6428110784106.2921032183

    92.226196435413.13688471857.928909812716.674.976100514413.409640079948.363349937691.198054774690.283046396381.7421401788107.6426712987110.3259988661100.0240211224103.0557669922

    96.410.63993131436.18.481.13078708977.696607573318.404687351386.289956934766.238692734270.6662820883106.4242803477111.8752133616100.763901805389.3963823523

    88.69.96.6786.16561205448.17100853679.620264427283.039164584660.169244236969.743293914101.8314546437105.6021913722105.932619046185.6532706677

    56.67.84115572925.308965429312.564.03241225478.02972009288.68409473580.679212909775.576305806873.665993654598.8238078623111.649294796497.695573033371.0267890763

    72.363922414675.576305806865.8785.0602317064111.306727347692.081543233569.8859743024

    MRC-5Fibroblasts

    SK-N-MCNeuroepithelioma

    DFO (S)

    311 (S)

    311m (S)

    NT (S)

    N2mT (S)

    N4mT (S)

    N44mT (S)

    DFO (F)

    311 (F)

    311m (F)

    NT (F)

    N2mT (F)

    N4mT (F)

    N44mT (F)

    Chelator Concentration (mM)

    Cellular Proliferation (% control)

    thymidine

    101.135.533.792.589.152.392.3104.198.8126.687.812378.683.4

    1414028.371.1109.372.279.747.6108.2100.352.5138.39386.2

    156.140.537.285.570.976.4113.538.183.589.852.852.6106106.2

    97.713.715.9111.964.676.449.2217.542.959.141.546.296.998.2

    69.26574400254.3768819453.756468381660.596057363630.761789735954.084582610913.94010688192.412154061520.500415930924.64916970637.037853881527.8998701254.109184387590.7221177789

    46.49504890713.4335932544.094484083885.15281598958.800784515837.39161791829.7933637532.5146677915.890397312321.22021843792.37744950264.699986616943.676176374112.5967714413

    DFO

    311

    NNH

    ST

    NT

    N2mT

    N4mT

    N44mT

    N4eT

    N4aT

    N4pT

    N44pH

    NOctH

    FeNT

    Chelator Concentration (mM)

    3H-Thymidine Incorporation (% control)

    logPvsIC50

    0.5

    0.5

    1.5

    1.5

    2.3

    3.3

    5.2

    0.3

    0.3

    10.5

    logP

    log P calc

    Chelator Antiproliferative Activity(IC50-mM)

    mtttable

    SK-N-MC neuroepitheliomaMRC-5 fibroblasts

    DFO22>25

    3110.3>25

    311m0.3*

    ST>12.5*

    NT0.5>25

    N2mT>12.5>25

    N4mT0.5>25

    N44mT1.5>25

    N4eT1.6*

    N4aT2.3*

    N4pT3.3*

    N44pH5.2*

    NoctH10.5*

    FeNT>12.5*

    * not determined

    logPtable

    3112.17

    311m2.17

    ST0.64

    NT1.76

    N2mT2.72

    N4mT2.19

    N44mT2.3

    N4eT2.4

    N4aT2.61

    N4pT3.75

    N44pH*

    NoctH5.19

    * Unable to be calculated by Broto's method

    Thytable

    DFO23

    3110.6

    311m0.6

    ST>25

    NT9.0

    N2mT15.5

    N4mT6.3

    N44mT1.5

    N4eT5.7

    N4aT7.9

    N4pT3.8

    N44pH4.3

    NoctH17.4

    FeNT>25

    mttforsem

    101.9404639287110.8621252644104.914193926898.370699989292.25754223394.317077289494.8043811677100.697396073197.690160858297.2354058355128.9206537078112.8960450374101.74330310970.1

    93.693476925772.002881808589.486296821382.651088282997.63388544990.439568596990.97111.306708766109.072633638690.138965084996.745117676594.386910045598.76955750860.2

    40.434940399633.705492360355.891796777655.5859507208102.855019961384.295716274584.113738050798.4125710762103.767383197692.5697.327809896781.5102.62351841920.39

    22.4613.773181107431.963311159926.499995097871.358426093773.682039329984.780730623990.2253281547112.957045914678.2686.812036236293.793.5352921497102.0386052855

    13.13688471857.928909812716.613.409640079948.363349937651.508125656967.946427408193.256533121687.832807529962.579814247274.976100514496.188644201996.766584406892.2261964354

    10.63993131436.18.47.696607573318.404687351328.33085988739.044121359250.921654156881.696716567161.681.130787089796.684.642359475896.4

    9.96.678.17100853679.620264427222.838642790626.887150449211.458283012129.643362877453.678089263986.165612054475.316085860566.931285689388.6

    7.84115572925.30896542935.47577989778.02972009288.68409473517.40481483217.891502760210.15569906769.958504008951.551527822264.032412254740.449390645961.896189521756.6

    311

    311m

    NT

    N4mT

    N44mT

    N4eT

    N4aT

    N4pT

    N44pH

    ST

    N2mT

    NOctH

    FeNT

    DFO

    Chelator Concentration (mM)

    Cellular Proliferation (% control)

    mtttabsem

    SK-N-MC neuroepithelioma

    3110.3

    311m0.3

    NT0.5

    N4mT0.5

    N44mT1.5

    N4eT1.6

    N4aT2.3

    N4pT3.3

    N44pH5.2

    NoctH10.5

    ST>12.5

    N2mT>12.5

    FeNT>12.5

    DFO22

    mtttablesem

    SK-N-MC neuroepitheliomaMRC-5 fibroblasts

    3110.3>25

    311m0.3>25

    NT0.5>25

    N4mT0.5>25

    N44mT1.5>25

    N2mT>12.5>25

    DFO22>25

    FeNT>12.5*

    * not determined

    NTpapMRC5sem

    101.9404639287110.8621252644104.914193926898.370699989292.257542233128.92065370780.192.617449664493.048745313100.058308952294.403198175896.7415732506107.12524357060.1

    93.693476925772.002881808589.486296821382.651088282997.63388544996.74511767650.289.115844762286.126334006397.1966868704103.283268587994.0952103656108.84985641520.2

    40.434940399633.705492360355.891796777655.5859507208102.855019961397.32780989670.3995.885614239993.5102394001101.553934862196.251645117898.3389849717107.1040349910.39

    22.4613.773181107431.963311159926.499995097871.358426093786.8120362362102.038605285583.746717245488.2030573983104.9878563539101.6428110784106.2921032183103.766943019390.2357141481

    13.13688471857.928909812716.613.409640079948.363349937674.976100514492.226196435490.283046396381.7421401788107.6426712987100.0240211224103.0557669922110.325998866191.1980547746

    10.63993131436.18.47.696607573318.404687351381.130787089796.466.238692734270.6662820883106.4242803477100.763901805389.3963823523111.875213361686.2899569347

    9.96.678.17100853679.620264427286.165612054488.660.169244236969.743293914101.8314546437105.932619046185.6532706677105.602191372283.0391645846

    7.84115572925.308965429312.58.02972009288.68409473564.032412254756.675.576305806873.665993654598.823807862397.695573033371.0267890763111.649294796480.6792129097

    75.576305806865.8785.060231706492.081543233569.8859743024111.306727347672.3639224146

    MRC-5Fibroblasts

    SK-N-MCNeuroepithelioma

    311 (S)

    311m (S)

    NT (S)

    N4mT (S)

    N44mT (S)

    N2mT (S)

    DFO (S)

    311 (F)

    311m (F)

    NT (F)

    N4mT (F)

    N44mT (F)

    N2mT (F)

    DFO (F)

    Chelator Concentration (mM)

    Cellular Proliferation (% control)

    efflux

    3.50693183770.1820638981

    7.41471903890.6185408672

    43.64671204811.0110996768

    41.27784325060.9172270926

    3.17903572130.2462487945

    12.93220284590.6142202956

    4.41681565450.3103442422

    28.98766324721.3863013841

    43.26727938140.9459199834

    34.85951462782.0327302508

    36.06188774611.8365922503

    37.26110391180.9013484931

    27.70191009140.1538082955

    35.69730951351.2223406338

    Fe uptake 22.2.00

    % Cellular Iron Released

    effluc TC

    2.731846317510.744623312964.969932998719.74343674864.41064018251.06669882491.06669882490.74804382540.74804382540.27096307170.27096307171.879076851.879076850.68985204990.6898520499

    3.839762840421.83607994571.830945954931.510971413869.99795298161.45610905611.45610905611.59483454661.59483454660.83750398930.83750398930.84334338580.84334338580.27517510430.2751751043

    6.962486854441.371010879378.468884731245.940835788276.43375906721.23248493921.23248493921.19167495611.19167495610.36256068640.36256068640.34061423560.34061423560.4365638650.436563865

    10.649926782654.875444758180.378032606753.907218528481.03815488653.73662454633.73662454631.28040970681.28040970680.64698383140.64698383141.06219697981.06219697980.99268792080.9926879208

    Control

    DFO

    311

    NT

    N44mT

    Time (h)

    % Cellular Iron Released

    uptake

    1004.2

    92.62165206144.28

    6.66700876320.94

    9.46381226840.56

    95.88901370359

    90.14222784212.8818577655

    90.98.1

    41.953.3712474269

    4.970.288267196

    19.642.6852675951

    18.742.0933098294

    25.352.1496329757

    25.358.5636126411

    38.82372683451.2023389055

    Internalized Iron Uptake (% control)

    uptake TC

    10090.50275466648.280279882989.04327844575.44336658426.28289349796.282893497913.608381911413.60838191145.15827503555.15827503550.30598498460.30598498461.25697177491.2569717749

    10084.74642645835.36161963982.3944966243.78245486985.66824721975.66824721976.92268937756.92268937753.21668697663.21668697660.59273445570.59273445570.86497459860.8649745986

    10075.91294755854.13544646375.11757666542.69832989465.95443972935.95443972932.58650513082.58650513089.03465428319.03465428310.12984510570.12984510570.29040154550.2904015455

    10047.63132696811.631420494560.62028416260.67914372042.30621680382.30621680381.44351145051.44351145051.86579133091.86579133090.0554884710.0554884710.48459841780.4845984178

    Control

    DFO

    311

    NT

    N44mT

    Time (h)

    Internalized Iron Uptake (% control)

    MTT

    0.1101.9404639287110.862125264497.2354058355104.9141939268128.920653707898.370699989292.25754223394.317077289494.8043811677100.6973960731112.896045037497.6901608582101.7433031097

    0.293.693476925772.002881808590.138965084989.486296821396.745117676582.651088282997.63388544990.439568596990.97111.30670876694.3869100455109.072633638698.7695575086

    0.3940.434940399633.705492360392.5655.891796777697.327809896755.5859507208102.855019961384.295716274584.113738050798.412571076281.5103.7673831976102.6235184192

    102.038605285522.4613.773181107478.2631.963311159986.812036236226.499995097871.358426093773.682039329984.780730623990.225328154793.7112.957045914693.5352921497

    92.226196435413.13688471857.928909812762.579814247216.674.976100514413.409640079948.363349937651.508125656967.946427408193.256533121696.188644201987.832807529996.7665844068

    96.410.63993131436.161.68.481.13078708977.696607573318.404687351328.33085988739.044121359250.921654156896.681.696716567184.6423594758

    88.69.96.653.6780892639786.16561205448.17100853679.620264427222.838642790626.887150449211.458283012175.316085860529.643362877466.9312856893

    56.67.84115572925.308965429351.55152782225.475779897764.03241225478.02972009288.68409473517.40481483217.891502760210.155699067640.44939064599.958504008961.8961895217

    DFO

    311

    NNH

    ST

    NT

    N2mT

    N4mT

    N44mT

    N4eT

    N4aT

    N4pT

    NOctH

    N44pH

    FeNT

    Chelator Concentration (mM)

    Cellular Proliferation (% control)

    MTTMRC51

    0.1101.9404639287104.9141939268128.920653707898.370699989292.2575422330.192.6174496644100.0583089522107.125243570694.403198175896.7415732506

    0.293.693476925789.486296821396.745117676582.651088282997.6338854490.289.115844762297.1966868704108.8498564152103.283268587994.0952103656

    0.3940.434940399655.891796777697.327809896755.5859507208102.85501996130.3995.8856142399101.5539348621107.10403499196.251645117898.3389849717

    102.038605285522.4631.963311159986.812036236226.499995097871.358426093790.235714148183.7467172454104.9878563539103.7669430193101.6428110784106.2921032183

    92.226196435413.136884718516.674.976100514413.409640079948.363349937691.198054774690.2830463963107.6426712987110.3259988661100.0240211224103.0557669922

    96.410.63993131438.481.13078708977.696607573318.404687351386.289956934766.2386927342106.4242803477111.8752133616100.763901805389.3963823523

    88.69.9786.16561205448.17100853679.620264427283.039164584660.1692442369101.8314546437105.6021913722105.932619046185.6532706677

    56.67.841155729212.564.03241225478.02972009288.68409473580.679212909775.576305806898.8238078623111.649294796497.695573033371.0267890763

    72.363922414675.576305806885.0602317064111.306727347692.081543233569.8859743024

    MRC-5Fibroblasts

    SK-N-MCNeuroepithelioma

    DFO (S)

    311 (S)

    NT (S)

    N2mT (S)

    N4mT (S)

    N44mT (S)

    DFO (F)

    311 (F)

    NT (F)

    N2mT (F)

    N4mT (F)

    N44mT (F)

    Chelator Concentration (mM)

    Cellular Proliferation (% control)

    NTpapMRC52

    0.1101.9404639287110.8621252644104.9141939268128.920653707898.370699989292.2575422330.192.617449664493.048745313100.0583089522107.125243570694.403198175896.7415732506

    0.293.693476925772.002881808589.486296821396.745117676582.651088282997.6338854490.289.115844762286.126334006397.1966868704108.8498564152103.283268587994.0952103656

    0.3940.434940399633.705492360355.891796777697.327809896755.5859507208102.85501996130.3995.885614239993.5102394001101.5539348621107.10403499196.251645117898.3389849717

    102.038605285522.4613.773181107431.963311159986.812036236226.499995097871.358426093790.235714148183.746717245488.2030573983104.9878563539103.7669430193101.6428110784106.2921032183

    92.226196435413.13688471857.928909812716.674.976100514413.409640079948.363349937691.198054774690.283046396381.7421401788107.6426712987110.3259988661100.0240211224103.0557669922

    96.410.63993131436.18.481.13078708977.696607573318.404687351386.289956934766.238692734270.6662820883106.4242803477111.8752133616100.763901805389.3963823523

    88.69.96.6786.16561205448.17100853679.620264427283.039164584660.169244236969.743293914101.8314546437105.6021913722105.932619046185.6532706677

    56.67.84115572925.308965429312.564.03241225478.02972009288.68409473580.679212909775.576305806873.665993654598.8238078623111.649294796497.695573033371.0267890763

    72.363922414675.576305806865.8785.0602317064111.306727347692.081543233569.8859743024

    MRC-5Fibroblasts

    SK-N-MCNeuroepithelioma

    DFO (S)

    311 (S)

    311m (S)

    NT (S)

    N2mT (S)

    N4mT (S)

    N44mT (S)

    DFO (F)

    311 (F)

    311m (F)

    NT (F)

    N2mT (F)

    N4mT (F)

    N44mT (F)

    Chelator Concentration (mM)

    Cellular Proliferation (% control)

    thymidine

    101.135.533.792.589.152.392.3104.198.8126.687.812378.683.4

    1414028.371.1109.372.279.747.6108.2100.352.5138.39386.2

    156.140.537.285.570.976.4113.538.183.589.852.852.6106106.2

    97.713.715.9111.964.676.449.2217.542.959.141.546.296.998.2

    69.26574400254.3768819453.756468381660.596057363630.761789735954.084582610913.94010688192.412154061520.500415930924.64916970637.037853881527.8998701254.109184387590.7221177789

    46.49504890713.4335932544.094484083885.15281598958.800784515837.39161791829.7933637532.5146677915.890397312321.22021843792.37744950264.699986616943.676176374112.5967714413

    DFO

    311

    NNH

    ST

    NT

    N2mT

    N4mT

    N44mT

    N4eT

    N4aT

    N4pT

    N44pH

    NOctH

    FeNT

    Chelator Concentration (mM)

    3H-Thymidine Incorporation (% control)

    logPvsIC50

    0.5

    0.5

    1.5

    1.5

    2.3

    3.3

    5.2

    0.3

    0.3

    10.5

    logP

    log P calc

    Chelator Antiproliferative Activity(IC50-mM)

    mtttable

    SK-N-MC neuroepitheliomaMRC-5 fibroblasts

    DFO22>25

    3110.3>25

    311m0.3*

    ST>12.5*

    NT0.5>25

    N2mT>12.5>25

    N4mT0.5>25

    N44mT1.5>25

    N4eT1.6*

    N4aT2.3*

    N4pT3.3*

    N44pH5.2*

    NoctH10.5*

    FeNT>12.5*

    * not determined

    logPtable

    3112.17

    311m2.17

    ST0.64

    NT1.76

    N2mT2.72

    N4mT2.19

    N44mT2.3

    N4eT2.4

    N4aT2.61

    N4pT3.75

    N44pH*

    NoctH5.19

    * Unable to be calculated by Broto's method

    Thytable

    DFO23

    3110.6

    311m0.6

    ST>25

    NT9.0

    N2mT15.5

    N4mT6.3

    N44mT1.5

    N4eT5.7

    N4aT7.9

    N4pT3.8

    N44pH4.3

    NoctH17.4

    FeNT>25

    mttforsem

    101.9404639287110.8621252644104.914193926898.370699989292.25754223394.317077289494.8043811677100.697396073197.690160858297.2354058355128.9206537078112.8960450374101.74330310970.1

    93.693476925772.002881808589.486296821382.651088282997.63388544990.439568596990.97111.306708766109.072633638690.138965084996.745117676594.386910045598.76955750860.2

    40.434940399633.705492360355.891796777655.5859507208102.855019961384.295716274584.113738050798.4125710762103.767383197692.5697.327809896781.5102.62351841920.39

    22.4613.773181107431.963311159926.499995097871.358426093773.682039329984.780730623990.2253281547112.957045914678.2686.812036236293.793.5352921497102.0386052855

    13.13688471857.928909812716.613.409640079948.363349937651.508125656967.946427408193.256533121687.832807529962.579814247274.976100514496.188644201996.766584406892.2261964354

    10.63993131436.18.47.696607573318.404687351328.33085988739.044121359250.921654156881.696716567161.681.130787089796.684.642359475896.4

    9.96.678.17100853679.620264427222.838642790626.887150449211.458283012129.643362877453.678089263986.165612054475.316085860566.931285689388.6

    7.84115572925.30896542935.47577989778.02972009288.68409473517.40481483217.891502760210.15569906769.958504008951.551527822264.032412254740.449390645961.896189521756.6

    311

    311m

    NT

    N4mT

    N44mT

    N4eT

    N4aT

    N4pT

    N44pH

    ST

    N2mT

    NOctH

    FeNT

    DFO

    Chelator Concentration (mM)

    Cellular Proliferation (% control)

    mtttabsem

    SK-N-MC neuroepithelioma

    3110.3

    311m0.3

    NT0.5

    N4mT0.5

    N44mT1.5

    N4eT1.6

    N4aT2.3

    N4pT3.3

    N44pH5.2

    NoctH10.5

    ST>12.5

    N2mT>12.5

    FeNT>12.5

    DFO22

    mtttablesem

    SK-N-MC neuroepitheliomaMRC-5 fibroblasts

    3110.3>25

    311m0.3>25

    NT0.5>25

    N4mT0.5>25

    N44mT1.5>25

    N2mT>12.5>25

    DFO22>25

    FeNT>12.5*

    * not determined

    NTpapMRC5sem

    101.9404639287110.8621252644104.914193926898.370699989292.257542233128.92065370780.192.617449664493.048745313100.058308952294.403198175896.7415732506107.12524357060.1

    93.693476925772.002881808589.486296821382.651088282997.63388544996.74511767650.289.115844762286.126334006397.1966868704103.283268587994.0952103656108.84985641520.2

    40.434940399633.705492360355.891796777655.5859507208102.855019961397.32780989670.3995.885614239993.5102394001101.553934862196.251645117898.3389849717107.1040349910.39

    22.4613.773181107431.963311159926.499995097871.358426093786.8120362362102.038605285583.746717245488.2030573983104.9878563539101.6428110784106.2921032183103.766943019390.2357141481

    13.13688471857.928909812716.613.409640079948.363349937674.976100514492.226196435490.283046396381.7421401788107.6426712987100.0240211224103.0557669922110.325998866191.1980547746

    10.63993131436.18.47.696607573318.404687351381.130787089796.466.238692734270.6662820883106.4242803477100.763901805389.3963823523111.875213361686.2899569347

    9.96.678.17100853679.620264427286.165612054488.660.169244236969.743293914101.8314546437105.932619046185.6532706677105.602191372283.0391645846

    7.84115572925.308965429312.58.02972009288.68409473564.032412254756.675.576305806873.665993654598.823807862397.695573033371.0267890763111.649294796480.6792129097

    75.576305806865.8785.060231706492.081543233569.8859743024111.306727347672.3639224146

    MRC-5Fibroblasts

    SK-N-MCNeuroepithelioma

    311 (S)

    311m (S)

    NT (S)

    N4mT (S)

    N44mT (S)

    N2mT (S)

    DFO (S)

    311 (F)

    311m (F)

    NT (F)

    N4mT (F)

    N44mT (F)

    N2mT (F)

    DFO (F)

    Chelator Concentration (mM)

    Cellular Proliferation (% control)

  • EPR of ribonucleotide reductase activityControl cells311 (25mM)Triapine (25mM)

  • Mechanisms of activity - effect on cell cycle control molecules

  • Fe chelation decreases nuclear p21 proteinCONDFO 311 Cis-platinAct DMCF-7CONDFO 311 Cis-platinAct DMRC-5p53p21-actinp53p21-actinLe and Richardson (2003) Carcinogenesis 24:1045-1058NUCLEAR LYSATES

  • Decrease in nuclear p21 expression not due to cytoplasmic localizationp21CONDFO311Act Dp53MCF-7Le and Richardson (2003) Carcinogenesis 24:1045-1058

  • Gene Array Analysis

  • The increase in NDRG1 mRNA is Fe dependentNDRG1-actinCONCON + FACDFODFO + MEMDFO + FAC311 311 + MEM311 + FACMCF-7 Breast Cancer CellsTfR1

  • In Vivo Experimental DesignM109 lung carcinoma animal modelTumor weightbody weightBlood cell count, RBC, WBC, Platelet, HbOutcome assessmentTumor H&E staining and TUNEL assayTumor implantation 4Drugs injection9Stop injectionHarvesting11Day 0

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

  • IntercellularAdhesionScavenging Extracellular FeMetalloproteaseActivityZnMTfTfR???POSSIBLE FUNCTIONS OF MELANOTRANSFERRIN

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

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

  • E PE PMTf 2MTf 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 16Long MTf1 transcript (2.6 kb) exons 1 to 16 + exon 16bThe relative positions of the originally identified melanotransferrin 1 (MTf1) gene compared to the newly identified MTf2 gene.

  • Total MTf Knockout Vector 5 arm3 armPGKneoTargeting VectorPGKneo5 Probe3 ProbeHomologous RecombinationWTTargetedDeletion Arm= Nhe I Restriction Enzyme site= Eco47II Restriction Site= Hind III Restriction SiteExpected Restriction Enzyme Fragments From WT and Targeted Gene5 ProbeEco47 II /Hind III double digestWT = 15 kbTargeted = 11.43 ProbeNhe I digest WT = 12.6 kbTargeted = 8.3

  • Examination of MTf Knockout MouseHistopathology/Blood Counts/Clinical Chemistry/X-ray

    Morphology/Morphometry

    Behaviour

    Conditional Knockout