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Molecular Pharmacology

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Research ArticleArticle

Targeting the Proton-Coupled Folate Transporter for Selective Delivery of 6-Substituted Pyrrolo[2,3-d]Pyrimidine Antifolate Inhibitors of De Novo Purine Biosynthesis in the Chemotherapy of Solid Tumors

Sita Kugel Desmoulin, Yiqiang Wang, Jianmei Wu, Mark Stout, Zhanjun Hou, Andreas Fulterer, Min-Hwang Chang, Michael F. Romero, Christina Cherian, Aleem Gangjee and Larry H. Matherly
Molecular Pharmacology October 2010, 78 (4) 577-587; DOI: https://doi.org/10.1124/mol.110.065896
Sita Kugel Desmoulin
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Yiqiang Wang
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Jianmei Wu
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Mark Stout
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Zhanjun Hou
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Andreas Fulterer
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Min-Hwang Chang
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Michael F. Romero
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Christina Cherian
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Aleem Gangjee
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Larry H. Matherly
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Abstract

The proton-coupled folate transporter (PCFT) is a folate-proton symporter with an acidic pH optimum, approximating the microenvironments of solid tumors. We tested 6-substituted pyrrolo[2,3-d]pyrimidine antifolates with one to six carbons in the bridge region for inhibition of proliferation in isogenic Chinese hamster ovary (CHO) and HeLa cells expressing PCFT or reduced folate carrier (RFC). Only analogs with three and four bridge carbons (N-{4-[3-2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]-pyrimidin-6-yl)propyl]benzoyl}-l-glutamic acid (compound 2) and N-{4-[4-2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]-pyrimidin-6-yl)butyl]benzoyl}*-l-glutamic acid (compound 3), respectively) were inhibitory, with 2 ≫ 3. Activity toward RFC-expressing cells was negligible. Compound 2 and pemetrexed (Pmx) competed with [3H]methotrexate for PCFT transport in PCFT-expressing CHO (R2/hPCFT4) cells from pH 5.5 to 7.2; inhibition increased with decreasing pH. In Xenopus laevis oocytes microinjected with PCFT cRNA, uptake of 2, like that of Pmx, was electrogenic. Cytotoxicity of 2 toward R2/hPCFT4 cells was abolished in the presence of adenosine or 5-amino-4-imidazolecarboxamide, suggesting that glycinamide ribonucleotide formyltransferase (GARFTase) in de novo purine biosynthesis was the primary target. Compound 2 decreased GTP and ATP pools by ∼50 and 75%, respectively. By an in situ GARFTase assay, 2 was ∼20-fold more inhibitory toward intracellular GARFTase than toward cell growth or colony formation. Compound 2 irreversibly inhibited clonogenicity, although this required at least 4 h of exposure. Our results document the potent antiproliferative activity of compound 2, attributable to its efficient cellular uptake by PCFT, resulting in inhibition of GARFTase and de novo purine biosynthesis. Furthermore, they establish the feasibility of selective chemotherapy drug delivery via PCFT over RFC, a process that takes advantage of a unique biological feature of solid tumors.

Footnotes

  • ↵Embedded Image The online version of this article (available at http://molpharm.aspetjournals.org) contains supplemental material.

  • This study was supported by the National Institutes of Health National Cancer Institute [Grants CA53535, CA125153]; the National Institutes of Health National Eye Institute [Grant EY017732]; the Barbara Ann Karmanos Cancer Institute; the Mesothelioma Applied Research Foundation; and the Canadian Institutes of Health Research [Doctoral Research Award (to S.K.D.)].

  • Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.

    doi:10.1124/mol.110.065896.

  • ABBREVIATIONS:

    RFC
    reduced folate carrier
    FR
    folate receptor
    PCFT
    proton-coupled folate transporter
    PT523
    Nα-(4-amino-4-deoxypteroyl)-Nδ-hemiphthaloyl-l-ornithine
    GW1843U89
    (S)-2-(5-(((1,2-dihydro-3-methyl-1-oxo-benzo(f)quinazolin-9-yl) methyl) amino)1-oxo-2-isoindolinyl) glutaric acid
    GARFTase
    glycinamide ribonucleotide formyltransferase
    hPCFT
    human protein-coupled folate transporter
    Mtx
    methotrexate
    Pmx
    pemetrexed
    Lmx
    lometrexol
    Rtx
    raltitrexed
    hRFC
    human reduced folate carrier
    LCV
    leucovorin
    HPLC
    high-performance liquid chromatography
    CHO
    Chinese hamster ovary
    MEM
    α-minimal essential media
    DPBS
    Dulbecco's phosphate-buffered saline
    MES
    2-(N-morpholino)ethanesulfonic acid
    DMSO
    dimethyl sulfoxide
    dFBS
    dialyzed fetal bovine serum
    AICA
    5-amino-4-imidazolecarboxamide
    GAR
    glycinamide ribonucleotide
    R2 cells
    MtxRIIOuaR2-4 CHO cells.

  • Received April 27, 2010.
  • Accepted July 2, 2010.
  • Copyright © 2010 The American Society for Pharmacology and Experimental Therapeutics
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Molecular Pharmacology: 78 (4)
Molecular Pharmacology
Vol. 78, Issue 4
1 Oct 2010
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Research ArticleArticle

Targeting the Proton-Coupled Folate Transporter for Selective Delivery of 6-Substituted Pyrrolo[2,3-d]Pyrimidine Antifolate Inhibitors of De Novo Purine Biosynthesis in the Chemotherapy of Solid Tumors

Sita Kugel Desmoulin, Yiqiang Wang, Jianmei Wu, Mark Stout, Zhanjun Hou, Andreas Fulterer, Min-Hwang Chang, Michael F. Romero, Christina Cherian, Aleem Gangjee and Larry H. Matherly
Molecular Pharmacology October 1, 2010, 78 (4) 577-587; DOI: https://doi.org/10.1124/mol.110.065896

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Research ArticleArticle

Targeting the Proton-Coupled Folate Transporter for Selective Delivery of 6-Substituted Pyrrolo[2,3-d]Pyrimidine Antifolate Inhibitors of De Novo Purine Biosynthesis in the Chemotherapy of Solid Tumors

Sita Kugel Desmoulin, Yiqiang Wang, Jianmei Wu, Mark Stout, Zhanjun Hou, Andreas Fulterer, Min-Hwang Chang, Michael F. Romero, Christina Cherian, Aleem Gangjee and Larry H. Matherly
Molecular Pharmacology October 1, 2010, 78 (4) 577-587; DOI: https://doi.org/10.1124/mol.110.065896
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