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
↵ 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.
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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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