PT - JOURNAL ARTICLE AU - Sita Kugel Desmoulin AU - Yiqiang Wang AU - Jianmei Wu AU - Mark Stout AU - Zhanjun Hou AU - Andreas Fulterer AU - Min-Hwang Chang AU - Michael Romero AU - Christina Cherian AU - Aleem Gangjee AU - Larry Matherly TI - Targeting the proton-coupled folate transporter for selective delivery of 6-substituted pyrrolo[2,3-<em>d</em>]pyrimidine antifolate inhibitors of <em>de novo</em> purine biosynthesis in the chemotherapy of solid tumors AID - 10.1124/mol.110.065896 DP - 2010 Jul 02 TA - Molecular Pharmacology PG - mol.110.065896 4099 - http://molpharm.aspetjournals.org/content/early/2010/07/02/mol.110.065896.short 4100 - http://molpharm.aspetjournals.org/content/early/2010/07/02/mol.110.065896.full AB - 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 1 to 6 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 3 and 4 bridge carbons (compounds 2 and 3, respectively) were inhibitory with 2 &gt;&gt; 3. Activity was negligible toward RFC-expressing cells. Compound 2 and pemetrexed (Pmx) competed with [3H]methotrexate for PCFT transport in PCFT-expressing CHO (R2/hPCFT4) cells from pH 5.5-7.2; inhibition increased with decreasing pH. In Xenopus oocytes microinjected with PCFT cRNA, uptake of 2 like 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 hours of exposure. Our results document the potent anti-proliferative activity of compound 2, attributable to its efficient cellular uptake by PCFT, resulting in inhibition of GARFTase and de novo purine biosynthesis. Further, they establish the feasibility of selective chemotherapy drug delivery via PCFT over RFC, a process which takes advantage of a unique biological feature of solid tumors.