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

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-, respectively) were inhibitory, with 2 Ͼ Ͼ 3. Activity toward RFC-expressing cells was negligible. Compound 2 and pemetrexed (Pmx) competed with [ 3 H]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 ϳ50 and 75%, respectively. By an in situ GARFTase assay, 2 was ϳ20-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.

Identification and Functional Impact of Homo-oligomers of the Human Proton-coupled Folate Transporter

“…5A). Again, both wt FLAG hPCFT Myc-His10 and wthPCFT HA were expressed at the cell surface either singly or in combination, consistent with our surface biotinylation results, although there was some staining of intracellular structures, as reported previously (24). There was no staining of untransfected R1-11 cells (not shown).…”

“…Generation of hPCFT and Other Plasmid Constructs-An expression construct for deglycosylated hPCFT with a Myc epitope tag (dghPCFT ) and Gln substituted for Asn at positions 58 and 68 was generated by site-directed mutagenesis, using wild-type (WT) Myc-His 6 -tagged hPCFT (wth-PCFT ) in pCDNA3.1 (24) as template. To prepare WT HA-tagged hPCFT (wthPCFT HA ) with an HA epitope insertion (YPYDVPDYA) at amino acid 459, preceded by a 3-amino acid (GTQ) linker, the hPCFT cDNA fragment was excised from the wthPCFT Myc-His6 construct by digestion with BamHI and KpnI and cloned upstream of the HA tag in pCDNA3 after deleting the hRFC cDNA fragment from a carboxyl-terminal HA-tagged hRFC construct (41).…”

“…3 H]Mtx (0.5 M) was measured over 2 min at 37°C in 60-mm dishes in MES-buffered saline (20 mM MES, 140 mM NaCl, 5 mM KCl, 2 mM MgCl 2 , and 5 mM glucose) at pH 5.5, as described (24). Levels of intracellular radioactivity were expressed as pmol/mg protein, calculated from direct measurements of radioactivity and protein contents (50) of the cell homogenates.…”

Inhibition of the Proton-Coupled Folate Transporter (PCFT-SLC46A1) by Bicarbonate and Other Anions

“…In this case, PCFT-mediated antifolate uptake into tumor cells will be much more rapid than occurs in normal tissues. This is the rationale for the development of antifolate compounds with high affinity for PCFT but very low affinity for RFC as a way of minimizing toxicity to normal tissues mediated by the latter transporter (Kugel Desmoulin et al, 2010Wang et al, 2011). It is also important to point out that cells that express only PCFT are sensitive to pemetrexed in cell culture containing the physiologic level of bicarbonate, so residual transport is sufficient to sustain pharmacological activity (Zhao et al, 2004b).…”

Functional Loss of the Reduced Folate Carrier Enhances the Antitumor Activities of Novel Antifolates with Selective Uptake by the Proton-Coupled Folate Transporter

“…and C2 for comparison with MTX, lometrexol (LMX), raltitrexed (RTX), and PMX, classic antifolates that are transported by both RFC and PCFT (Goldman et al, 2010;Kugel Desmoulin et al, 2010 and with N ␣ -(4-amino-4-deoxypteroyl)-N ␦ -hemiphthaloyl-L-ornithine (PT523), which is transported by RFC but not by PCFT (Zhao and Goldman, 2007;Kugel Desmoulin et al, 2010 (Fig. 5A; Supplemental Table 5S).…”