Targeting Nonsquamous Nonsmall Cell Lung Cancer via the Proton-Coupled Folate Transporter with 6-Substituted Pyrrolo[2,3-d]Pyrimidine Thienoyl Antifolates

Abstract

Pemetrexed (PMX) is a 5-substituted pyrrolo [2,3-d]pyrimidine antifolate used for therapy of nonsquamous nonsmall cell lung cancer (NS-NSCLC). PMX is transported by the reduced folate carrier (RFC) and proton-coupled folate transporter (PCFT). Unlike RFC, PCFT is active at acidic pH levels characterizing the tumor microenvironment. By real-time reverse-transcription polymerase chain reaction (RT-PCR) and immunohistochemistry, PCFT transcripts and proteins were detected in primary NS-NSCLC specimens. In six NS-NSCLC cell lines (A549, H1437, H460, H1299, H1650, and H2030), PCFT transcripts and proteins were detected by real-time RT-PCR and western blots, respectively. 6-Substituted pyrrolo [2,3-d] H]C2 transport and decreased growth inhibition by C1 and C2, and to a lesser extent by PMX. In vivo efficacy of C1 was seen toward H460 tumor xenografts in severe-combined immunodeficient mice. Our results suggest that 6-substituted pyrrolo [2,3-d] pyrimidine thienoyl antifolates offer significant promise for treating NS-NSCLC by selective uptake by PCFT.

Fluorine-Substituted Pyrrolo[2,3-d]Pyrimidine Analogues with Tumor Targeting via Cellular Uptake by Folate Receptor α and the Proton-Coupled Folate Transporter and Inhibition of de Novo Purine Nucleotide Biosynthesis

“…Notably, in vitro efficacies of these fluorinated compounds toward the EOC cells were equal to or exceeded those for 1 , previously the most potent 6-substituted pyrrolo[2,3- d ]-pyrimidine analogue we have discovered. 11,19,30,67 For 9 and 11 , selectivity toward PCFT and FR α over RFC (as reflected in IC 50 values toward engineered HeLa cells expressing only RFC versus PCFT or FR α ) far exceeded that for 1 . While fluorine-substituted analogues, like their nonfluorinated counterparts, all inhibited GARFTase with nanomolar IC 50 values, with the exception of compounds 3 and 9 , fluorine substitution in itself had no apparent impact on the potency of GARFTase inhibition as the potencies of the fluorinated and non-fluorinated analogues toward cellular GARFTase were similar.…”

“…11,19,25,27,28,30,42,45,50,51 To confirm FR-mediated drug uptake, 200 nM folic acid was added to parallel incubations for RT16 and D4 cells. After 96 h, viable cells were assayed with Cell-Titer Blue reagent (Promega, Madison, WI), and fluorescence was measured with a fluorescence plate reader.…”

“…11,19,25,27,28,30,42,45,50,51 For de novo purine biosynthesis inhibitors, additional protection experiments used AICA (320 μ M) to distinguish inhibitory effects at GARFTase from those at AICARFTase.…”

“…19,26,30,42,73 Results were normalized to cell proteins. To calculate the IC 50 values, results for drug-treated samples were normalized to untreated controls.…”

“…17 PCFT is also expressed in the liver and kidney. 3,4,18 PCFT is expressed in human tumors with particularly high levels in NSCLC, 19 EOC, 11 and malignant pleural mesothelioma. 20 PCFT functions in the cellular uptake of folates and antifolates at acidic pHs characterizing the microenvironments of many solid tumors.…”