Panel A: PC43-10 cells ectopically expressing wild type RFC but no FR31 were assayed for [3H]MTX in the presence of the pyrrolopyrimidine antifolates 1–5, MTX, PMX, LMX, LCV, or folic acid (each at 10 µM). Panel B: Data are shown for the effects of the unlabeled ligands with FRα-expressing RT16 CHO cells and FRβ-expressing D4 CHO cells. Relative binding affinities for assorted folate/antifolate substrates were determined over a range of ligand concentrations and were calculated as the inverse molar ratio of unlabeled ligands required to inhibit 3H-folic acid binding by 50%. By definition, the relative affinity of folic acid is 1. Details for the transport and binding assays are provided in the Experimental Section. Abbreviations: MTX, methotrexate; PMX, pemetrexed; LMX, lometrexol; LCV, LCV; FA, folic acid. Results are presented as mean values plus/minus SEM from 3 experiments.