Figure 2. Folate transporters, folate metabolic pathways and intracellular enzyme targets of antifolates. Folate and antifolate transport across biological membranes is mediated by the reduced folate carrier (RFC), the proton-coupled folate transporter (PCFT) and folate receptors (FR). While RFC and PCFT are facilitative transporters, FRs mediate transport by a non-classical endocytosis involving formation of endosomes that migrate to the cytoplasm. Exit of the folate ligand from the endosome involves diffusion or a mediated process. PCFT has been proposed to facilitate endosomal exit,³⁸ however this does not appear to be obligatory.¹⁶ Intracellular folates including tetrahydrofolate (THF), dihydrofolate (DHF), 10-formyl tetrahydrofolate (10-CHO-THF), 5, 10-methylene tetrahydrofolate (5,10-CH₂-THF), 5,10-methenyl tetrahydrofolate (5,10-CH⁺-THF), and 5-methyl tetrahydrofolate (5-CH3-THF) participate in folate interconverting and biosynthetic reactions. Reactions 1–4 are in both the cytoplasmic and mitochodrial compartments. Reactions 1 and 9 are also present in the nucleus. Reaction 1 designates serine hydroxymethyltransferase. In the mitochondria, reactions 2 and 3 are catalyzed by bifunctional 5,10-CH₂-THF dehydrogenase (MTHFD) 2 or MTHFD2L and 4 is catalyzed by monofunctional MTHFD1L. In the cytoplasm, reactions 4, 3 and 2 are catalyzed by the 10-CHO-THF synthetase, 5,10-CH⁺-THF cyclohydrolase and MTFD activities, respectively, of the trifunctional C1-THF synthase (MTHFD1). Other steps shown are catalyzed by β-glycinamide ribonucleotide formyltransferase (GARFTase; reaction 5), 5-amino-4-imidazolecarboxamide ribonucleotide formyltransferase (AICARFTase; reaction 6), thymidylate synthase (TS; reaction 7), dihydrofolate reductase (DHFR; reaction 8), 5,10-methyleneTHF reductase (MTHFR; reaction 9), and methionine synthase (MS; reaction 10). Antifolates inhibit folate biosynthetic reactions as shown and include aminopterin (AMT), methotrexate (MTX), pralatrexate (PDX), raltitrexed (RTX), lometrexol (LMX), pemetrexed (PMX), ONX-0801 (ONX), and compounds 3, 16 and 17.