The
folate compound
10-formyldihydrofolate (H2folate) has not been found as a component of intracellular folates in normal tissues but has been identified in the cytosol of
methotrexate (MTX)-treated MCF-7
breast cancer cells and normal human myeloid precursor cells. Its identity was verified by coelution of this compound with a synthetic marker on high pressure liquid chromatography, its reduction to
10-formyltetrahydrofolate (H4folate) in the presence of
dihydrofolate reductase, and its enzymatic deformylation to
dihydrofolate in the presence of aminoimidazolecarboxamide
ribonucleotide (
AICAR) transformylase. Chemically synthesized monoglutamated or pentaglutamated 10-formyl-H2folate was examined for its interaction with three
folate-dependent
enzymes:
AICAR transformylase, glucinamide ribotide (
GAR) transformylase, and thymidylatesynthase. 10-Formyl-H2folate-Glu5 was a competitive inhibitor of
thymidylate synthase (Ki = 0.16 microM with 5,10-methylene-H4folate-Glu1 as substrate and 1.6 microM with 5,10-methylene-H4folate-Glu5) and inhibited
GAR transformylase (Ki = 2.0 microM). It acted as a substrate for
AICAR transformylase (Km = 5.3 microM), and its efficiency was equal to that of the natural substrate 10-formyl-H4folate-Glu5. The inhibition of
thymidylate synthase by 10-formyl-H2folate was highly dependent on the inhibitor's polyglutamation state, the -Glu5 derivative having a 52-85-fold greater affinity as compared to the affinity of -Glu1. Polyglutamation of 10-formyl-H2folate did not affect its inhibition of
GAR transformylase. While the actual role of 10-formyl-H2folate contributing to the cytotoxicity of MTX has not been determined, this compound has the potential to enhance inhibition of
GAR transformylase and
thymidylate synthase, and at the same time provides additional substrate for
AICAR transformylase. The MTX-induced intracellular accumulation of 10-formyl-H2folate and H2folate may play a role in the
drug-related cytotoxicity through the contribution of these folates to the inhibition of
thymidylate synthase and de novo
purine synthesis.