In order to determine the biochemical basis for the cytotoxicity of homofolates, poly-γ-glutamyl derivatives of homofolate (HPteGlu) and tetrahydrohomofolate (H4HPteGlu) were synthesized and tested as inhibitors of glycinamide ribonucleotide formyltransferase (GARFT), aminoimidazolecarboxamide ribonucleotide formyltransferase (AICARFT), thymidylate synthase, and serine hydroxymethyltransferase (SHMT) in extracts of Manca human lymphoma and L1210 murine leukemia cells. The most striking inhibitions are that of GARFT by (6R,S)-H4HPte Glu4–6 with IC50 values from 1.3 to 0.3 εM. Both diastereomers, (6R)-H4HPteGlu4 and (6S)-H4HPteGlu6, inhibit GARFT activity. In Manca cell extracts, the (6S) form is more potent than the (6R) form whereas in the murine system the reverse is true. The (6R,S)-H4HPteGlu polyglutamates are weak inhibitors of human AICARFT (IC50, 6–10 εM). Polyglutamates of HPteGlu, however, are more inhibitory to AICARFT, with HPteGlu4–6 having IC50 values close to 2 εM. Polyglutamates of HPteGlu and of H4HPteGlu are weaker inhibitors of thymidylate synthase (IC50, 8 εM for HPteGlu5–6 and > 20 εM for H4HPteGlu1–5). Polyglutamates of HPteGlu and of H4HPteGlu are poor inhibitors of SHMT (IC50, >20 εM). Manca cell growth is inhibited 50% by HPteGlu and (6R,S)-5-methyl-H4HPteGlu at 6 and 8 εM, respectively. Both of these effects are reversed by 0.1 mM inosine. Trimetrexate at a subinhibitory concentration, 10 nM, antagonizes growth inhibition by HPteGlu, raising the IC50 from 6 to 64 εM, but enhances inhibition by (6R,S)-5-methyl-H4HPteGlu, lowering the IC50 from 8 to 5 εM. Our results support the view that homofolates become toxic after conversion to H4HPteGlu polyglutamates which block GARFT, a step in purine biosynthesis.

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This work was supported by NCI Grants CA 10914 (R. L. K.), CA 22764, CA 08748, and CA 18856 (F. M. S.), CA 32687 (M. G. N.), and CA 25236 (J. R. P.) from the National Cancer Institute, USPHS/Department of Health and Human Services.

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