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Inhibition of Glycinamide Ribonucleotide Formyltransferase and Other Folate Enzymes by Homofolate Polyglutamates in Human Lymphoma and Murine Leukemia Cell Extracts¹

Department of Biochemistry, Tufts University Health Science Campus, Boston, Massachusetts 02111 [J. T., Y. G., R. L. K.]; Memorial Sloan-Kettering Cancer Center, New York, New York 10021 [F. M. S.]; Department of Biochemistry, University of South Alabama, Mobile, Alabama 36688 [B. R. M., M. G. N.]; and Drug Synthesis Section, Southern Research Institute, Birmingham, Alabama 35255 [J. R. P.]

In order to determine the biochemical basis for the cytotoxicity of homofolates, poly--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 Glu_4–6 with IC₅₀ values from 1.3 to 0.3 M. 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 M). Polyglutamates of HPteGlu, however, are more inhibitory to AICARFT, with HPteGlu_4–6 having IC₅₀ values close to 2 M. Polyglutamates of HPteGlu and of H₄HPteGlu are weaker inhibitors of thymidylate synthase (IC₅₀, 8 M for HPteGlu_5–6 and > 20 M for H₄HPteGlu_1–5). Polyglutamates of HPteGlu and of H₄HPteGlu are poor inhibitors of SHMT (IC₅₀, >20 M). Manca cell growth is inhibited 50% by HPteGlu and (6R,S)-5-methyl-H₄HPteGlu at 6 and 8 M, 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 M, but enhances inhibition by (6R,S)-5-methyl-H₄HPteGlu, lowering the IC₅₀ from 8 to 5 M. Our results support the view that homofolates become toxic after conversion to H₄HPteGlu polyglutamates which block GARFT, a step in purine biosynthesis.

¹ 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.

² To whom requests for reprints should be addressed, at Department of Biochemistry, Tufts University Health Science Campus, Boston MA 02111.

Received 6/24/88. Revised 9/20/88. Accepted 10/ 5/88.