| Abstract: |
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), ami-noimidazolecarboxamide 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 Gli4-6 with IC50 values from 1.3 to 0.3 μM. Both diastereomers, (6R)-H4HPteGlu6 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 poly-glutamates are weak inhibitors of human AICARFT (IC50, 6-10 μM). Polyglutamates of HPteGlu, however, are more inhibitory to AICARFT, with HPteGliu4-6 having IC50 values close to 2 μM. Polyglutamates of HPteGlu and of H4HPteGlu are weaker inhibitors of thymidylate synthase (IC50, 8 μM for HPteGlu5-6 and >20 μM for H4HPteGlu1-5). Polyglutamates of HPteGlu and of H4HPteGlu are poor inhibitors of SHMT (IC50, >20 μm).Manca cell growth is inhibited 50% by HPteGlu and (6R,S)-5-methyl-H4HPteGlu at 6 and 8 μm, respectively. Both of these effects are reversed by 0.1 mM inosine. Trimetrexate at a subinhi-bitory concentration, 10 nM, antagonizes growth inhibition by HPteGlu, raising the IC50 from 6 to 64 μm, but enhances inhibition by (6R,S)-5-methyl-H4HPteGlu, lowering the IC50 from 8 to 5 μM. Our results support the view that homofolates become toxic after conversion to H4HPteGlu polyglutamates which block GARFT, a step in purine biosynthesis. © 1989, American Association for Cancer Research. All rights reserved. |
