The Critical Role of Polyglutamylation Reductase and Inhibitors of Other Folate-requiring Enzymes: Synergy between Inhibitors of Dihydrofolate in Vitro

The combined action among polyglutamylatable and nonpolyglutamylatable antifolates, directed against dihydrofolate reducÃ-ase (DHFR), glycinamide ribonucleotide formyltransferase (GARFT), 5-aminoimidazole4-carboxamide ribonucleotide formyltransferase (AICARFT), and thymidylate synthase (TS), in human ileocecal HCT-8 cells was examined in a 96-well plate growth inhibition assay (96-h continuous drug expo sure). An interaction parameter, a, was estimated for each of 95 experi ments by fitting a seven-parameter model to data with weighted nonlinear regression. In a representative experiment, raising the folie acid concen tration in the medium dramatically increased the Loewe synergy for the combination of trimetrexate (TMTX) and the GARFT inhibitor AG2034 (from a mean a ±SE of 1.50 ±0.25 at 2.3 /IM folie acid to 146 ±20 at 78 JIM folie acid). Enhancements were also found for combinations of TMTX with the GARFT inhibitors AG2032, Lometrexol, and LY309887, the AICARFT inhibitor AG2009, and the TS inhibitors LY231514 and Tomudex but not with the GARFT inhibitor LL95509 or with the TS inhibitors AG337, ZD9331, and BW1843U89. Replacing TMTX with methotrexate in two-drug mixtures decreased the intensity of Loewe synergy. Examination of isobolograms at different effect levels revealed informative reproducible changes in isobol patterns. No two-drug combi nations among inhibitors of GARFT, AICARFT, and TS exhibited Loewe synergy at either 2.3 or 78 /¿Mfolie acid. Thus, the ideal requirement for the folie acid-enhanced synergy is that a nonpolyglutamylatable DHFR inhibitor be combined with a polyglutamylatable inhibitor of another folate-requiring enzyme. A hypothesis to explain this general phenomenon involves the critical role of folylpoly-y-glutamate synthetase and the effect of the DHFR inhibitor in decreasing the protection by folie acid of cells to the other antifolates.