Tight Binding of Folate Substrates and Inhibitors to Recombinant Mouse Glycinamide Ribonucleotide Formyltransferase

Abstract

The binding of the prototypical folate inhibitor of de novo purine synthesis, 5,10-dideazatetrahydrofolate (DDATHF), and its hexaglutamate to recombinant trifunctional mouse glycinamide ribonucleotide formyltransferase (rmGARFT) was studied by equilibrium dialysis and by steady-state kinetics using sensitive assays that allowed initial rate calculations. rmGARFT was expressed in insect cells infected with a recombinant baculovirus and purified by a two-step procedure that allowed production of about 25 mg of pure protein/L of culture. The binding of DDATHF to GARFT was approximately 50-fold tighter than previously reported, with Kd and Ki values of 2-9 nM, making the parent form of this antifolate a tight-binding inhibitor. The binding of the hexaglutamate of DDATHF to rmGARFT had Kd and Ki values of 0.1-0.3 nM, consistent with the view that polyglutamation enhances binding of antifolates to GARFT. Kinetic analyses using either mono- or hexaglutamate substrate did not yield different values for the Ki for the hexaglutamate form of DDATHF, in contradiction with previous reports. Both the folate substrate commonly used to study GARFT, 10-formyl-5,8-dideazafolate, and its hexaglutamate were found to have very low Km values, namely, 75 and 7.4 nM, respectively, and the folate reaction products for these substrates were equally potent inhibitors, results which modify the interpretation of previous kinetic experiments. The product analog DDATHF and beta-glycinamide ribonucleotide bound to enzyme equally well in the presence and absence of the other, an observation at variance with the concept that GARFT obeys an ordered sequential binding of the substrates. We conclude that the kinetics of mouse GARFT are most consistent with a random order of substrate binding, that both the inhibitor DDATHF and the folate substrate are tight-binding ligands, and that polyglutamate forms enhance the affinity of both substrate and inhibitor by an order of magnitude.

Molecular Analysis of Murine Leukemia Cell Lines Resistant to 5,10-Dideazatetrahydrofolate Identifies Several Amino Acids Critical to the Function of Folylpolyglutamate Synthetase

“…The more marked resistance of ZD1694 in these cell lines likely reflects the fact that the polyglutamates of ZD1694 bind to thymidylate synthase about 100 times tighter than the ZD1694 monoglutamate. On the other hand, DDATHF and LY309887 are tight binding inhibitors of GART prior to metabolism (31), and become better enzyme inhibitors with polyglutamation (31), but to a lesser degree than does ZD1694 (28).…”

Pemetrexed: biochemical and cellular pharmacology, mechanisms, and clinical applications

“…The structure of lometrexol had been suggested by G. Peter Beardsley to his former mentor Ted Taylor as a potential antifolate, initially thought to be a likely inhibitor of thymidylate synthase; to their surprise, it was not, but it was a potent inhibitor of tumor cell growth (28). Cell culture experiments (28,29), then direct enzymology on isolated (29,30) and recombinant (31) proteins in the laboratory of one of the authors, showed that lometrexol was the first potent antifolate inhibitory to purine synthesis due to its direct suppression of glycinamide ribonucleotide transferase (GARFT) activity. Because this was not a nucleotide analogue, it was considered not to pose a mutagenic nor carcinogenic threat.…”

Cellular Folates Prevent Polyglutamation of 5,10-Dideazatetrahydrofolate

“…The content of H 4 PteGlu n and 5,10-CH 2 -H 4 PteGlu n was determined by this method, then the cellular levels of 10-CHO-H 4 PteGlu n were determined indirectly by quantitating the additional H 4 PteGlu n formed in the presence of excess GARFT and glycinamide ribonucleotide. Briefly, cell pellets containing 4 ϫ 10 6 cells were resuspended in 200 l of boiling 10 mM sodium phosphate buffer, pH 7.5, containing 0.1% Triton X-100, 1% 2-ME, and 1% freshly prepared sodium ascorbate.…”

“…Kinetic analysis of GART inhibition was performed on GART purified by affinity chromatography from both cell lines. K i values were obtained from Dixon plots using K m values of 75 nM (4). Values are expressed as mean Ϯ S.D.…”

“…2 Compared with methotrexate (MTX), the classical antifolate targeted toward dihydrofolate reductase, (6R)-DDATHF, ZD-1694, and MTA are metabolized more rapidly and extensively to long chain polyglutamates by the enzyme folylpolyglutamate synthetase (FPGS) (2, 9, 14 -16). The metabolism of these drugs to their polyglutamate derivatives is essential for their cellular retention and these polyglutamates have been reported to be substantially more potent inhibitors of their respective target enzymes (4,6,7,14,15). These new antifolates, therefore, probably function as "pro-drugs," with the synthesis of polyglutamates being a requisite step for the development of cytotoxicity (17).…”