DL-beta,beta-difluoroglutamic acid mediates position-dependent enhancement or termination of pteroylpoly(gamma-glutamate) synthesis catalyzed by folylpolyglutamate synthetase.
Poly(gamma-glutamylation) of glutamate (L-Glu)-containing antifolates and natural folates is important in pharmacological mechanisms and in physiological processes. Based on previous work from our laboratories, we hypothesized that replacement of the L-Glu moiety in parent molecules with DL-beta,beta-difluoroglutamic acid (DL-beta,beta-F2Glu) might be a generic means of increasing polyglutamylation by both increasing the synthesis rate and decreasing the degradation rate (J. J. McGuire et al., J. Biol. Chem. 265, 14073-14079 (1990)); thus biological potency might be increased without other biochemical properties being altered. DL-beta,beta-F2Glu, synthesized by an improved route (B. P. Hart and J. K. Coward, Tetrahedron Lett. 34, 4917-4920 (1993)), has been incorporated into a methotrexate (MTX) homolog, beta,beta-difluoromethotrexate (beta,beta-F2MTX), and a folic acid (PteGlu) homolog, beta,beta-difluorofolic acid (beta,beta-PteF2Glu). Biochemical properties of beta,beta-F2MTX (e.g., inhibition of isolated dihydrofolate reductase, transport in whole cells) are similar to those of MTX except that, in accord with our hypothesis, apparent substrate efficiency for rat and human folylpolyglutamate synthetase (FPGS) is 4- to 7.5-fold higher, respectively, for beta,beta-F2MTX than for MTX. Analysis of the products synthesized by purified FPGS, however, suggests that while addition of the first gamma-Glu to beta,beta-F2MTX is highly efficient, subsequent additions occur at a negligible rate; this premise was confirmed by directly comparing the in vitro FPGS substrate activity of MTX-gamma-Glu and beta,beta-F2MTX-gamma-Glu. Furthermore, the dramatically diminished in vitro growth inhibitory potency of beta,beta-F2MTX as compared to MTX when exposure time to drug is decreased (despite otherwise similar biochemical properties) suggests that polyglutamylation is also impaired in intact cells. Similar results with FPGS have been obtained with oxidized and reduced forms of beta,beta-PteF2Glu. These data suggest that the effect of beta,beta-F2Glu on polyglutamylation by FPGS is dependent on its position relative to the point of L-Glu ligation. When beta,beta-F2Glu is the acceptor amino acid (i.e., point of attachment), ligation of Glu is enhanced; however, if beta,beta-F2Glu is one residue distal to the acceptor amino acid, further elongation is blocked.
McGuire
J J
JJ
Grace Cancer Drug Center, Roswell Park Cancer Institute, Buffalo, New York 14263, USA.
Hart
B P
BP
Haile
W H
WH
Rhee
M S
MS
Galivan
J
J
Coward
J K
JK
D000818:Animals; D002384:Catalysis; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D004351:Drug Resistance; D005971:Glutamates / Q000494:pharmacology*; D006801:Humans; D007202:Indicators and Reagents; D007700:Kinetics; D008114:Liver Neoplasms, Experimental; D008727:Methotrexate / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000378:metabolism / Q000494:pharmacology; D010453:Peptide Synthases / Q000378:metabolism*; D011624:Pteroylpolyglutamic Acids / Q000096:biosynthesis*; D051381:Rats; D013379:Substrate Specificity; D014407:Tumor Cells, Cultured
D005971:Glutamates; D007202:Indicators and Reagents; D011624:Pteroylpolyglutamic Acids; C097863:beta,beta-difluoromethotrexate; C065405:3,3-difluoroglutamate; D010453:Peptide Synthases; C023103:folylpolyglutamate synthetase; D008727:Methotrexate