gamma-Fluoromethotrexate: synthesis and biological activity of a potent inhibitor of dihydrofolate reductase with greatly diminished ability to form poly-gamma-glutamates.

Abstract

A methotrexate (MTX) analog containing fluorine at the rcarbon of the glutamate moiety, -fluoromethotrexate (FMTX), has been synthesized and evaluated for its biochemical and pharmacological properties. FMTX inhibition of dihydrofolate reductase from several sources is nearly equivalent to that shown by MTX. Most important, FMTX is an exceedingly poor substrate for folylpoly (-glutamate) Utilizing the H35 hepatoma cell culture system (3, 5, 6), we are attempting to definitively establish the role of glutamylation in the activity of MTX. The approach has been to employ a MTX derivative that is accumulated by cells and inhibits DHFR as effectively as does MTX but cannot be glutamylated. The derivative we have employed, t-fluoromethotrexate (FMTX), is an analog of MTX containing fluorine in place of hydrogen on the y carbon of glutamate (Fig. 1). Recent studies with partially purified folylpoly(-glutamate) synthetase (FPGS; EC 6.3.2.17) isolated from rat liver have shown that DL-threo-4-fluoroglutamate (threo-FGlu) acts as a chain-terminating inhibitor of the FPGS reaction (15). The data support the hypothesis that the incorporation of a molecule of FGlu into growing polyglutamate chains leads to a product, R-Glu-y-FGlu, that undergoes further glutamylation at a markedly reduced rate. These results suggest that the synthesis of reduced folates or antifolates (e.g., MTX) containing FGlu in place of glutamate would lead to molecules with greatly diminished ability to form poly(-glutamate) derivatives. The topic of this report is the influence of the fluorine substitution on the toxicity of MTX in cultured hepatic cells. In particular, short-term exposure to FMTX causes growth inhibition that is several orders of magnitude weaker than that obtained with MTX. To establish that limited glutamylation is a major reason for these results, it is necessary to demonstrate (i) that both agents exert similar inhibitory effects toward the target enzyme DHFR, (ii) that FMTX and MTX are accumulated to a similar extent in H35 cells, and (iii) that FMTX is poorly glutamylated by H35 cells. These observations are evaluated with Abbreviations: MTX, methotrexate; FMTX, y-fluoromethotrexate; FGlu, DL-4-fluoroglutamate; DHFR, dihydrofolate reductase; FPGS, folylpoly(y-glutamate) synthetase. 2598The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Human Cytosolic and Mitochondrial Folylpolyglutamate Synthetase Are Electrophoretically Distinct

“…Similar to our studies with acquired MTX resistance through decreased MTX polyglutamate (MTXGn) accumulation (15,16) and with the nonpolyglutamylatable analog ␥-FMTX (17,18), the authors (14) conclude that MTXGn and, thus, FPGS are not required during continuous MTX exposure. Furthermore, the extent of metabolism to MTXGn is dependent on FPGS level; thus, in pulse exposure, where MTXGn allows intracellular retention in the absence of extracellular drug, MTX sensitivity is directly related to FPGS level.…”

Increase of dihydrofolate reductase in peripheral blood lymphocytes of rheumatoid arthritis patients treated with low‐dose oral methotrexate

“…FMTX is an MTX analog that is very similar to MTX in terms of transport and DHFR inhibition properties, but it cannot be polyglutamylated because of the fluorine in the gamma position (12). Cells that have been exposed to a concentration of FMTX that is at least equitoxic to 1 pM MTX would thus be expected to have DNA synthesis activity equal to that of untreated control cells after the 6-hour efflux period.…”

Exploitation of folate and antifolate polyglutamylation to achieve selective anticancer chemotherapy

“…For example, MTX does not require polyglutamylation when it is used in continuous exposure because its monoglutamate is a tight-binding inhibitor of the target enzyme DHFR [12,13]. If exposure time is short, however, polyglutamylation substantially enhances potency as a result of retention of MTX polyglutamates after o NH 2…”

“…removal of extracellular drug [12,14,15]. With drugs that are potent inhibitors of their targets only after polyglutamylation, such as DDATHF [16] and D1694 (Tomudex) [ 17], a requirement for polyglutamate synthesis is evident in both continuous and short-term exposure.…”