1843U89 is a potent inhibitor (Ki = 0.09 nM) of
thymidylate synthase (TS; EC 2.1.1.45) that is in clinical trial for the treatment of solid
tumors. Although it is an excellent substrate for the
folate anabolizing
enzyme folylpolyglutamate synthetase (FPGS),
1843U89 differs from other
folate-based inhibitors of TS (e.g., CBC3717,
D1694, and
LY231514), in that the parent compound is as potent an
enzyme inhibitor as its polyglutamated analogues. As reported (D.S. Duch et at.,
Cancer Res., 53:810-818, 1993),
1843U89 is 10-80-fold more cytotoxic than the close structural analogue 1031U89, which is an equipotent inhibitor of TS but is a less efficient substrate for FPGS. This correlation between substrate efficiency for FPGS and cytotoxicity suggests that polyglutamation of
1843U89 contributes to its cytotoxicity. In the current study, we measured intracellular levels of polyglutamated anabolites of 1031U89,
1843U89, and three other benzoquinazoline inhibitors of TS as well as anabolites of
D1694 in HCT-8 ileocecal
carcinoma cells. Each TS inhibitor was anabolized to polyglutamated analogues with one to five added glutamyl residues after exposure for 24 h to IC90 concentrations (those that inhibit growth by 90% after 72 h of constant exposure).
D1694, which requires polyglutamation for potent
enzyme inhibition as well as for cytotoxicity, was anabolized mostly to
penta- and hexaglutamates, whereas approximately 80% of intracellular
1843U89 was the diglutamate analogue. The substrate efficiency of the benzoquinazolines for FPGS was predictive of the extent of intracellular anabolism. The diglutamate analogue of
1843U89 was only 1/100 as efficient a substrate for further glutamation as was
1843U89 itself. The efficient anabolism to the diglutamate analogue and the lack of dependence on further polyglutamation for
enzyme inhibition or cytotoxicity provide a rationale for the reported
1843U89 sensitivity of cells with impaired FPGS activity. As part of an investigation of the effects of polyglutamation, we measured the retention of intracellular
1843U89 and
D1694 anabolites after 24 h of exposure to 20 nM of each compound. After 48 h in
drug-free medium, 7% of intracellular
1843U89 (mostly diglutamate analogue) and 36% of
D1694 (mostly
penta- and hexaglutamates) remained in the cells. Because prolonged retention (associated with tissue storage of polyglutamates) can contribute to clinical toxicities,
1843U89 may present fewer long-term toxicities than
D1694.