CARRIER-MEDIATED AND RECEPTOR-MEDIATED TRANSPORT OF FOLATE ANTAGONISTS TARGETING FOLATE-DEPENDENT ENZYMES - CORRELATES OF MOLECULAR-STRUCTURE AND BIOLOGICAL-ACTIVITY

The transport properties and growth-inhibitory potential of 37 classic and novel antifolate compounds have been tested in vitro against huma n and murine cell lines expressing different levels of the reduced fol ate carrier (RFC), the membrane-associated folate binding protein (mFB P), or both. The intracellular targets of these drugs were dihydrofola te reductase (DHFR), glycinamide ribonucleotide transformylase (GARTF) , folylpolyglutamate synthetase (FPGS), and thymidylate synthase (TS). Parameters that were investigated included the affinity of both folat e-transport systems for the antifolate drugs, their growth-inhibitory potential as a function of cellular RFC/mFBP expression, and the prote ctive effect of either FA or leucovorin against growth inhibition. Met hotrexate, aminopterin, N-10-propargyl-5,8-dideazafolic acid (CB3717), ZD1694, 5,8-dideazaisofolic acid (IAHQ), 5,10-dideazatetrahydrofolic acid (DDATHF), and 5-deazafolic acid (efficient substrate for FPGS) we re used as the basic structures in the present study, from which modif ications were introduced in the pteridine/quinazoline ring, the C-9-N- 10 bridge, the benzoyl ring, and the glutamate side chain. It was obse rved that RFC exhibited an efficient substrate affinity for all analog ues except CB3717, 2-NH2-ZD1694, and glutamate side-chain-modified FPG S inhibitors. Substitutions at the a-position (e.g., 2-CH3) improved t he RFC substrate affinity for methotrexate and aminopterin. Other good substrates included PT523 -deoxypteroyl)-N-delta-hemiphthaloyl-L-orri thine), 10-ethyl-10-deazaaminopterin, and DDATHF. With respect to mFBP , modifications at the N-3 and 4-oxo positions resulted in a substanti al loss of binding affinity. Modifications at other sites of the molec ule were well tolerated. Growth-inhibition studies identified a series of drugs that were preferentially transported via RFC (2,4-diamino st ructures) or mFBP (CB3717, 2-NH2-ZD1694, or 5,8-dideazaisofolic acid), whereas other drugs were efficiently transported via both transport p athways (e.g., DDATHF, ZD1694, BW1843U89, or LY231514). Given the fact that for an increasing number of normal and neoplastic cells and tiss ues, different expression levels of RFC and mFBP are being recognized, this folate antagonist structure-activity relationship can be of valu e for predicting drug sensitivity and resistance of tumor cells or dru g-related toxicity to normal cells and for the rational design and dev elopment of novel antifolates.