Biochemical factors determining tumor response to 5,10-dideazatetrahydrofolate, a folate antimetabolite inhibitory to de novo purine biosynthesis

5,10-dideazatetrahydrofolate (DDATHF) represents the lead compound of a class of folate antimetabolites that exhibit potent anti-tumor activity via selective inhibition of de novo purine biosynthesis. It has been shown that intracellular metabolism of DDATHF to long chain polyglutamates is central to drug cytotoxicity. The focus of this dissertation is to define the biochemical factors that determine tumor sensitivity or resistance to DDATHF. Two approaches were undertaken: (1) Using a series of DDATHF analogs as inhibitors of glycinamide ribonucleotide formyltransferase (GARFT), the cellular target of DDATHF, we have identified the structural features of this folate antagonist that are necessary for GARFT inhibition. Thus, isosteric replacement of nitrogen by carbon at the 5-position of tetrahydrofolate is sufficient for GARFT inhibition. Although the glutamic acid side chain of DDATHF does not seem to play a role in binding to GARFT, the polyglutamate derivatives of DDATHF were substantially more potent inhibitors of the enzyme than the parent compound. (2) Biochemical mechanisms of drug resistance by tumors were studied in variants of mouse leukemic cells selected for resistance to DDATHF. The underlying mechanism of resistance has been determined to be due to mutations in two highly conserved amino residues in the reduced folate transporter, causing an enhanced translocation of folic acid across the plasma membrane, an expansion of total folate pools, and, subsequently, blockade of DDATHF polyglutamation. This novel mechanism of resistance to a ntifolates has profound implications on the conceptual understanding of folate homeostasis as well as the clinical use of DDATHF for cancer chemotherapy.