Analogues of folate antagonists as affinity and fluorescent probes of dihydrofolate reductase structure and function.

Dihydrofolate reductase (DHFR; tetrahydrofolate; NADP' oxidoreductase, EC 1.5.1.3) catalyses the NADPH-dependent reduction of 7,8-dihydrofolate (FAH,) to 5,6,7,8-tetrahydrofolate (FAH,). The coenzyme FAH, serves as a carrier of one-carbon fragments in a number of biosynthetic transfer reactions (Rader & Huennekens, 1973). It is apparent that DHFR is the intracellular target for the anti-cancer drug methotrexate (MTX) as well as the anti-bacterial drug trimethoprim. As a target site for chemotherapeutic agents, DHFR is intimately involved in the continuous synthesis of thymidylate (dTMP). Thymidylate synthase (EC 2.1.1.45) catalyses the reductive methylation of 2'-deoxyuridylate (dUMP) to dTMP in the presence of N5,N"-methylene FAH,. The latter compound not only supplies the onecarbon unit to the 5'-position of the uracil ring of dUMP, but also serves as a reductant supplying a hydrogen from C-6 of the reduced pyrazine ring of FAH,, yielding FAH,. Thus, FAH, must be regenerated via the DHFRcatalysed reaction in order to maintain the intracellular pool of FAH, one-carbon derivatives for both dTMP and purine nucleotide biosynthesis. The inhibition of DHFR by folate antagonists such as MTX results in a deficiency in the cellular pools of dTMP and purines and thus in a decrease in nucleic acid synthesis. It is the latter property of folate antagonists that has found clinical applications in the treatment of a number of neoplastic disorders and microbial infections. Structural studies of this enzyme from both bacterial and animal sources have been aided by the fact that the M, of many prokaryotic and eukaryotic DHFRs is in the range 1800CL22000. Investigations by a number of laboratories have contributed to our understanding of the structural and functional