Abstract 4543: Targeting the NADPH binding site of dihydrofolate reductase for cancer therapy

Abstract

Abstract Dihydrofolate reductase (DHFR) catalyzes the reduction of dihydrofolate into tetrahydrofolate using NADPH as the cofactor. Folate-dependent one carbon metabolism is essential for the de novo synthesis of purines, pyrimidines and certain amino acids. Thus, DHFR plays an essential role in production and maintaining reduced folates for cellular proliferation, and consequently it has been a critical target for chemotherapy. Methotrexate (MTX), an antifolate that targets DHFR, has been used for decades in the clinic. However, both intrinsic and acquired resistance occurs due to decreased uptake, gene amplification and protein mutation. We have initiated studies to find an inhibitor that targets the cofactor NADPH binding site that would bypass these resistance mechanisms to MTX in contrast to analogs that target the dihydrofolate binding site. Fourteen NAD/NADP analogues were screened by the MTT assay in Chinese hamster ovary (CHO) cells which lack endogenous DHFR expression but were stably transfected with a DHFR-EGFP fusion construct driven by a CMV promoter (DG44 DHFR-EGFP). Two compounds, thionicotinamide adenine dinucleotide (NAD-S) and thionicotinamide adenine dinucleotide phosphate (NADP-S), had IC50 values of 1 μM. NAD-S and NADP-S were also found to be potent inhibitors of two other cancer cell lines, C85 (metastatic human colon cancer cell line) and CCRF-CEM (human T-cell lymphoblastic leukemia cell line). Surprisingly, both NAD-S and NADP-S decreased the levels of DHFR in CEM-CCRF cells. Pulse-chase experiments showed that the half life of DHFR is significantly reduced upon treatment of cells with 10 μM NADP-S. Furthermore, this NADP analog caused an increase in cells at the G1 phase (11% at 10 μM and 44% at 40 μM NADP-S treatment) that was reversible by removing NADP-S from the cell culture. Because NADP-S decreased DHFR levels, the combination of NADP-S with MTX was tested and found to significantly reduce colony formation, and was synergetic in vitro (Chou-Talalay analysis). We conclude that NADPS has a novel mechanism of action, i.e., it causes degradation of DHFR, likely by competing with NADPH binding, known to stabilize DHFR. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4543.