De novo pyrimidine biosynthesis in Jurkat T-cells is inhibited by leflunomide's primary metabolite A77-1726 at the level of dihydroorotate-dehydrogenase (DHODH)

Leflunomide and its active metabolite A77-1726 share no apparent chemical o r structural relationship with existing immunomodulating drugs, and are dis tinct in their mechanism of action from any other clinically used immunosup pressant. They are considered to exert their beneficial effects by inhibiti on of de novo pyrimidine biosynthesis, thereby blocking proliferation in a variety of cells from different species. In vitro, A77-1726 directly binds to and inhibits the mitochondrial enzyme dihydroorotate-dehydrogenase (DHOD H), which is responsible for the conversion of dihydroorotate (DHO) to orot ate during de novo pyrimidine synthesis. Therefore, inhibition of DHODH sho uld result in an accumulation of its substrate DHO in cell cultures. For de termination of DHO in a human T-cell line in this study, a new sensitive an d specific chromatographic separation method with detection of changes in c onductivity was used. Jurkat cells, derived from an acute leukemia, were ob tained from ATCC (TIB-182). Our experimental data demonstrate that prolifer ation of Jurkat cells was inhibited by leflunomide's active metabolite A77- 1726 in a dose-dependent manner with IC50 values of 20-25 mu M. This antipr oliferative effect could be reversed by the addition of exogenous uridine ( 100 mu M). Incubation of the Jurkat T-cell line with leflunomide's active m etabolite also resulted in a rapid accumulation of DHO in these cells, due to inhibition of DHODH. The intracellular DHO concentrations correlated wit h the cell number and they were dose- and time-dependent Cell cycle analysi s undertaken in parallel demonstrated that Jurkat cells were arrested predo minantly at the early S-phase, and entry into the G2- and M-phases was inhi bited, without any signs of causing cell death. Using brequinar (BQR) a wel l-known inhibitor of de novo pyrimidine biosynthesis, at the level of DHODH has shown comparable results in our assay systems. Based on these results we conclude that leflunomide and its active metabolite A77-1726 strongly in hibit the mitochondrial enzyme DHODH in the human Jurkat T-cell line, and t hereby induce a rapid dose-and time-dependent intracellular accumulation of DHO in these cells. DHODH plays a key role in de novo pyrimidine biosynthe sis, and inhibition of the pyrimidine ribonucleotide uridine-monophosphate leads to a cell cycle arrest at the G0/G1/S phase boundary in Jurkat cells, when incubated in vitro with A77-1726.