MALONONITRILAMIDES (MNAs) EXERT THEIR IMMUNOSUPPRESSIVE ACTIVITY PRIMARILY BY INHIBITION OF DIHYDROOROTATE DEHYDROGENASE (DHODH).

Abstract

216 Purpose: MNAs, a new generation of low molecular weight immunosuppressants belong to the derivatives of leflunomide's active metabolite A771726 and have been shown to prevent and reverse acute allograft rejection and effectively control xenograft survival in various rodent transplantation models. In this study, we examined the molecular mechanism of action of the MNAs' immunosuppressive activity. Methods: In vitro, MNAs bind to and inhibit the mitochondrial enzyme DHODH, which is responsible for the conversion of dihydroorotate (DHO) to orotate during the de novo pyrimidine synthesis. The resultant accumulation of DHO levels in cell cultures was determined by a new sensitive and specific chromatographic separation method as described by Milbert et al. (Rheumatol. Eur. 27:109, 1998) . Results: Various MNAs (A77 1726, HMR 1279, HMR 1715 and HR 325) inhibited the proliferation of different cell lines in a dose-dependent manner, and concomitantly reduced the pyrimidine ribonucleotide pools, because this anti-proliferative effect could be reversed by addition of uridine or cytidine to replenish the nucleotides. In parallel experiments, a rapid accumulation of DHO, due to DHODH inhibition could be demonstrated in the cell cultures after incubation with MNAs. These intracellular DHO concentrations correlated with the cell numbers and were time and dose dependent on the used concentrations of MNAs. Using brequinar (BQR), a well known inhibitor of the de novo pyrimidine biosynthesis at the level of DHODH, has shown comparable results, although significant differences between the MNAs and BQR could be demonstrated in "washing out" experiments. Conclusion: MNAs primarily inhibit DHODH, which plays a key role in the de novo pyrimidine synthesis. The resultant lowering of pyrimidine ribonucleotides causes inhibition of further cell cycle progression and inhibited the proliferation of various cell lines. Furthermore, inhibition of DHODH, which is responsible for the conversion of DHO to orotate during the de novo pyrimidine biosynthesis, results in a rapid, time, and dose dependent accumulation of DHO in cell cultures.