Complexed Crystal Structure of the Dihydroorotase Domain of Human CAD Protein with the Anticancer Drug 5-Fluorouracil.

Dihydroorotase (DHOase) is the third enzyme in the pathway used for the biosynthesis of pyrimidine nucleotides. In mammals, DHOase is active in a trifunctional enzyme, CAD, which also carries out the activities of carbamoyl phosphate synthetase and aspartate transcarbamoylase. Prior to this study, it was unknown whether the FDA-approved clinical drug 5-fluorouracil (5-FU), which is used as an anticancer therapy, could bind to the DHOase domain of human CAD (huDHOase). Here, we identified huDHOase as a new 5-FU binding protein, thereby extending the 5-FU interactome to this human enzyme. In order to investigate where 5-FU binds to huDHOase, we solved the complexed crystal structure at 1.97 Å (PDB ID 8GVZ). The structure of huDHOase complexed with malate was also determined for the sake of comparison (PDB ID 8GW0). These two nonsubstrate ligands were bound at the active site of huDHOase. It was previously established that the substrate N-carbamoyl-L-aspartate is either bound to or moves away from the active site, but it is the loop that is extended towards (loop-in mode) or moved away (loop-out mode) from the active site. DHOase also binds to nonsubstrate ligands via the loop-out mode. In contrast to the Escherichia coli DHOase model, our complexed structures revealed that huDHOase binds to either 5-FU or malate via the loop-in mode. We further characterized the binding of 5-FU to huDHOase using site-directed mutagenesis and the fluorescence quenching method. Considering the loop-in mode, the dynamic loop in huDHOase should be a suitable drug-targeting site for further designing inhibitors and clinical chemotherapies to suppress pyrimidine biosynthesis in cancer cell lines.

Anticonvulsive and antioxidant effects of curcumin on pilocarpine-induced seizures in rats.

BACKGROUND: Curcumin, an active ingredient of turmeric with antioxidant and anti-inflammatory properties has recently been reported to have anticonvulsant effects in several animal models of epilepsy. This study aimed to investigate the effects of curcumin on the pilocarpine rat model of status epilepticus. METHODS: The effect of intraperitoneal administration of curcumin (30, 100, and 300 mg/kg) on pilocarpine-induced seizures in rats was tested. The correlation between seizure activity and hippocampal levels of nitric oxide synthase and free radicals was quantified. Whether curcumin treatment modulated these parameters was also investigated. RESULTS: Curcumin significantly increased seizure threshold at doses of 100 and 300 mg/kg. Rats with pilocarpine- induced seizures showed significantly elevated levels of malonaldehyde, nitric oxide synthase, and lactate dehydrogenase, but decreased levels of superoxide dismutase and glutathione compared with normal control rats. At doses of 100 and 300 mg/kg, curcumin reversed the effects of pilocarpine-induced seizures on nitric oxide synthase, lactate dehydrogenase, glutathione, and superoxide dismutase. However, curcumin did not restore the elevated malonaldehyde levels. CONCLUSION: Curcumin has anticonvulsant activity in the pilocarpine rat model of seizures, and that modulation of free radicals and nitric oxide synthase may be involved in this effect.