Pyrimidine nucleobase ligands of orotate phosphoribosyltransferase from Toxoplasma gondii

Sixty-seven pyrimidine nucleobase analogues were evaluated as ligands of Toxoplasma gondii orotate phosphoribosyltransferase (OPRTase, EC 2.4.2.10) by measuring their ability to inhibit this enzyme in vitro. Apparent Ki values were determined for compounds that inhibited T. gondii OPRTase by greater than 20% at a concentration of 400 microM. 1-Deazaorotic acid (0.47 microM) and 5-azaorotic acid (2.1 microM) were found to bind better (8.3- and 1.9-fold, respectively) to T. gondii OPRTase than orotic acid, the natural substrate of the enzyme. Based on these results, a structure-activity relationship of ligand binding to OPRTase was formulated using uracil, barbituric acid, and orotic acid as reference compounds. It was concluded that the following structural features of pyrimidine nucleobase analogues were required or strongly preferred for binding: (i) an endocyclic pyridine-type nitrogen or methine at the 1-position; (ii) exocyclic oxo groups at the 2- and 4-positions; (iii) a protonated endocyclic pyridine-type nitrogen at the 3-position; (iv) an endocyclic pyridine-type nitrogen or methine at the 5-position; (v) an exocyclic hydrogen or fluorine at the 5-position; (vi) an endocyclic pyridine-type nitrogen or methine at the 6-position; and (vii) an exocyclic negatively charged or electron-withdrawing group at the 6-position. A comparison of the results from the present study with those from a previous study on mammalian OPRTase [Niedzwicki et al., Biochem Pharmacol 33: 2383-2395, 1984] identified four compounds (6-chlorouracil, 5-azaorotic acid, 1-deazaorotic acid, and 6-iodouracil) that may bind selectively to T. gondii OPRTase.