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L-651,582 (5-amino-[4-(4-chlorobenzoyl)-3,5-dichlorobenzyl]-1,2,3-triazole-4- carboxamide), which is active in vivo against coccidiosis caused by the intracellular parasitic protozoan Eimeria tenella, is also effective against this organism in tissue culture in Maden Darby bovine kidney (MDBK) host cells. L-651,582 inhibited 45Ca++ uptake, as well as [3H]hypoxanthine incorporation into soluble nucleotide pools, in MDBK cells at concentrations similar to those required for antiparasitic activity (IC50 = 0.3 microgram/ml). However, the drug did not inhibit the [3H] hypoxanthine incorporation into the nucleotide pools of the intracellular E. tenella under the same conditions. The antiparasitic activity of several L-651,582 analogs paralleled their ability to inhibit [3H]hypoxanthine incorporation in MDBK cells. L-651,582 similarly inhibited the growth of the intracellular parasitic protozoan Toxoplasma gondii in vitro with either HeLa cells or Normal Human Fibroblasts as host cells. The IC50 for inhibition of T. gondii growth in normal human fibroblasts was similar to that for [3H]hypoxanthine incorporation inhibition. In HeLa cells, however, 40-fold higher levels were required for the inhibition of [3H]hypoxanthine incorporation than were required to inhibit parasite growth, showing that antiparasitic activity was not a consequence of the alteration in host nucleotide metabolism. This was also consistent with the observation that the effect of L-651,582 against E. tenella in MDBK cells was not reversed by the addition of nutrients involved in nucleotide biosynthesis. This study suggests that L-651,582 has antiparasitic activity which correlates with host cell effects and which may be a result of blocking Ca++ entry in the host cells, but is not the result of an alteration of host nucleotide biosynthesis.

Mycophenolic acid (MA) was demonstrated to be an effective inhibitor of the growth of the intracellular parasitic protozoan Eimeria tenella in tissue culture and guanine was shown to reverse this inhibition as expected for an inhibitor of IMP dehydrogenase (IMP:NAD+ oxidoreductase, EC 1.1.1.205). A high performance liquid chromatography study of the intracellular nucleotide pools labeled with [3H]hypoxanthine was carried out in host cells lacking hypoxanthine-guanine phosphoribosyltransferase, and the depletion of guanine nucleotides demonstrated that the intracellular parasite enzyme was being inhibited by the drug. Kinetic studies carried out on the enzyme derived from E. tenella oocysts demonstrated substrate inhibition by NAD and mycophenolic acid inhibition similar to that found for mammalian enzymes, but different from that for bacterial enzymes. The inhibition by mycophenolic acid was not time-dependent and was immediately reversed upon dilution. As found previously for other IMP dehydrogenases, an Ordered Bi-Bi mechanism prevails with IMP on first followed by NAD, NADH off first, and then XMP. The kinetic patterns are consistent with substrate inhibition at high concentrations of NAD due to the formation of an E X XMP X NAD complex. Uncompetitive inhibition by MA versus IMP, NAD, and K+ was found and this was interpreted as evidence for the formation of an E X XMP X MA complex. A speculative mechanism for the inhibition of the enzyme is offered which is consistent with the fact that E X XMP X MA readily forms, whereas E X IMP X MA does not.