AN ANALYSIS OF THE LACK OF DRUG SYNERGISM DURING SEQUENTIAL BLOCKADE OF DE NOVO PYRIMIDINE BIOSYNTHESIS.

Summary Combinations of drugs known to be inhibitors of enzymes in a common metabolic sequence have been previously shown to result in a synergistic response in terms of inhibition of growth of a living cell. The enzyme kinetics of such situations has not been previously investigated. Combinations of 5-azaorotate, which is a competitive inhibitor of the conversion of orotic acid to orotidylic acid, and 6-azauridine, which, following its metabolic conversion to 6-azauridylic acid, is a competitive inhibitor of the conversion of orotidylic acid to uridylic acid, were tested for their capacity to limit the formation of uridine nucleotides from orotic acid in an isolated enzyme system. Combinations of the two analogs, over a wide range of concentrations, resulted in an inhibition of the over-all sequence no greater than that produced by a single inhibitor alone. The same results were observed in a comparable experiment in which intact leukocytes, isolated from a dog, were used. The possibility that 5-azaorotate blocked the activation of 6-azauridine to 6-azauridylic acid, or decreased the affinity of 6-azauridylic acid for its enzyme site, was ruled out by direct experimentation. It was concluded that the lack of even an additive effect of sequential inhibitors could be explained on the basis of an analysis of the kinetics of sequential enzymes. The results indicate that inhibitors of sequential enzymes cannot result in a synergistic response. Thus, the analysis of the enzyme kinetics of sequential inhibition in a cell-free system in vitro does not appear to offer a satisfactory explanation for the synergistic response seen with sequential inhibitors in certain whole cell systems in vivo .