177 TWO DISTINCT TARGET SITES ON IMP DEHYDROGENASE IN CHEMOTHERAPY

Abstract

The inhibitory mechanisms of ribavirin 5′-monophosphate, (RMP), SM-108 nucleotide (bredinin 5′-monophosphate, BMP) and thiazole-4-carboxamide adenine dinucleotide (TAD), the active forms of the antimetabolites, ribavirin, SM-108 and tiazofurin, were studied in IMP dehydrogenase purified to homogeneity from rat hepatoma 3924A. RMP and BMP as well as XMP inhibited competitively with IMP and noncompetitively with NAD+. The inhibition by TAD was similar to that of NADU: uncompetitive with IMP and mixed type with NAD+. Ki values for RMP (0.8 uM), BMP (0.11 uM) and TAD (0.13 uM) were markedly lower than those for XMP (136 uM) and NADH (210 uM), and also the Km for IMP (23 uM) and NAD (65 uM). Thus, the drugs interact with IMP dehydrogenase with higher affinities than the natural metabolites, RMP or BMP with IMP-XMP and TAD with NADH site. Preincubation of the purified enzyme with RMP enhanced its inhibitory effect in a time-dependent manner. The enzyme was protected from inactivation by IMP or XMP but not by NAD or NADH. Ribavirin and tiazofurin together provided synergistic killing of hepatoma cells in clonogenic assays and the metabolic flux of de novo guanylate synthesis was also synergistically inhibited. The results provide a biochemical basis for combination chemotherapy with tiazofurin and ribavirin against the 2 different ligand sites of IMP dehydrogenase. (Supported by Outstanding Investigator Grant CA-42510 to G.W.)