inosinic-acid and mizoribine

There is an alarming rate of human African trypanosomiasis recrudescence in many parts of sub-Saharan Africa. Yet, the disease has no successful chemotherapy. Trypanosoma lacks the enzymatic machinery for the de novo synthesis of purine nucleotides, and is critically dependent on salvage mechanisms. Inosine 5'-monophosphate dehydrogenase (IMPDH) is responsible for the rate-limiting step in guanine nucleotide metabolism. Here, we characterize recombinant Trypanosoma brucei IMPDH (TbIMPDH) to investigate the enzymatic differences between TbIMPDH and host IMPDH. Size-exclusion chromatography and analytical ultracentrifugation sedimentation velocity experiments reveal that TbIMPDH forms a heptamer, different from type 1 and 2 mammalian tetrameric IMPDHs. Kinetic analysis reveals calculated K m values of 30 and 1300 μ m for IMP and NAD, respectively. The obtained K m value of TbIMPDH for NAD is approximately 20-200-fold higher than that of mammalian enzymes and indicative of a different NAD binding mode between trypanosomal and mammalian IMPDHs. Inhibition studies show K i values of 3·2 μ m, 21 nM and 3·3 nM for ribavirin 5'-monophosphate, mycophenolic acid and mizoribine 5'-monophosphate, respectively. Our results show that TbIMPDH is different from its mammalian counterpart and thus may be a good target for further studies on anti-trypanosomal drugs.

Topics: Amino Acid Sequence; Animals; Binding Sites; Escherichia coli; Humans; Hydrogen-Ion Concentration; IMP Dehydrogenase; Inosine Monophosphate; Kinetics; Mycophenolic Acid; NAD; Nucleotides; Protein Multimerization; Recombinant Proteins; Ribonucleosides; Sequence Alignment; Trypanosoma brucei brucei

Inosine is absorbed via a N1 transporter that is selective for purine nucleosides. It is conceivable that inosine and inosinic acid might affect the intestinal absorption of mizoribine, an imidazole nucleoside that inhibits the de novo production pathway of guanine ribonucleotide. An in situ loop experiment was performed using four intestinal loop segments prepared by ligation: segment 1, about 6 to 9 cm from the end of the pylorus; segment 2, about 10 to 13 cm; segment 3, about 14 to 17 cm; and segment 4, about 18 to 21 cm. Mizoribine (0.1 mg/ml) or mizoribine+inosine (1 or 10 mg/ml) were infused into each loop. The absorption rate in the most proximal segment of intestinal loop was the highest. In the presence of inosine, this rate decreased significantly. Urinary recovery rates of mizoribine were significantly decreased by pretreatment with inosine or inosinic acid. The Cmax in the group given mizoribine+inosinic acid was significantly lower than that in the group given mizoribine alone. These results strongly indicate that (I) the N1 transporter in the intestine might act to absorb mizoribine; and (II) inosine and inosinic acid might competitively inhibit the absorption of mizoribine via the N1 transporter.

Topics: Animals; Area Under Curve; Chromatography, High Pressure Liquid; Dose-Response Relationship, Drug; Immunosuppressive Agents; Inosine; Inosine Monophosphate; Intestinal Absorption; Male; Metabolic Clearance Rate; Rats; Rats, Sprague-Dawley; Ribonucleosides