Inhibition Of IMP Dehydrogenase Activity Research Articles

Physiological Analysis of the Stringent Response Elicited in an Extreme Thermophilic Bacterium,Thermus thermophilus

Guanosine tetraphosphate (ppGpp) is a key mediator of stringent control, an adaptive response of bacteria to amino acid starvation, and has thus been termed a bacterial alarmone. Previous X-ray crystallographic analysis has provided a structural basis for the transcriptional regulation of RNA polymerase activity by ppGpp in the thermophilic bacterium Thermus thermophilus. Here we investigated the physiological basis of the stringent response by comparing the changes in intracellular ppGpp levels and the rate of RNA synthesis in stringent (rel(+); wild type) and relaxed (relA and relC; mutant) strains of T. thermophilus. We found that in wild-type T. thermophilus, as in other bacteria, serine hydroxamate, an amino acid analogue that inhibits tRNA(Ser) aminoacylation, elicited a stringent response characterized in part by intracellular accumulation of ppGpp and that this response was completely blocked in a relA-null mutant and partially blocked in a relC mutant harboring a mutation in the ribosomal protein L11. Subsequent in vitro assays using ribosomes isolated from wild-type and relA and relC mutant strains confirmed that (p)ppGpp is synthesized by ribosomes and that mutation of RelA or L11 blocks that activity. This conclusion was further confirmed in vitro by demonstrating that thiostrepton or tetracycline inhibits (p)ppGpp synthesis. In an in vitro system, (p)ppGpp acted by inhibiting RNA polymerase-catalyzed 23S/5S rRNA gene transcription but at a concentration much higher than that of the observed intracellular ppGpp pool size. On the other hand, changes in the rRNA gene promoter activity tightly correlated with changes in the GTP but not ATP concentration. Also, (p)ppGpp exerted a potent inhibitory effect on IMP dehydrogenase activity. The present data thus complement the earlier structural analysis by providing physiological evidence that T. thermophilus does produce ppGpp in response to amino acid starvation in a ribosome-dependent (i.e., RelA-dependent) manner. However, it appears that in T. thermophilus, rRNA promoter activity is controlled directly by the GTP pool size, which is modulated by ppGpp via inhibition of IMP dehydrogenase activity. Thus, unlike the case of Escherichia coli, ppGpp may not inhibit T. thermophilus RNA polymerase activity directly in vivo, as recently proposed for Bacillus subtilis rRNA transcription (L. Krasny and R. L. Gourse, EMBO J. 23:4473-4483, 2004).

6-Azauracil inhibition of GTP biosynthesis in Saccharomyces cerevisiae.

The addition of 6-azauracil to the growth medium causes a strong reduction of the GTP level in the nucleotide pool of Saccharomyces cerevisiae. In-vitro experiments show a strong inhibition of IMP dehydrogenase activity by 6-azaUMP explaining the preceding effect. PPR2 mutants, previously characterized by an increased sensitivity to 6-azauracil compared to the wild-type, are specifically susceptible to the lowering of the GTP pool, and are able to grow in presence of 6-azauracil when guanine is added to the medium.

Clinical and molecular impact of inhibition of IMP dehydrogenase activity by tiazofurin

The impact of tiazofurin on inhibition of IMP dehydrogenase was discussed at the clinical and molecular levels. 1. 1. Evidence was provided for the role of IMP dehydrogenase and guanylates in the expression of the neoplastic program in cancer cells with particular relevance to human leukemic cells. 2. 2. The argument for expecting an impact of tiazofurin in human myelocytic cells was provided. 3. 3. Similarity of the kinetics of human leukemic cell IMP dehydrogenase to the rat hepatoma enzyme was documented. 4. 4. New evidence was provided for the role of salvage in chemotherapy and the function of hypoxanthine in inhibiting guanine salvage. 5. 5. The action of tiazofurin and retinoic acid was reported in HL-60 leukemic cells. 6. 6. The effect of tiazofurin and retinoic acid on proliferation and cytotoxicity was outlined for hepatoma 3924A cells. 7. 7. The effect of guanine on induced differentiation by tiazofurin and retinoic acid was examined. 8. 8. Biochemical basis was provided for the lack of development of resistance in patients treated with tiazofurin. 9. 9. Presumptive evidence was provided that tiazofurin treatment induced differentiation of leukemic cells in the patients. 10. 10. The molecular biology of tiazofurin-induced differentiation in K-562 cells was reviewed with the possible relevance to clinical treatment that tiazofurin might also act through down-regulation of ras oncogene.

Induced maturation of the human promyelocytic leukemia cell line, HL-60, by 2-β-D-ribofuranosylselenazole-4-carboxamide

The new synthetic nucleoside analogue, 2-beta-D-ribofuranosylselenazole-4-carboxamide, was evaluated for its effects upon the growth and maturation of the human promyelocytic leukemia cell line, HL-60. At a concentration of greater than or equal to 1 nm, this agent was found both to decrease HL-60 cell proliferation and to cause the cells to acquire an ability to phagocytose opsonized yeast and to reduce nitroblue tetrazolium dye, functions characteristic of mature myeloid cells. In addition, this agent at similar concentrations caused a marked depression of intracellular guanosine nucleotide pools and a reduction in the incorporation of [14C] hypoxanthine into guanylates. These results suggested that the selenazole nucleoside caused an inhibition of inosinate monophosphate dehydrogenase, a key enzyme of guanylate biosynthesis. We therefore measured the activity of this enzyme indirectly by simultaneous-UV-radioactivity HPLC as well as by a direct radiometric method and demonstrated markedly reduced enzyme activities by both assays in drug treated cells. Dose response studies indicated that concentrations of drug which caused greater than 30% inhibition of IMP dehydrogenase activity induced greater than 50% maturation of the cells. These observations with this new nucleoside analogue provide further support for the concept that production of guanosine nucleotides and the activity of IMP dehydrogenase have a role in regulating the terminal maturation of myeloid cells.

Elucidation of aberrant purine metabolism. Application to hypoxanthine-guanine phosphoribosyltransferase- and adenosine kinase-deficient mutants, and IMP dehydrogenase- and adenosine deaminase-inhibited human lymphoblasts

We propose that the ratio of [ 14C]formate-labelled purine nucleosides and bases (both intra and extracellular) to nucleic acid purines provides, in exponentially growing cultures, a sensitive index for comparative studies of purine metabolism. This ratio was 4-fold greater for an HGPRT − mutant than for the parental HGPRT + human lymphoblast line. The major components of the labelled nucleoside and base fraction were hypoxanthine and inosine. By blocking adenosine deaminase activity with coformycin we found that approx. 90% of inosine was formed directly from IMP rather than the route IMP → AMP → adenosine → inosine. The ratio of labelled base + nucleosides to nucleic acids was essentially unchanged for an AK − lymphoblast line and 2-fold greater than control for an HGPRT −-AK − line, demonstrating that a deficiency of adenosine kinase alone has little effect on the accumulation of purine nucleosides and bases. Although adenosine was a minor component of the nucleoside and base fraction, the adenosine fraction increased from 3 to 13% with the addition of coformycin to the HGPRT −-AK − line. In the parental and HGPRT − lines, adenosine was shown to be primarily phosphorylated rather than deaminated at concentrations less than 5 μM. Inhibition of IMP dehydrogenase activity by mycophenolic acid caused a 12- and 3-fold increase in the rate of production of labelled base and nucleoside in the parent and HGPRT − cells respectively. These results suggest that a mutationally induced partial deficiency in the activities converting IMP to guanine nucleotides may result in an increased catabolism of IMP.