Biological Significance of Guanylate Synthesis and IMP

Chapter 3 Biological Significance of Guanylate Synthesis and IMP Dehydrogenase Isoforms Yutaka Natsumeda

Downloaded by CORNELL UNIV on July 27, 2012 | http://pubs.acs.org Publication Date: March 3, 2003 | doi: 10.1021/bk-2003-0839.ch003

Clinical Development Institute, Banyu Pharmaceutical Company, Ltd., AIG Kabutocho Building, 5-1 Nihombashi Kabutocho, Chuo-ku, Tokyo 103-0026, Japan

I M P dehydrogenase ( I M P D H ) is the rate-limiting enzyme for de novo guanine nucleotide synthesis and a potential target for anticancer and immunosuppressive chemotherapy. Human I M P D H was regarded as a single molecular species until the discovery in 1989 of two isoforms derived from different genes. The two isoforms showed striking differences in expression during neoplastic transformation and proliferation, lymphocytic activation and cell maturation. The type II I M P D H expression is stringently linked with immature characteristics and type I appears to be expressed constitutively. Selective inhibition of the inducible type II I M P D H may mitigate toxicity caused by inhibition of the type I isoform. Although mycophenolic acid has a slight selectivity to type II in its inhibition, the selectivity may not be sufficient. Further studies will be required to elucidate biological, biochemical and structural differences between type I and type II IMPDHs and to better understand the selective inhibition of these two isoforms in anticancer and immunosuppressive chemotherapy.

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© 2003 American Chemical Society

In Inosine Monophosphate Dehydrogenase; Pankiewicz, K., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2003.

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Introduction I M P dehydrogenase ( I M P D H ) catalyzes the conversion of I M P to X M P at the I M P metabolic branch point and appears to be the rate-limiting enzyme for biosynthesis of guanine nucleotides which play important roles in many anabolic and regulatory processes ( i - 6 ) . Weber et al. conducted extensive studies using rat transplantable hepatomas with different growth rates and demonstrated quantitative and qualitative changes in key purine metabolic enzymes in tumors as compared to those in normal rat liver (7). A m o n g these enzymes, I M P D H has attracted great interest as a target for anticancer chemotherapy because a positive correlation was demonstrated between the rate of tumor proliferation and the activity of I M P D H . The specific activity of I M P D H , which is the least in rat normal liver among key purine metabolic enzymes, was increased the most in rat rapidly growing hepatoma (7). In fact, inhibition of I M P D H has been proved to have a potent antiproliferative effect against various tumor cells (8-21) and also lymphocytes (22-24). The mechanism of the antiproliferative effect has been attributed to the decrease in intracellular concentrations of guanine nucleotides, especially G T P and d G T P in the target cells (8, 12-14, 22-25). Sensitivities to the I M P D H inhibitors such as tiazofurin and ribavirin vary with cell types depending on the level of drug activation to their active metabolites and on requirement of de novo guanine nucleotide biosynthesis for proliferation in the cells. A n exciting discovery was made in 1989 and reported in 1990 (26) that there exist two distinct c D N A s (type I and type II) encoding human I M P D H with 84% amino acid sequence identity (26, 27). The type I I M P D H gene is located on chromosome 7 (28), and the type II gene is on chromosome 3 (29). The type II enzyme is regarded as an important chemotherapy target because type II m R N A expression is specifically up-regulated during neoplastic transformation and lymphocytic activation and down-regulated during cancer cell differentiation (30-34). On the other hand, type I I M P D H m R N A appears to be expressed constitutively i n the various states of proliferation and differentiation (31, 32). The striking differences in regulation of the expression of I M P D H isoforms during transformation and cell proliferation may open a novel approach to isozyme-targeted chemotherapy.


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Biological Significance of Guanine Nucleotide Synthesis in Proliferating Cells Guanine nucleotides are required not only i n nucleic acid biosynthesis but also in many anabolic processes such as synthesis of protein, phospholipids, adenine nucleotides and biopterines, protein glycosylation, cytoskeletal organization, and transmembrane signaling (2-3). Intracellular concentrations of guanine nucleotides are one order of magnitude lower than those of adenine nucleotides (8, 12, 13, 24, 25). A t the I M P metabolic branch point, I M P is predominantly utilized to adenine nucleotide synthesis in the resting condition (35). When cells start growing, I M P utilization for guanine nucleotide synthesis is preferentially increased and that for adenine nucleotide synthesis inversely decreased (35). The link between preferential re-direction of metabolic switching at the I M P branch point toward guanylate synthesis and growth stimulation supports the potential significance of the guanylate pathway and I M P D H as targets of chemotherapy. In a strict sense, type II I M P D H should be the crucial target enzyme, because the expression of this isozyme is selectively and inherently linked with cell proliferation and immature characteristics. Since guanine nucleotides are synthesized not only by the de novo pathway but also by the salvage pathway, it is relevant to elucidate relative contributions of both synthetic pathways for guanylate production in cells. Hypoxanthine-guanine phosphoribosyltransferase ( H G P R T ) catalyzes one step production of I M P and G M P from hypoxanthine and guanine, respectively, with the co-substrate 5-phosphoribosyl 1-pyrophosphate ( P R P P ) (36). Extensive studies comparing specific activities of the purine salvage enzyme, H G P R T , and de novo enzymes involved in I M P and G M P synthesis, amidophosphoribosyltransferase (amidoPRT) and I M P D H , showed that potential capacities of the salvage pathway were much higher than those of the de novo pathways in all the tissues and cells examined (37-39) except HGPRT-deficient mutants. A l s o the affinity to the common substrate P R P P of H G P R T was orders of magnitude higher than that of amidoPRT (37). This fact explains why the drug action associated with guanine nucleotide depletion induced by I M P D H inhibitors is circumvented by adding the salvage precursor guanine or guanosine (12, 17, 21, 22, 24, 40). The high capacity of the salvage pathway is biologically reasonable, because reutilization of a preformed purine ring can save three molecules of A T P to produce one molecule of purine nucleotide. Nevertheless, the salvage synthesis of guanine nucleotides


51 seems to be limited in vivo in humans because the salvage precursor, guanine or guanosine, is not fully available in plasma (41, 42). Although hypoxanthine is available in plasma (41, 42) and is converted to I M P , guanine nucleotide synthesis could still be blocked in the presence of an inhibitor of I M P D H or G M P synthase.


Biological Significance of Depletion in Guanine Nucleotides In rats carrying subcutaneously transplanted hepatoma 3 9 2 4 A solid tumors, a single intraperitoneal injection of tiazofurin, which is bioactivated to a potent I M P D H inhibitor, thiazole-4-carboxamide adenine dinucleotide ( T A D ) , depleted G D P , G T P and d G T P pools i n the tumor; and concurrently, I M P and P R P P pools expanded (8). The increase of P R P P concentration was attributed to the enhancement of the inosinate cycle consisting of 5'-nucleotidase, inosine phosphorylase, phosphoribomutase, P R P P synthase and H G P R T (43). In normal liver the effect of tiazofurin was less pronounced than i n the hepatoma (8). The differential response in normal liver and hepatoma might be attributable to different metabolic turnover rates of guanylate production and its utilization in those tissues rather than the different metabolic rates producing the active metabolite T A D . In fact I M P D H was inhibited i n normal liver to much lower activity than the remaining activity i n the tumor after tiazofurin injection. The decrease in G T P pool was closely correlated with the cytotoxic effect of tiazofurin in hepatoma 3 9 2 4 A cells (8). It has also been shown that depletion of the G T P pool caused by an I M P D H inhibitor results in a decrease in glycosylation of protein (17, 44) including adhesion molecules (45, 46), biopterin synthesis (5), R N A - p r i m e d D N A synthesis (47), IgE receptor-mediated degranulation (48, 49), secretion of serotonin (50), and cellular ras-GTP complex (52). It also results in an increase in type II I M P D H m R N A level (52) and purine salvage pathway capacity (53). Recently mycophenolic acid, an inhibitor of I M P D H , was demonstrated also to suppress cytokine-induced nitric oxide production in vascular endothelial cells (54, 55). Treatment of immature proliferating cells with an inhibitor of I M P D H induces differentiation and maturation of the cells (13, 14, 17, 18, 40, 56-59). Differentiation induced by a known inducer such as retinoic acid,


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12-0-tetradecanylphorbol-13-acetate does not inhibit I M P D H activity, is type II I M P D H (31, 32). Thus, type cell proliferation and should be a immunosuppressive chemotherapy.

( T P A ) or dimethyl sulfoxide, which associated with down regulation of II I M P D H is inherently linked with crucial target for anticancer and


Structural Similarities and Differences between IMPDH Isoforms Human type II I M P D H has a similar primary structure to mouse and Chinese hamster I M P D H s in which only 6 and 7 amino acids out of 514 are different, respectively (26, 27, 60). The difference between the human type I and type II I M P D H sequences is much more extensive: 84 out of 514 amino acids differ (26). Among these changes, 52 are conservative amino acid substitutions and 32 diverge with respect to their chemical properties. The consensus nucleotide-binding motif of |3-a-|3 has been predicted in Escherichia coli I M P D H on the basis of steric and physicochemical properties of amino acids from pattern searches of protein-sequence databases (61). The domains are located from Asp-319 to Lys-349 in human I M P D H s and the alignments are w e l l conserved i n I M P D H s through evolution from bacteria to mammals (26, 27, 60, 62-65). The domain includes a cysteine which is the only sulfhydryl group conserved in various I M P D H s (64, 66) and appears to be involved in IMP-binding (66-69).

Comparison of Kinetic Properties of Human Type I and Type II IMPDHs To elucidate differences in kinetic properties of human I M P D H isoforms, unmodified recombinant sequences of each isoform were overexpressed in an IMPDH-deficient strain of E.coli and purified to homogeneity (70). Both recombinant I M P D H s were tetramers which was in agreement with the subunit structure of the native mammalian I M P D H (70, 71). Recombinant I M P D H fusion proteins were a mixture of monomer, dimer, tetramer and various sizes of aggregated forms, even though the enzyme was purified to homogeneity as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.



53 In the studies using the recombinant enzymes, human type I and II I M P D H s both exhibited an ordered B i B i kinetic mechanism in which I M P binds to the free enzyme first, followed by X M P (70). Both I M P D H isoforms showed similar affinities for the substrates. The Km values determined from the steady-state fit for type I and type II I M P D H s were in agreement with kinetic data previously reported for murine (71, 72) and human I M P D H (75), which presumably included a mixture of the two isoforms, and were 18 and 9 p M , respectively, for I M P , and 46 and 32 p,M, respectively, for N A D (70). The isoforms had similar fccat values of 1.5 and 1.3 turnovers/molecule of enzyme/second at 3 7 ° C for types I and II, respectively (70). Two alternative substrates, nicotinamide hypoxanthine dinucleotide and nicotinamide guanine dinucleotide were able to act as hydride acceptors, although their affinities were 20- to 150-fold lower than the natural substrate, N A D (70). In spite of the significant changes in Km values for these alternative hydride acceptors, &cat values remained unchanged from those obtained with N A D , suggesting that the ratelimiting step of the reaction does not involve binding of N A D to the enzyme (70). Inhibitory mechanisms by the products, X M P and N A D H , were identical for human type I and type II I M P D H s . X M P inhibited i n a competitive manner against I M P and Ki values of type I and II isoforms were 80 and 94 p M , respectively (70). N A D H inhibited in an uncompetitive manner against I M P and with a mixed-type pattern against N A D and Ki values were 102 p M for type I and 90 p M for type II I M P D H , respectively (70).

Inhibition of IMPDH Isozymes by Mycophenolic Acid Mycophenolate mofetil, a morpholinoethyl ester of mycophenolic acid, is clinically used as an immunosuppressive agent for the prevention of graft rejection after organ transplantation. The esterification to the prodrug improves the bioavailability of mycophenolic acid, a potent inhibitor of I M P D H (74). Pharmacokinetic studies showed that oral administration of mycophenolate mofetil provided plasma concentrations sufficient to show immunosuppressive effect with limiting toxicity, even when higher doses than the therapeutic dose are given to non-human primates for long periods (75). The mechanism of inhibition by mycophenolic acid was the same for both type I and type II I M P D H s (70). The drug inhibited i n an uncompetitive manner with respect to I M P and N A D , indicating that


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mycophenolic acid interacts with the enzyme after both substrates bind to it. Uncompetitive inhibition by this drug is of particular benefit due to the substrate accumulation, which potentiates inhibition. The results of kinetic studies suggest that mycophenolic acid binds to either the enzyme-IMP ( X M P ) - N A D ( N A D H ) ternary complex or the e n z y m e - X M P complex (70, 76). The affinity of mycophenolic acid to type II I M P D H was 4.8-fold higher than that to the type I isoform (70). Further analyses and attempts are in progress to design more selective compounds to type II I M P D H (77).

Acknowledgments The author is indebted to D r . T. Ikegami for his dedication in the early stage of this work especially for developing the radioassay (78) and the affinity column chromatography for I M P D H purification (79). Special thanks go to D r . S. Ohno for his guidance on c D N A cloning and discovery of human I M P D H isoforms. The author is also grateful to D r . G . Weber, Dr. K . Suzuki and D r . K . Tsushima for their continuous encouragement and discussions for this work and to M s . Yasuko Asano for assistance i n preparing this manuscript.

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Biological Significance of Guanylate Synthesis and IMP

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