Enzymatic formation of inosine 3',5'-monophosphate and of 2'-deoxyguanosine 3',5'-monophosphate. Inosinate and deoxyguanylate cyclase activity.

Enzymes in particulate fractions from sea urchin sperm and in soluble fractions from rat lung were shown to catalyze the formation of inosine 3',5'-monophosphate (cyclic IMP) and of 2'-deoxyguanosine 3',5'-monophosphate (cyclic dGMP) from ITP and dGTP, respectively. With sea urchin sperm particulate fractions, Mn2+ was an essential metal cofactor for inosinate, deoxyguanylate, guanylate and adenylate cyclase activities. Heat-inactivation studies differentiated inosinate and deoxyguanylate cyclase activities from adenylate cyclase, but indicated an association of these activities with guanylate cyclase. Preincubation of sea urchin sperm particulate fractions with trypsin altered in a very similar manner guanylate, inosinate, and deoxyguanylate cyclase activities, and various metals and metal-nucleotide combinations protected the three cyclase activities to comparable degrees against trypsin. The relative guanylate, deoxyguanylate and inosinate cyclase activities at 0.1 mM nucleoside triphosphate were 1.0, 0.5 and 0.08, respectively. With these three cyclase activities, plots of reciprocal velocities against reciprocal Mn2+-nucleoside triphosphate concentrations were concave upward, suggesting positive homotropic effects. With rat lung soluble preparations, relative guanylate, deoxyguanylate, inosinate and adenylate cyclase activities at 0.09 mM nucleoside triphosphate were 1.0, 1.7, 0.1 and 0, respectively. MnGTP was a competitive inhibitor of deoxyguanylate cyclase activity (Ki equals 12.2 muM) and MndGTP was a competitive inhibitor of guanylate cyclase activity (Ki equals 16.2 muM). Inhibition studies using ITP were not conducted. When soluble fractions from rat lung were applied to Bio-Gel A 1.5 m columns, elution profiles of guanylate, deoxyguanylate and inosinate cyclase activities were similar. These results suggest that deoxyguanylate, guanylate and inosinate cyclase activities reside within the same protein molecule.     

Alterations of inosinate branchpoint enzymes in cultured human lymphoblasts

The specific activities of the three enzymes of the inosinate branchpoint are independently regulated when lymphoblasts are grown under various tissue culture conditions. In comparison to rapidly dividing cells, lymphoblasts at high cell density with no cellular division have decreased activity of the enzymes which commit inosinate to adenylate or guanylate, while cytoplasmic 5'-nucleotidase is relatively preserved. A linear relationship between inosinate dehydrogenase activity and growth rate (r = 0.92) exists in lymphoblasts with slowed growth rates. In contrast, in dividing cells adenylosuccinate synthetase and 5'-nucleotidase do not vary with growth rate. Adenylosuccinate synthetase and inosinate dehydrogenase activities appear to be related to the presence or rate of cellular division, as opposed to the presence or degree of neoplastic transformation. Lymphoblast lines with alterations of specific purine metabolic enzymes have characteristic alteration of the inosinate utilizing enzymes. Deficiencies of purine nucleoside phosphorylase or hypoxanthine phosphoribosyltransferase, abnormalities which render the cell unable to salvage purine effectively, are associated with depressed inosinate dehydrogenase activity. Insertion of the hypoxanthine phosphoribosyltransferase gene into hypoxanthine phosphoribosyltransferase-deficient cells normalizes inosinate dehydrogenase activity, while a hypoxanthine phosphoribosyltransferase-deficient mutant selected from a hypoxanthine phosphoribosyltransferase-containing line has depressed inosinate dehydrogenase activity. In contrast, overactivity of phosphoribosylpyrophosphate synthetase, with enhanced excretion of purines due to excessive production, is associated with elevated inosinate dehydrogenase activity. Inosinate dehydrogenase appears to be regulated according to the availability of purine nucleotides. Patients who overproduce uric acid and potentially have undescribed purine metabolic defects are now being screened for abnormalities in the inosinate branchpoint enzymes.     

Inhibitors of IMP dehydrogenase stimulate the phosphorylation of the antiviral nucleoside 2' ,3'-dideoxyguanosine

The inosinate dehydrogenase (IMPD) inhibitors ribavirin, tiazofurin and mycophenolic acid were found to stimulate by as much as 20-fold the anabolism of the anti-HIV agent 2' ,3'dideoxyguanosine to its 5'-diphosphate (ddGDP) in a human T-cell culture system (Molt-4 cells). Stimulation of the further conversion to ddGTP (the active form of the drug) was lesser in magnitude but still highly significant (up to 4-fold at appropriate concentrations of ribavirin or tiazofurin). In parallel with these increases, the inhibitors also produced increases of up to 35-fold in IMP levels. These results support the proposal that the initial phosphorylation of ddGuo is catalyzed by a phosphotransferase (5'-nucleotidase) which utilizes IMP as its phosphate donor (Johnson and Fridland, [1989] Molec. Pharmacol. 36, 291-295). Concomitant with this increase in 5'-phosphorylation of ddGuo, an increase in its anti-HIV activity of up to 6.5-fold was observed when this agent was combined with ribavirin (5 microM) in the H9 [corrected] cell assay system.     

Carbanicotinamide adenine dinucleotide: synthesis and enzymological properties of a carbocyclic analogue of oxidized nicotinamide adenine dinucleotide

The dinucleotide carbanicotinamide adenine dinucleotide (carba-NAD), in which a 2,3-dihydroxycyclopentane ring replaces the beta-D-ribonucleotide ring of the nicotinamide ribonucleoside moiety of NAD, has been synthesized and characterized enzymologically. The synthesis begins with the known 1-aminoribose analogue (+/-)-4 beta-amino-2 alpha,3 alpha-dihydroxy-1 beta-cyclopentanemethanol. The pyridinium ring is first introduced and the resultant nucleoside analogue specifically 5'-phosphorylated. Coupling the racemic carbanicotinamide 5'-mononucleotide with adenosine 5'-monophosphate produces two diastereomeric carba-NAD analogues which are chromatographically separable. Only one diastereomer is a substrate for alcohol dehydrogenase and on this basis is assigned a configuration analogous to D-ribose. The reduced dinucleotide carba-NADH was characterized by fluorescence spectroscopy and found to adopt a "stacked" conformation similar to that of NADH. The analogue is reduced by both yeast and horse liver alcohol dehydrogenase with Km and Vmax values for the analogue close to those observed for NAD. Carba-NAD is resistant to cleavage by NAD glycohydrolase, and the analogue has been demonstrated to noncovalently inhibit the soluble NAD glycohydrolase from Bungarus fasciatus venom at low concentrations (less than or equal to 100 microM).     

IMP dehydrogenase and action of antimetabolites in human cultured blast cells

Tiazofurin was demonstrated to be an effective inhibitor of the growth of human cultured blast cells, and the high specific activities of IMP dehydrogenase (EC 1.1.1.205) were observed in all the cell extracts tested. IMP dehydrogenase has been purified to homogeneity from MOLT 4F human T-lymphoblast, and the Km values for IMP and NAD were 29 and 54 microM, respectively. The inhibitory mechanisms of thiazole-4-carboxamide adenine dinucleotide (TAD) and ribavirin 5'-monophosphate (RMP), the active forms of the antimetabolites tiazofurin and ribavirin, were investigated on the purified enzyme. RMP inhibits competitively with respect to IMP as well as XMP, and the inhibition by TAD was similar to that by NADH, which was uncompetitive with NAD. However, the Ki values of RMP (0.58 microM) and TAD (0.075 microM) were several orders of magnitude lower than those of XMP (85 microM) and NADH (94 microM). Thus, the drugs interact with the two distinct sites of IMP dehydrogenase with much higher affinities than the natural substrates and products. Preincubation of the purified enzyme with RMP enhanced its inhibitory effect in a time-dependent manner, and the enhancement was further increased by the addition of TAD. The combination of tiazofurin and ribavirin exerted a synergistic effect on the growth inhibition in MOLT 4F cells.     

Action of the active metabolites of tiazofurin and ribavirin on purified IMP dehydrogenase

The inhibitory mechanisms of ribavirin 5'-monophosphate (RMP) and thiazole-4-carboxamide adenine dinucleotide (TAD), the active forms of the antimetabolites ribavirin and tiazofurin, were investigated in IMP dehydrogenase purified to homogeneity from rat hepatoma 3924A. The hepatoma IMP dehydrogenase has a tetrameric structure with a subunit molecular weight of 60,000. For the substrates IMP and NAD+, Km's were 23 and 65 microM, respectively. Product-inhibition patterns showed an ordered Bi-Bi mechanism for the enzyme reaction where IMP binds to the enzyme first, followed by NAD+; NADH dissociates from the ternary complex first and then XMP is released. XMP interacts with the free enzyme and competes for the ligand site with IMP, while NADH binds to the enzyme-XMP complex. RMP exerted the same inhibitory mechanisms as XMP, and the inhibition by TAD was similar to that by NADH. However, the Ki values for RMP (0.8 microM) and TAD (0.13 microM) were orders of magnitude lower than those of XMP (136 microM) and NADH (210 microM). Thus, the drugs interact with IMP dehydrogenase with higher affinities than the natural substrates and products, RMP with the IMP-XMP site and TAD with the NADH site. Preincubation of the purified enzyme with RMP enhanced its inhibitory effect in a time-dependent manner. The enzyme was protected from this inactivation by IMP or XMP. These results provide a biochemical basis for combination chemotherapy with tiazofurin and ribavirin targeted against the two different ligand sites of IMP dehydrogenase.     

Novel mycophenolic adenine bis(phosphonate)s as potent anticancer agents and inducers of cells differentiation.

An effective treatment of myeloid leukemias would rely on inducing myeloid cells to undergo differentiation. It has been demonstrated that inhibition of IMPDH with mycophenolic acid or tiazofurin resulted in inhibition of cell growth as well as induction of differentiation. We synthesized a number of bis(phosphonate) analogues of tiazofurin-, benzamide-, and mycophenolic-adenine dinucleotide which were found to be cytotoxic as well as effective inducers of cell differentiation.     

Novel Mycophenolic Adenine bis(Phosphonate)S as Potent Anticancer Agents and Inducers of Cells Differentiation

Abstract

An effective treatment of myeloid leukemias would rely on inducing myeloid cells to undergo differentiation. It has been demonstrated that inhibition of IMPDH with mycophenolic acid or tiazofurin resulted in inhibition of cell growth as well as induction of differentiation. We synthesized a number of bis(phosphonate) analogues of tiazofurin-, benzamide-, and mycophenolic-adenine dinucleotide which were found to be cytotoxic as well as effective inducers of cell differentiation.     

Studies of the regulation of purine nucleotide catabolism.

Ehrlich ascites tumor cells containing radioactive ATP were incubated in vitro with a range of concentrations of 2-deoxyglucose in order to produce different rates of ATP catabolism. Concentrations of all radioactive products of ATP catabolism were measured, and apparent rates of adenylate deaminase and inosinate dehydrogenase and of adenylate and inosinate dephosphorylation were calculated. It was concluded that these processes were reggulated primarily by the rate of formation of substrate, and to a lesser extent in some cases, by substrate concentration. No evidence was obtained for regulation of these processes by the concentration of ATP. The deoxyglucose-induced catabolism of radioactive GTP was also studied. When ATP catabolism was induced by incubation with 2,4-dinitrophenol, time courses of accumulation of purine nucleoside monophosphates and rates of alternative pathways of their metabolism were quite different than when deoxyglucose was used.     

Inhibitors of inosinate dehydrogenase

Inosinate dehydrogenase (IMP-dehydrogenase) from Chinese Hamster Ovary cells was found to be inhibited by two types of inhibitors. One type of inhibitor binds competitively at IMP-binding site and other type at NAD-binding site of the enzyme. Some new inhibitors of both classes have been illustrated here and their inhibition constants and type of inhibition determined. NAD-analog of tiazofurin was found to bind with both the sites and this type of inhibitors were found to be non-competitive with IMP and NAD.     

Studies of purine and tiazofurin metabolism in drug sensitive human chronic myelogenous leukemia K 562 cells

Antineoplastic activity of tiazofurin (2-beta-D-ribofuranosylthiazole-4-carboxamide) is mediated by an anabolite of the drug thiazole-4-carboxamide adenine dinucleotide (TAD), an analog of NAD which inhibits IMP dehydrogenase activity resulting in the depletion of guanylate pools and cell death. Human chronic myelogenous leukemia K 562 cells were found to be sensitive to tiazofurin with an IC50 of 19.2 microM. TAD content in K 562 cells (1.3 nmol/10(9)/h) was in the range found in susceptible murine and human tumor cells. Studies were conducted to relate tiazofurin toxicity with biochemical effects by examining nucleotide pools. Among the nucleotides, only guanylate pools were significantly depleted by the drug. To further study the effect of the drug on the purine nucleotide de novo and salvage biosynthetic pathways, flux of radiolabelled formate and guanine was employed. The results showed that de novo synthesis of guanylates was curtailed primarily by the drug's action without influencing adenylate biosynthesis or salvage of guanine to guanylates. These studies show that K 562 cells are sensitive to selective inhibition of de novo guanylate pathway indicating that human chronic myelogenous leukemia in blast crisis might be a good candidate for Phase II clinical trials with tiazofurin.     

Inhibition of gluconeogenesis and lactate formation from pyruvate by N6, O2'-dibutyryl adenosine 3':5'-monophosphate.

N6,O2-Dibutyryl adenosine 3':5'-monophosphate (Bt2cAMP) inhibits gluconeogenesis and lactate formation but increases ketogenesis by isolated liver cells incubated with high concentrations of pyruvate. The inhibitory effects can not be explained on the basis of an inhibition of the pyruvate dehydrogenase complex nor by a change in the NAD+ oxidation-reduction potential of the mitochondrial compartment. Both oleate and 3-hydroxybutyrate substantially increase the rates of gluconeogenesis and lactate formation from pyruvate but do not overcome the inhibition caused by Bt2cAMP. A decreased effectiveness of pyruvate kinase is proposed to account for the inhibition of both gluconeogenesis and lactate formation by Bt2cAMP. This enzyme catalyzes a step required in the transfer of reducing equivalents from the mitochondrial compartment to the cytoplasm and participates in the formation of glucose and lactate from pyruvate by the overall reaction: 2 pyruvate- + 2 NADHmito + 4 ATP4- + 4 H2O leads to 1/2 glucose + lactate- + 2 NAD+ mito + 4 ADP3- + 4 HPO4(2)- + H+. Inhibition of pyruvate kinase promotes gluconeogenesis with most substrates but inhibits gluconeogenesis from pyruvate for want of cytoplasmic reducing equivalents.     

Cryptosporidium parvum IMP dehydrogenase: identification of functional, structural, and dynamic properties that can be exploited for drug design

The protozoan parasite Cryptosporidium parvum causes severe enteritis with substantial morbidity and mortality among AIDS patients and young children. No fully effective treatment is available. C. parvum relies on inosine 5'-monophosphate dehydrogenase (IMPDH) to produce guanine nucleotides and is highly susceptible to IMPDH inhibition. Furthermore, C. parvum obtained its IMPDH gene by lateral transfer from an epsilon-proteobacterium, suggesting that the parasite enzyme might have very different characteristics than the human counterpart. Here we describe the expression of recombinant C. parvum IMPDH in an Escherichia coli strain lacking the bacterial homolog. Expression of the parasite gene restores growth of this mutant on minimal medium, confirming that the protein has IMPDH activity. The recombinant protein was purified to homogeneity and used to probe the enzyme's mechanism, structure, and inhibition profile in a series of kinetic experiments. The mechanism of the C. parvum enzyme involves the random addition of substrates and ordered release of products with rate-limiting hydrolysis of a covalent enzyme intermediate. The pronounced resistance of C. parvum IMPDH to mycophenolic acid inhibition is in strong agreement with its bacterial origin. The values of Km for NAD and Ki for mycophenolic acid as well as the synergistic interaction between tiazofurin and ADP differ significantly from those of the human enzymes. These data suggest that the structure and dynamic properties of the NAD binding site of C. parvum IMPDH can be exploited to develop parasite-specific inhibitors.     

Induced maturation of the human promyelocytic leukemia cell line, HL-60, by 2-beta-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.     

NAD kinase interaction with the activator--glutamate dehydrogenase

An electrophoretically homogeneous preparation of the NAD kinase activating factor was isolated from rabbit liver and its physico-chemical properties were investigated. The similarity of molecular weights of the activator subunit and hexamer, pI values, the number of SH-groups to the corresponding parameters for glutamate dehydrogenase and the glutamate dehydrogenase activity demonstrated by this factor allowed for the identification of the NAD kinase activating factor as glutamate dehydrogenase. Using three independent methods, the formation of the NAD kinase--glutamate dehydrogenase complex was shown. Both the oligomeric and monomeric (subunit) forms of NAD kinase were found to be able to form complexes with glutamate dehydrogenase.     

Mycophenolic acid and thiazole adenine dinucleotide inhibition of Tritrichomonas foetus inosine 5'-monophosphate dehydrogenase: implications on enzyme mechanism

Inosine 5'-monophosphate dehydrogenase (IMPDH) catalyzes the oxidation of inosine 5'-monophosphate (IMP) to xanthosine 5'-monophosphate (XMP) with the conversion of NAD to NADH. An ordered sequential mechanism where IMP is the first substrate bound and XMP is the last product released was proposed for Tritrichomonas foetus IMPDH on the basis of product inhibition studies. Thiazole adenine dinucleotide (TAD) is an uncompetitive inhibitor versus IMP and a noncompetitive inhibitor versus NAD, which suggests that TAD binds to both E-IMP and E-XMP. Mycophenolic acid is also an uncompetitive inhibitor versus IMP and noncompetitive versus NAD. Multiple-inhibitor experiments show that TAD and mycophenolic acid are mutually exclusive with each other and with NADH. Therefore, mycophenolic acid most probably binds to the dinucleotide site of T. foetus IMPDH. The mycophenolic acid binding site was further localized to the nicotinamide subsite within the dinucleotide site: mycophenolic acid was mutually exclusive with tiazofurin, but could form ternary enzyme complexes with ADP or adenosine diphosphate ribose. NAD inhibits the IMPDH reaction at concentrations greater than 3 mM. NAD substrate inhibition is uncompetitive versus IMP, which suggests that NAD inhibits by binding to E-XMP. TAD is mutually exclusive with both NAD and NADH in multiple-inhibitor experiments, which suggests that there is one dinucleotide binding site. The ordered mechanism predicts that multiple-inhibitor experiments with XMP and TAD, mycophenolic acid, or NAD should have an interaction constant (alpha) between 0 and 1. However, alpha was greater than 1 in all cases.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cell cycle dependent regulation of IMP dehydrogenase activity and effect of tiazofurin.

The activity of IMP dehydrogenase (IMP DH), the rate-limiting enzyme of de novo GTP biosynthesis, was shown to be increased in cancer cells. Tiazofurin, an inhibitor of IMP dehydrogenase, proved to be an effective agent in the treatment of refractory granulocytic leukemia. To examine the cell cycle dependent alterations of GTP synthesis and sensitivities to tiazofurin, we measured IMP DH activities and GTP pools, as well as the effects of tiazofurin on cell cycle phase enriched HL-60 cells. We now show that IMP DH activities and GTP concentrations are increased in S-phase enriched fractions of HL-60 cells. Moreover, the depletion of GTP concentrations by tiazofurin is most effective in S-phase enriched HL-60 cells. These results may be utilized in cancer chemotherapy to combine tiazofurin with biologic response modifiers which recruit quiescent leukemic cells into the cell cycle.     

Evidence for two species of glutamate dehydrogenases in Thiobacillus novellus

When grown autotrophically in a thiosulfate-mineral salts medium, cells of the facultative chemoautotrophic bacterium, Thiobacillus novellus, produced two distinct glutamate dehydrogenases, one specific for nicotinamide adenine dinucleotide phosphate (NADP) and the other specific for nicotinamide adenine dinucleotide (NAD). When glutamate was supplied exogenously as the sole carbon source, the NAD-specific glutamate dehydrogenase was fully induced. Lower levels of the enzyme were found in bacteria grown in l-arginine, l-alanine, glucose, glycerol, lactate, citrate, or succinate. Arginine, histidine, and aspartate, on the other hand, caused a marked repression of the NADP-specific glutamate dehydrogenase activity. The NAD-dependent glutamate dehydrogenase was allosteric. Adenosine-5'-monophosphate and adenosine-5'-diphosphate acted as positive effectors. Both glutamate dehydrogenases were purified about 250-fold and were shown to be distinct protein with different physical properties.     

Structure of human nicotinamide/nicotinic acid mononucleotide adenylyltransferase. Basis for the dual substrate specificity and activation of the oncolytic agent tiazofurin.

Nicotinamide/nicotinate mononucleotide (NMN/ NaMN)adenylyltransferase (NMNAT) is an indispensable enzyme in the biosynthesis of NAD(+) and NADP(+). Human NMNAT displays unique dual substrate specificity toward both NMN and NaMN, thus flexible in participating in both de novo and salvage pathways of NAD synthesis. Human NMNAT also catalyzes the rate-limiting step of the metabolic conversion of the anticancer agent tiazofurin to its active form tiazofurin adenine dinucleotide (TAD). The tiazofurin resistance is mainly associated with the low NMNAT activity in the cell. We have solved the crystal structures of human NMNAT in complex with NAD, deamido-NAD, and a non-hydrolyzable TAD analogue beta-CH(2)-TAD. These complex structures delineate the broad substrate specificity of the enzyme toward both NMN and NaMN and reveal the structural mechanism for adenylation of tiazofurin nucleotide. The crystal structure of human NMNAT also shows that it forms a barrel-like hexamer with the predicted nuclear localization signal sequence located on the outside surface of the barrel, supporting its functional role of interacting with the nuclear transporting proteins. The results from the analytical ultracentrifugation studies are consistent with the formation of a hexamer in solution under certain conditions.