Adenosine analogue inhibitors of S-adenosylhomocysteine hydrolase

Elevated plasma homocysteine (Hcy) levels are an independent risk factor for the onset and progression of Alzheimer’s disease. Reduction of Hcy to normal levels therefore presents a new approach for disease modification. Hcy is produced by the cytosolic enzyme S-adenosylhomocysteine hydrolase (AHCY), which converts S-adenosylhomocysteine (SAH) to Hcy and adenosine. Herein we describe the design and characterization of novel, substrate-based S-adenosylhomocysteine hydrolase inhibitors with low nanomolar potency in vitro and robust activity in vivo.     

Quantitation of S-adenosylhomocysteine hydrolase in mouse L929 cells using the inhibitor neplanocin A

S-Adenosylhomocysteine (AdoHcy) hydrolase (EC 3.3.1.1) catalyzes the reversible hydrolysis of AdoHcy to adenosine and homocysteine. Neplanocin A, a cyclopentyl analog of adenosine, has previously been shown to act as a tight-binding inhibitor of the purified bovine liver enzyme, binding with a stoichiometry of one molecule per tetramer of enzyme (R.T. Borchardt, B.T. Keller, and U.G. Patel-Thombre, 1984, J. Biol. Chem. 259, 4353-4358). In the current study neplanocin A was also shown to act as a stoichiometric inhibitor of the L929 cell enzyme having Ki = 0.2 nM. Using this inhibitor to titrate the AdoHcy hydrolase, the concentration of the enzyme in intact L929 cells was calculated to be 0.8 microM, assuming a 1:1 inhibitor:protein stoichiometry. It was observed that the specific activity of AdoHcy hydrolase as measured in the hydrolytic direction increased 270% over a 12-h period after L929 cells were given fresh serum-free medium or when the cell extract was dialyzed first against phosphate buffer. Using the neplanocin A titration technique, it was found that the enzyme concentration in L929 cells remained constant over a 48-h period after refeeding the cultures. These results suggest the presence of an endogenous inhibitor or a readily reversible-type enzyme modification which is responsible for regulating AdoHcy hydrolase in vivo.     

The mechanism of inhibition of Alcaligenes faecalis S-adenosylhomocysteine hydrolase by neplanocin A

Neplanocin A, a cyclopentenyl analog of adenosine, has been reported by S. Yaginuma, N. Muto, M. Tsujino, Y. Sudate, M. Hayashi, and M. Otari (1981) J. Antibiot. 34, 359-366 to exhibit antibacterial activity against Alcaligenes faecalis. Since neplanocin A (NpcA) is a known inhibitor of eukaryotic S-adenosylhomocysteine (AdoHcy) hydrolase (EC 3.3.1.1) (R. T. Borchardt, B. T. Keller, and U. Patel-Thombre (1984) J. Biol. Chem. 259, 4353-4358), the present study was undertaken to determine the effects of this carbocyclic nucleoside on AdoHcy hydrolase isolated from a prokaryotic source (A. faecalis). AdoHcy hydrolase was purified to homogeneity by affinity chromatography on an AdoHcy-agarose matrix from A. faecalis. Neplanocin A inactivated the purified AdoHcy hydrolase in a time- and concentration-dependent manner and the enzyme activity could not be recovered by dialysis. The inactivation of this bacterial enzyme by neplanocin A is accompanied by a reduction of three of the six enzyme-bound NAD+s to NADHs. These results suggest that the prokaryotic enzyme, like the eukaryotic AdoHcy hydrolase, is susceptible to inhibition by neplanocin A. The mechanism of inactivation in both cases appears to be a Kcat mechanism involving the reduction of the enzyme-bound NAD+ to NADH. The fact that total inhibition of the prokaryotic AdoHcy hydrolase by NpcA results in a reduction of only three of the six enzyme-bound NAD+s to NADHs suggests that the enzyme shows half-site reactivity (i.e., only three of the six subunits are catalytically active).     

The role of nicotinamide adenine dinucleotide in the inhibition of bovine liver S-adenosylhomocysteine hydrolase by neplanocin A

Neoplanocin A, a cyclopentenyl analog of adenosine, has been shown recently to be a tight binding inhibitor of S-adenosylhomocysteine (AdoHcy) hydrolase (EC 3.3.1.1), exhibiting a stoichiometry of one molecule of inhibitor per molecule of the enzyme tetramer (Borchardt, R. T., Keller, B. T., and Patel-Thombre, U. (1984) J. Biol. Chem. 259, 4353-4358). In the present study a detailed analysis was performed of the possible role of the enzyme-bound NAD+ in the inactivation of AdoHcy hydrolase by neplanocin A. The NAD+/NADH content was quantitated using a fluorescence technique. The native enzyme showed intrinsic fluorescence with an emission maximum at 460 nm when excited at 340 nm, partially due to NADH bound to the enzyme. It was found that the content of NAD+ and NADH in freshly prepared, native enzyme is equal, having a stoichiometry of two nucleotides per enzyme molecule (tetramer). In addition, it was observed that the enzymatic activity of the native enzyme can be increased by about 30% following preincubation with NAD+. Furthermore, it was demonstrated that the mechanism of inhibition of AdoHcy hydrolase by neplanocin A involves the reduction of enzymatically bound NAD+ to NADH. Catalytic activity of the inactivated enzyme could be fully recovered in a time-dependent manner by further incubation with NAD+ (but not NADH). It was also found that inhibition by neplanocin A does not involve dissociation of the bound NAD+ or NADH from the enzyme, but simply reduction of the NAD+ to NADH.     

Role of S-adenosylhomocysteine hydrolase in adenosine-induced apoptosis in HepG2 cells

Adenosine has been shown to initiate apoptosis through different mechanisms: (i) activation of adenosine receptors, (ii) intracellular conversion to AMP and stimulation of AMP-activated kinase, (iii) conversion to S-adenosylhomocysteine (AdoHcy), which is an inhibitor of S-adenosylmethionine (AdoMet)-dependent methyltransferases. Since the pathways involved are still not completely understood, we further investigated the role of AdoHcy hydrolase in adenosine-induced apoptosis. In HepG2 cells, adenosine induced caspase-like activity and DNA fragmentation, a marker of apoptosis. These effects were potentiated by co-incubation with homocysteine or adenosine deaminase inhibitor, pentostatin, and were mimicked by inhibition of AdoHcy hydrolase by adenosine-2',3'-dialdehyde (Adox). Adenosine-induced effects were significantly inhibited by dipyridamole, an inhibitor of adenosine transporter, whereas inhibitors of adenosine kinase did not affect adenosine-induced changes. Various adenosine receptor agonists and AICAR, an activator of AMP-activated kinase, did not mimic the effect of adenosine. Thus, adenosine-induced apoptosis is likely due to intracellular action of AdoHcy and independent of AMP-activated kinase and adenosine receptors. Because elevated AdoHcy levels are associated with reduced mRNA methylation, we studied mRNA expression in Adox-treated cells by microarray analysis. Since several p53-target genes and other apoptosis-related genes were up-regulated by Adox, we conclude that AdoHcy is involved in adenosine-induced apoptosis by altering gene expression.     

Adenine nucleoside dialdehydes: potent inhibitors of bovine liver S-adenosylhomocysteine hydrolase

Various ribonucleoside 2',3'-dialdehydes, including adenosine dialdehyde, S-adenosylhomocysteine (AdoHcy) dialdehyde, and 5-(methylthio)-5'-deoxyadenosine (MTA) dialdehyde, were shown to be potent inhibitors of bovine liver AdoHcy hydrolase (EC 3.3.1.1). These ribonucleoside 2',3'-dialdehydes produce both time-dependent and concentration-dependent inactivation of the AdoHcy hydrolase. The inactivation appears to be irreversible since the enzyme activity cannot be recovered after prolonged dialysis against phosphate buffer. However, a substantial percentage of the enzyme activity could be recovered when the inactivated enzyme was dialyzed against a nitrogen buffer [e.g., tris(hydroxymethyl)aminomethane (Tris)]. This reversal of inhibition could be prevented, however, by pretreatment of the ligand-enzyme complex with sodium borohydride prior to dialysis in Tris buffer. Inclusion of substrates (e.g., adenosine or AdoHcy) afforded protection of the enzyme from the inactivation induced by the ribonucleoside 2',3'-dialdehydes. These data suggest that the bond formed between the enzyme and the inhibitor is probably a Schiff base linkage between the aldehydic functionality of the inhibitor and a protein lysinyl residue in or around the adenosine-AdoHcy binding site. When [2,8-3H]adenosine dialdehyde was used, a stoichiometry of 1.73 nmol of inhibitor bound per nmol of AdoHcy hydrolase was determined. Analysis of the kinetics of enzyme inactivation using the Ackermann-Potter approach indicates that adenosine dialdehyde is a tight-binding inhibitor, exhibiting a stoichiometry of one to two molecules of inhibitor bound to one molecule (tetramer) of enzyme and a Ki = 2.39 nM.     

Metabolism of S-adenosylhomocysteine and S-tubercidinylhomocysteine in neuroblastoma cells.

The metabolism of the methylase product inhibitor S-adenosylhomocysteine and its 7-deaza analogue S-tubercidinylhomocysteine has been studied in cultured N-18 neuroblastoma cells. The latter compound, designed to resist metabolic degradation, has been shown to be inert under the same conditions where S-adenosylhomocysteine is rapidly and extensively degraded. The product analyses elucidated by high-performance liquid chromatography indicate that the primary route of S-[8-(14)C]adenosylhomocysteine metabolism in these cells leads to adenosine. This product does not accumulate but is rapidly converted to nucleotides or oxypurines by the action of adenosine kinase and adenosine deaminase, respectively. The presence of the potent adenosine deaminase inhibitor coformycin leads to a pronounced inhibition of oxypurine formation, an increase in nucleotide formation, and a slight accumulation of the primary metabolic products adenosine and adenine.     

Synthesis and biological evaluation of halo-neplanocin A as novel mechanism-based inhibitors of S-adenosylhomocysteine hydrolase

Halogenated analogues of neplanocin A were synthesized from the key intermediate 1, among which fluoro-neplanocin A was found to be novel mechanism-based irreversible inhibitor of S-Adenosylhomocysteine hydrolase.     

Structure-activity relationship of 5'-substituted fluoro-neplanocin a analogues as potent inhibitors of S-adenosylhomocysteine hydrolase

Four 5'-substituted fluoro-neplanocin A analogues la-d were designed and synthesized, and the inhibitory activity against SAH was in the following order: NH2 > SH > F, N3, indicating a hydrogen bonding donor is essential for inhibitory activity.     

Exploration of a fundamental substituent effect of alpha-ketoheterocycle enzyme inhibitors: Potent and selective inhibitors of fatty acid amide hydrolase

A series of C4 substituted alpha-ketooxazoles were examined as inhibitors of the serine hydrolase fatty acid amide hydrolase in efforts that further define and generalize a fundamental substituent effect on enzyme inhibitory potency. Thus, a plot of the Hammett sigma(m) versus -logK(i) provided a linear correlation (R(2)=0.90) with a slope of 3.37 (rho=3.37), that is of a magnitude that indicates that of the electron-withdrawing character of the substituent dominates its effects (a one unit change in sigma(m) provides a >1000-fold change in K(i)).     

A Functional Screening of Adenosine Analogues at the Adenosine A2B Receptor: A Search for Potent Agonists

Abstract

Various adenosine analogues were tested at the adenosine A_2B receptor. Agonist potencies were determined by measuring the cyclic AMP production in Chinese Hamster Ovary cells expressing human A_2B receptors. 5′-.N-Substituted carboxamidoadenosines were most potent. 5′-N-Ethylcarboxamidoadenosine (NECA) was most active with an ECso value of 3.1 μM. Other ribose modified derivatives displayed low to negligible activity. Potency was reduced by substitution on the exocyclic amino function (N⁶) of the purine ring system. The most active N⁶-substituted derivative N⁶-methyl-NECA was 5 fold less potent than NECA. C8-and most C2-substituted analogues were virtually inactive. 1-Deaza-analogues had a reduced potency, 3-and 7-deazaanalogues were not active.     

APEC,An A2-Selective Adenosine Agonist,is a More Potent Locomotor Depressant Than N6-Cyclohexyladenosine

Abstract

The locomotor depressant effects of APEC are due to activation of central A2 adenosine receptors, while the depressant effects of NECA are dependent primarily on A₁-receptor activation. A variety of potent A₁-antagonists, including conjugates of the XAC, were screened as antagonists; 2-thio-CPX and CPT reversed effects of CHA, but not of APEC.     

Adenosine metabolism in a rat hippocampal slice preparation: incorporation into S-adenosylhomocysteine

Abstract: The incorporation of [¹⁴C]adenosine into various metabolites was studied in a hippocampal slice preparation in order to assess the extent of adenosine metabolism via synthesis of S -adenosylhomocysteine, a potent inhibitor of transmethylation reactions. Highest incorporation of ¹⁴C occurred into nucleotides, with only a few percent being recovered in inosine + hypoxanthine, S -adenosylhomocysteine, and the free adenosine pool. Labeling of S -adenosylhomocysteine did not significantly increase with higher concentrations of added adenosine despite greater accumulation of free [¹⁴C]adenosine in the tissue. Addition of l -homocysteine significantly increased the labelling of S -adenosylhomocysteine. The results indicate that S -adenosylhomocysteine synthesis is a minor pathway of adenosine metabolism in brain tissue under steady-state conditions. Further, changes in adenosine concentration, without a concomitant change in l -homocysteine availability, are unlikely to lead to a significant accumulation of S -adenosylhomocysteine. S -Adenosylhomocysteine is therefore not likely to play a significant role in mediating the biological effects of adenosine in the CNS via inhibition of transmethylations.     

Effects of the S-adenosylhomocysteine hydrolase inhibitors 3-deazaadenosine and 3-deazaaristeromycin on RNA methylation and synthesis

The effects of 3-deazaaristeromycin and 3-deazaadenosine on RNA methylation and synthesis were examined in the mouse macrophage cell line, RAW264. S-Adenosylhomocysteine accumulated in cells incubated with 3-deazaaristeromycin while S-3-deazaadenosylhomocysteine was the major product in cells incubated with 3-deazaadenosine and homocysteine thiolactone. RNA methylation was inhibited to a similar extent by the accumulation of either S-adenosylhomocysteine or S-3-deazaadenosylhomocysteine, with S-adenosylhomocysteine being a slightly better inhibitor. In mRNA, the synthesis of N6-methyladenosine and N6-methyl-2'-O-methyladenosine were inhibited to the greatest extent, while the synthesis of 7-methylguanosine and 2'-O-methyl nucleosides were inhibited to a lesser extent. Incubation of cells with 100 microM 3-deazaaristeromycin or with 10 microM 3-deazaadenosine and 50 microM homocysteine thiolactone produced little inhibition of mRNA synthesis, even though mRNA methylation was inhibited. In contrast, mRNA synthesis was greatly inhibited by treatment of cells with 100 microM 3-deazaadenosine and the inhibition of synthesis was not correlated with an inhibition of methylation.     

Genetics of human S-adenosylhomocysteine hydrolase. A new polymorphism in man

Summary A specific staining procedure for the demonstration of S-adenosylhomocysteine hydrolase (SAAH, EC 3.3.1.1) is given. The enzyme has a broad tissue distribution and is also present in erythrocytes. The SAHH gene is polymorphic in the population of southwest Germany with two common alleles: SAHH ^*1=0.96 and SAHH ^*1=0.04. Family studies resulted in the expected segregation ratios. No evidence for close linkage with a total of 25 marker loci was found. But information from human mouse somatic-cell hybrids led to the localization of the SAHH gene to human chromosome 20, thereby confirming the findings of Hershfield and Francke (1982).Dedicated to Professor Dr. P. E. Becker on the occasion of his 75th birthday     

Protection from MPTP-induced neurotoxicity in differentiating mouse N2a neuroblastoma cells

We have shown previously that subcytotoxic concentrations of MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) inhibit axon outgrowth and are associated with increased neurofilament heavy chain (NF-H) phosphorylation in differentiating mouse N2a neuroblastoma cells while higher doses (> 100 microM) cause cell death. In this work we assessed the ability of potential neuroprotective agents to alleviate both MPTP-induced cell death (cytotoxicity) and MPTP-induced NF-H phosphorylation/reduction in axon outgrowth (neurotoxicity) in N2a cells induced to differentiate by dbcAMP. The neurotoxic effects of MPTP occurred in the absence of significant alterations in energy status or mitochondrial membrane potential. The hormone oestradiol (100 microM) reduced the cytotoxic effect of MPTP, but blocked di-butyryl cyclic AMP (dbcAMP)-induced differentiation, i.e. axon outgrowth. Both the cytotoxic and neurotoxic effects of MPTP were reduced by the monoamine oxidase (MAO) inhibitors deprenyl and, to a lesser extent, clorgyline. Alleviation of both neurotoxicity and cytotoxicity was also achieved by conditioned medium derived from rat C6 glioma cells. In contrast, whilst the p38 MAP kinase inhibitor, SB202190, protected cells against MPTP-induced neurotoxicity, it could not maintain cell viability at high MPTP exposures. In each case neuroprotection involved maintenance of the differentiating phenotype linked with attenuation of NF-H hyper-phosphorylation; the latter may represent a mechanism by which neuronal cells can moderate MPTP-induced neurotoxicity. The use of a simplified neuronal cell model, which expresses subtle biochemical changes following neurotoxic insult, could therefore provide a valuable tool for the identification of potential neuroprotective agents.     

Growth and metabolism of fucosylated plasma-membrane glycoproteins in mouse neuroblastoma N2a cells

The presence of 1.0mm-dibutyryl cyclic AMP (N(6),O(2')-dibutyryladenosine 3':5'-cyclic monophosphate) and 1.5mm-theophylline completely inhibits the growth of mouse neuroblastoma N2a cells by 24-36h. When compared with N2a cultures without inhibitors (controls), the proportion of cells in S phase, measured by radioautography with [(3)H]-thymidine, was decreased from 55 to 12%. In addition, the presence of the inhibitors decreased apparent [(3)H]fucose incorporation into glycoproteins by 50%, and removing the inhibitors resulted in a rapid recovery of both DNA synthesis and glycoprotein metabolism. Measurement of intracellular acid-soluble radioactive fucose revealed that decreased fucose uptake could account for the apparent change in incorporation. Removing dibutyryl cyclic AMP and theophylline from the medium resulted in a rapid uptake of radioactive fucose to within control values, which illustrated that the inhibitors decreased transport of the carbohydrate, although the cells remained viable. Treatment with dibutyryl cyclic AMP and theophylline also reversibly inhibited glycoprotein degradation. Plasma membranes isolated from growing cells and from growth-inhibited cells labelled with [(14)C]fucose and [(3)H]fucose respectively were co-electrophoresed on sodium dodecyl sulphate/polyacrylamide gels. These displayed no apparent differences in synthesis of specific membrane glycoproteins. Electrophoresis of plasma membranes isolated from cultures pulse-chased with [(14)C]fucose and [(3)H]fucose was used to discern turnover patterns of specific plasma-membrane glycoproteins. High-molecular-weight glycoproteins exhibited rapid rates of turnover in membranes from growing cells, but moderate turnover rates in growth-inhibited cells and cells reversed from growth inhibition. These data indicate that growth arrest of N2a cells results in alterations in the metabolic turnover of plasma-membrane glycoproteins.     

Trypanosoma cruzi: molecular cloning and characterization of the S-adenosylhomocysteine hydrolase

S-Adenosylhomocysteine (AdoHcy) hydrolase has emerged as an attractive target for antiparasitic drug design because of its role in the regulation of all S-adenosylmethionine-dependent transmethylation reactions, including those reactions crucial for parasite replication. From a genomic DNA library of Trypanosoma cruzi, we have isolated a gene that encodes a polypeptide containing a highly conserved AdoHcy hydrolase consensus sequence. The recombinant T. cruzi enzyme was overexpressed in Escherichia coli and purified as a homotetramer. At pH 7.2 and 37 degrees C, the purified enzyme hydrolyzes AdoHcy to adenosine and homocysteine with a first-order rate constant of 1 s(-1) and synthesizes AdoHcy from adenosine and homocysteine with a pseudo-first-order rate constant of 3 s(-1) in the presence of 1 mM homocysteine. The reversible catalysis depends on the binding of NAD(+) to the enzyme. In spite of the significant structural homology between the parasitic and human AdoHcy hydrolase, the K(d) of 1.3 microM for NAD(+) binding to the T. cruzi enzyme is approximately 11-fold higher than the K(d) (0.12 microM) for NAD(+) binding to the human enzyme.     

Binding partners for the myelin-associated glycoprotein of N2A neuroblastoma cells

The myelin-associated glycoprotein (MAG) has been proposed to be important for the integrity of myelinated axons. For a better understanding of the interactions involved in the binding of MAG to neuronal axons, we performed this study to identify the binding partners for MAG on neuronal cells. Experiments with glycosylation inhibitors revealed that sialylated N-glycans of glycoproteins represent the major binding sites for MAG on the neuroblastoma cell line N2A. From extracts of [3H]glucosamine-labelled N2A cells several glycoproteins with molecular weights between 20 and 230 kDa were affinity-precipitated using immobilised MAG. The interactions of these proteins with MAG were sialic acid-dependent and specific for MAG.