Synthesis of carbocyclic nucleosides and their SAH hydrolase inhibitory activities.

The cellular enzyme S-adenosyl-L-homocysteine (SAH) hydrolase has emerged as a target enzyme for the molecular design of anti-viral agents. Recently, SAH hydrolase has been considered as an attractive target in parasite chemotherapy for malaria. We report synthesis of several carbocyclic purine nucleosides and their inhibitory activities against human and malaria recombinant SAH hydrolases.     

Confirmation of a ping-pong mechanism for S-adenosyl-L-methionine:magnesium protoporphyrin methyltransferase of etiolated wheat by an exchange reaction

An exchange reaction between unlabeled S-adenosyl-L-methionine and radiolabeled S-adenosyl-L-homocysteine has been used to confirm the occurrence of a ping-pong mechanism in S-adenosyl-L-methionine:magnesium protoporphyrin methyltransferase of etiolated wheat. The enzyme, S-adenosyl-L-homocysteine hydrolase, has been used to prepare radiolabeled S-adenosyl-L-homocysteine from labeled adenosine and DL-homocysteine. The exchange reaction was accomplished with a methyltransferase preparation purified by affinity chromatography on hemin-linked Sepharose 4B, and radioactivity was exchanged into unlabeled S-adenosyl-L-methionine to an extent of 70% of the theoretical maximum value.     

The mechanism of inhibition of S-adenosyl-L-homocysteine hydrolase by fluorine-containing adenosine analogs

(Z)-4',5'-Didehydro-5'-deoxy-5'-fluoroadenosine (I), 5'-deoxy-5'-difluoroadenosine (II), and 4',5'-didehydro-5'-deoxy-5'-fluoroarabinosyl-adenosine (III) are inhibitors of rat liver S-adenosyl-L-homocysteine hydrolase. Compounds I and II are time-dependent and irreversible inhibitors of the enzyme. Both I and II are oxidized by E.NAD to produce E.NADH, and fluoride anion is formed in the inactivation reaction (0.7 to 1.0 mole fluoride/mole of enzyme subunit, and 1.7 moles fluoride/mole of enzyme subunit from I and II, respectively). The enzyme is stoichiometrically labeled with [8-3H]-I, but the label is lost upon denaturation of the protein either with or without treatment of the labeled complex with sodium borohydride. The compound III, the arabino derivative of I, is a competitive inhibitor of the enzyme. The mechanism of the inhibition of S-adenosyl-L-homocysteine hydrolase by these inhibitors is discussed.     

Purification and properties of recombinant Plasmodium falciparum S-adenosyl-L-homocysteine hydrolase

Recombinant S-adenosyl-L-homocysteine (SAH) hydrolase of the malaria parasite Plasmodium falciparum was expressed in Escherichia coli, purified to homogeneity and characterized. Comparison of the malaria parasite SAH hydrolase with that derived from the human gene indicated marked differences in kcat values. The values of both forward and reverse reactions of P. falciparum SAH hydrolase are more than 21-fold smaller than those of the human enzyme. Km values of the parasite and human SAH enzymes are 1.2 and 7.8 microM, respectively. On the other hand, IC50 values of neplanocin A, a strong inhibitor of SAH hydrolase and a growth inhibitor of P. falciparum, are 101 nM for the parasite enzyme and 47 nM for human enzyme. P. falciparum SAH hydrolase has been thought to be a target for a chemotherapeutic agent against malaria. This study may make it possible to develop a specific inhibitor for the parasite SAH hydrolase.     

Synthesis of an alpha-aminophosphonate nucleoside as an inhibitor of S-adenosyl-L-homocysteine hydrolase

A phosphonic acid analogue of S-adenosyl-L-homocysteine was prepared by a novel method and the epimeric mixture separated. Preliminary studies indicate that each epimer causes time-dependent inactivation of S-adenosyl-L-homocysteine hydrolase, however each presented distinct kinetic characteristics.     

Purification and properties of S-adenosyl-L-methionine: caffeic acid O-methyltransferase from leaves of spinach beet (Beta vulgaris L).

1. An enzyme catalysing the methylation of caffeic acid to ferulic acid, using S-adenosyl-L-methionine as methyl donor, has been extracted from leaves of spinach beet and purified 75-fold to obtain a stable preparation. 2. The enzyme showed optimum activity at pH 6.5, and did not require the addition of Mg2+ for maximum activity. 3. It was most active with caffeic acid, but showed some activity with catechol, protocatechuic acid and 3,4-dihydroxybenzaldehyde. The Km for caffeic acid was 68 muM. 4. 4. The Km for S-adenosyl-L-methionine was 12.5 muM. S-Adenosyl-L-homocystein (Ki = 4.4 muM) was a competitive inhibitor of S-adenosyl-L-methionine. 5. The synthesis of S-adenosyl-L-homocysteine from adenosine and L-homocysteine and its consequent effect on caffeic acid methylation were demonstrated with a partially-purified preparation from spinach-beet leaves, which possessed both S-adenosyl-L-homocysteine hydrolase (EC 3.3.1.1) and adenosine nucleosidase (EC 3.2.2.7) activities. This preparation was also able to catalyse the rapid breakdown of S-adenosyl-L-homocysteine to adenosine and adenine; the possible significance of this reaction in relieving the inhibition of caffeic acid methylation by S-adenosyl-L-homocystein is discussed.     

Affinity-chromatographic purification of S-adenosyl-L-homocysteine hydrolase. Some properties of the enzyme from rat liver.

S-Adenosyl-L-homocysteine hydrolase has been purified to apparent homogeneity from rat liver by means of affinity chromatography on 8-(3-aminopropylamino)adenosine linked to Sepharose. The purified enzyme was free from adenosine kinase and adenosine deaminase activities and was homogeneous on SDS/polyacrylamide-gel electrophoresis which gave a subunit mol.wt. of 47 000. The native enzyme showed some microheterogeneity on polyacrylamide-gel electrophoresis under increased-resolution conditions but was homogeneous on isoelectric focusing (pI 5.6). The molecular weight of the native enzyme was about 220 000 as judged by pore-gradient electrophoresis. The native enzyme bound adenosine tightly and showed Km values of 0.6 microM, 0.9 microM and 60 microM for adenosine, S-adenosyl-L-homocysteine and L-homocysteine respectively. The enzyme was rapidly inactivated when incubated in the presence of adenosine, S-adenosyl-L-homocysteine or several adenosine derivatives or analogues. Inactivation took place both at 0 and 37 degrees C. Freezing in the absence of glycerol resulted in the appearance of dissociation products of the oligomeric protein. Multimer formation was observed at low thiol concentrations.     

Purification of S-adenosyl-L-homocysteine hydrolase from Dictyostelium discoideum: reversible inactivation by cAMP and 2'-deoxyadenosine

S-Adenosyl-L-homocysteine hydrolase from Dictyostelium discoideum has been purified to homogeneity. It is composed of four subunits, each with a molecular mass of 47,000. In the hydrolysis direction, the enzyme has a pH optimum of 7.5, a Km for S-adenosyl-L-homocysteine (SAH) of 6 microM, and a Vmax of 0.22 mumol min-1 mg-1. In the synthesis direction, the pH optimum is 8.0, the Km for adenosine is 0.4 microM, and the Vmax is 0.30 mumol min-1 mg-1. Although the enzyme binds beta-nicotinamide adenine dinucleotide, as well as adenosine 3',5'-cyclic monophosphate and 2'-deoxyadenosine, these ligands have no effect on enzymatic activity when added to the assay mixture. However, preincubation of SAH hydrolase with NAD+ results in a 25% activation of the enzyme. In addition, this ligand has a striking effect on subunit-subunit interactions, as shown by stabilization of quaternary structure during polyacrylamide gel electrophoresis. Preincubation with cAMP or 2'-deoxyadenosine inactivates the enzyme. Although in both cases the activity is restored upon further incubation with NAD+, we show that inactivation by these two ligands proceeds by different mechanisms. NAD+-reversible inactivation by cAMP and 2'-deoxyadenosine was also observed with the SAH hydrolase from rabbit erythrocytes. Thus, these previously unreported properties of SAH hydrolase also occur with mammalian enzymes and are not restricted to D. discoideum.     

Synthesis of S-adenosyl-L-homocysteine hydrolase inhibitors and their biological activities.

Several nucleosides have been prepared as a possible inhibitor of human S-adenosyl-L-homocysteine (SAH) hydrolase for the development of anti-viral agents. Recently, SAH hydrolase has been considered as an attractive target for parasite chemotherapy for malaria. We report synthesis of several nucleosides including carbocyclic nucleosides and their inhibitory activities against recombinant malaria and human SAH hydrolases.     

9-[(2'S,3'S)-3'-formyl-2',3'-dihydroxypropyl]adenine: a facile affinity-labeling probe of human S-adenosyl-L-homocysteine hydrolase.

Treatment of human recombinant S-adenosyl-L-homocysteine (SAH) hydrolase with 9-[(2'S,3'S)-3'-formyl-2',3'-dihydroxypropyl]adenine (FDHPA) caused irreversible inactivation in a time- and concentration-dependent manner (Ki = 8.8 microM, k(inact) = 0.09 min(-1)). FDHPA behaved as a facile affinity-labeling probe of SAH hydrolase.     

Synthesis and antiviral evaluation against Vaccinia virus of new N¹-oxide analogues of 5'-noraristeromycin

New N1-benzyl esters of N1-oxide analogues of 5'-noraristeromycin were synthesized and tested as potential inhibitors of S-adenosyl-L-homocysteine hydrolase in Vaccinia virus infected cell systems.     

Synthesis of novel 4'-modified neplanocin A analogues and their inhibitory activity against S-adenosyl-L-l-homocysteine hydrolase

A new approach was developed for the synthesis of 4'-modified neplanocin A analogues, as potential inhibitors against S-adenosyl-L-homocysteine hydrolase. The vinylstannane 13, a key intermediate in the present approach, was prepared by radical-mediated sulfur-extrusive stannylation.     

Synthesis of 2-modified aristeromycins and their analogs as potent inhibitors against Plasmodium falciparum S-adenosyl-L-homocysteine hydrolase

2-Modified aristeromycin derivatives and their related analogs were synthesized to investigate their inhibitory activity against human and Plasmodium falciparum S-adenosyl-L-homocysteine hydrolase (PfSAHH). 2-Fluoroaristeromycin showed a strong inhibitory activity against PfSAHH selectively and complete resistance to adenosine deaminase.     

Synthesis of 4'-modified noraristeromycins to clarify the effect of the 4'-hydroxyl groups for inhibitory activity against S-adenosyl-L-homocysteine hydrolase

4'-Modified noraristeromycin (NAM) analogs, 4'-sulfo-, 4'-sulfamoy, 4'-azido and 4'-amino-NAM, were systematically synthesized. The inhibitory activities of these analogs and related compounds against Plasmodium falciparum and human S-adenosyl-L-homocysteine hydrolase were investigated.     

Role of Adenosine Kinase in the Biological (Antiviral and Anticellular) Activities of Adenosine Analogues

Abstract

A paired adenosine kinase-positive/adenosine kinase-negative cell system is proposed to distinguish those adenosine analogues that need to be phosphorylated to exert their biological effects from those that are mainly targeted at S-adenosyl-L-homocysteine hydrolase.     

A colorimetric assay for S-adenosylhomocysteine hydrolase

A colorimetric method for S-adenosyl-L-homocysteine hydrolase (SAHase) which uses S-adenosyl-L-homocysteine (SAH) as substrate is described. This method involves the hydrolytic conversion of SAH into adenosine (ADO) and L-homocysteine (HCY). The formation of HCY is quantified using Ellman's reagent and spectrophotometrical measured at 412 nm. Under these assay conditions, the product was followed continuously in a facile and quantitative manner until substrate conversion was complete. This method is an easy, cheap and shorter alternative to more complex methods and it is applicable to routine clinical analysis and in the assay and development of new S-nucleosidylhomocysteines to be used as therapeutic compounds.     

The antiviral drug ribavirin is a selective inhibitor of S-adenosyl-L-homocysteine hydrolase from Trypanosoma cruzi

Ribavirin (1,2,4-triazole-3-carboxamide riboside) is a well-known antiviral drug. Ribavirin has also been reported to inhibit human S-adenosyl-L-homocysteine hydrolase (Hs-SAHH), which catalyzes the conversion of S-adenosyl-L-homocysteine to adenosine and homocysteine. We now report that ribavirin, which is structurally similar to adenosine, produces time-dependent inactivation of Hs-SAHH and Trypanosoma cruzi SAHH (Tc-SAHH). Ribavirin binds to the adenosine-binding site of the two SAHHs and reduces the NAD(+) cofactor to NADH. The reversible binding step of ribavirin to Hs-SAHH and Tc-SAHH has similar K(I) values (266 and 194 microM), but the slow inactivation step is 5-fold faster with Tc-SAHH. Ribavirin may provide a structural lead for design of more selective inhibitors of Tc-SAHH as potential anti-parasitic drugs.     

Unexpected inhibition of S-adenosyl-L-homocysteine hydrolase by a guanosine nucleoside

A series of shape-modified flexible nucleosides ('fleximers', 1, 2, and 3) was modeled, synthesized and subsequently assayed against S-adenosyl-L-homocysteine hydrolase (SAHase). No inhibitory activity was observed for the adenosine fleximer, which served as a substrate, but moderate inhibitory activity was exhibited by the guanosine fleximers. This is the first known report of a guanosine nucleoside analogue possessing activity against SAHase.