Inactivation of S-adenosylhomocysteine hydrolase by nucleosides

The irreversible inactivation of S-adenosylhomocysteine hydrolase purified from hamster and bovine liver by adenosine analogs substituted in the 5' and 2 positions has been investigated in detail. 5'-Cyano-5'-deoxyadenosine inactivates as potently as 9-beta-D-arabinofuranosyladenine (Ara-A). Substitution of the Ara-A at the 2 position by halogens or deleting N at the 3 position decreases its potency. Although weak, 2',3'-dideoxyadenosine can also inactivate the enzyme. The irreversible inactivation of the hydrolase in rat hepatocytes incubated with 2-chloroadenosine or 3-deaza-Ara-A could be demonstrated, concomitant with increases in 35S-labeled S-adenosylhomocysteine and S-adenosylmethionine in the hepatocytes.     

INACTIVATION OF S-ADENOSYL-L-HOMOCYSTEINE HYDROLASE WITH FLUORINATED ANALOGS OF 2′- AND 3′-DEOXY-5′-METHYLTHIOADENOSINE

Fluorinated analogs of 2′- and 3′-deoxy-5′-methylthioadenosine 1–4 caused irreversible inactivation of AdoHcy hydrolase. Based on the ESI-Mass spectra analysis of the inactivated enzyme with the fluorinated analog 1 a mechanism of inactivation is proposed.     

Rational Approaches to the Design of Mechanism-Based Inhibitors of S-Adenosylhomocysteine Hydrolase

Abstract

Crucial to the rational design of inhibitors of S-adenosyl-L-homocysteine (AdoHcy) hydrolase was the elucidation of its mechanism of catalysis by Palmer and Abeles (J. Biol. Chem. 254, 1217–1226, 1979). This mechanism involves an NAD⁺-dependent oxidation (oxidative activity) of the 3′-hydroxyl group of AdoHcy followed by elimination of homocysteine (Hcy) to form 4′,5′-didehydro-3′-keto-Ado. Addition of water at the 5′-position (hydrolytic activity) of this tightly bound intermediate followed by an NADH-dependent reduction results in the formation of adenosine (Ado). Many inhibitors of this enzyme have been shown to serve as substrates [e.g., 9-(trans-2-trans-3-dihydroxycyclopent-4-en-1-yl)adenine, DHCeA)] for the oxidative activity of AdoHcy hydrolase, affording the 3′-keto-derivative (e.g., 3′-keto-DHCeA), which is tightly bound to the enzyme, and converting the enzyme from its active form (NAD⁺) to its inactive form (NADH) (Type I mechanism-based inhibitors; Wolfe and Borchardt, J. Med. Chem. 34, 1521–1530, 1991). More recently, substrates [e.g., (E)-5.,6′-didehydro-6′-deoxy-6′-fluorohomoadenosine, EDDFHA] for the hydrolytic activity of AdoHcy hydrolase have been identified by our laboratories. Identification of hydrolytic substrates affords a new strategy for the design of more potent and more specific inhibitors of AdoHcy hydrolase.     

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.     

MECHANISM OF INACTIVATION OF S-ADENOSYL-L-HOMOCYSTEINE HYDROLASE BY 5′-DEOXY-5′-S-ALLENYLTHIOADENOSINE AND 5′-DEOXY-5′-S-PROPYNYLTHIOADENOSINE

5′-Deoxy-5′-S-allenylthioadenosine 1 and 5′-deoxy-5′-S-propnylthioadenosine 2, derived from adenosine, were prepared. 1 and 2 caused irreversible inactivation of AdoHcy hydrolase. ESI mass spectra analysis of the inactivated enzyme demonstrated that 1 and 2 were type II “mechanism-based” inhibitors.     

Mechanism of inactivation of S-adenosyl-L-homocysteine hydrolase by 5'-deoxy-5'-S-allenylthioadenosine and 5'-deoxy-5'-S-propynylthioadenosine

5'-Deoxy-5'-S-allenylthioadenosine 1 and 5'-deoxy-5'-S-propnylthioadenosine 2, derived from adenosine, were prepared. 1 and 2 caused irreversible inactivation of AdoHcy hydrolase. ESI mass spectra analysis of the inactivated enzyme demonstrated that 1 and 2 were type II "mechanism-based" inhibitors.     

Carbocyclic Adenosine Analogues as S-Adenosylhomocysteine Hydrolase Inhibitors and Antiviral Agents: Recent Advances

Abstract

Various carbocyclic analogues of adenosine, including aristeromycin (carbocyclic adenosine), carbocyclic 3-deazaadenosine, neplanocin A, 3-deazaneplanocin A, the 5′-nor derivatives of aristeromycin, carbocylic 3-deazaadenosine, neplanocin A and 3-deazaneplanocin A, and the 2-halo (i.e., 2-fluoro) and 6′-R-alkyl (i.e., 6′-R-methyl) derivatives of neplanocin A have been recognized as potent inhibitors of S-adenosylhomocysteine (AdoHcy) hydrolase. This enzyme plays a key role in methylation reactions depending on S-adenosylmethionine (AdoMet) as methyl donor. AdoHcy hydrolase inhibitors have been shown to exert broad-spectrum antiviral activity against pox-, paramyxo-, rhabdo-, filo-, bunya-, arena-, and reoviruses. They also interfere with the replication of human immunodeficiency virus through inhibition of the Tat transactivation process.     

Inactivation of S-adenosyl-L-homocysteine hydrolase with fluorinated analogs of 2'- and 3'-deoxy-5'-methylthioadenosine

Fluorinated analogs of 2'- and 3'-deoxy-5'-methylthioadenosine 1-4 caused irreversible inactivation of AdoHcy hydrolase. Based on the ESI-Mass spectra analysis of the inactivated enzyme with the fluorinated analog 1 a mechanism of inactivation is proposed.     

Recognition of Ribose Moiety by Adenosine (Phosphate) Deaminase from Squid Liver

An adenosine (phosphate) deaminase from the squid liver had much lower activity for 5′-deoxyadenosine than that for adenosine, 2′-, or 3′-deoxyadenosine. 3′-IMP and inosine as well as purine riboside and adenine competitively inhibited the deamination of adenosine 3′ phenylphosphonate by the enzyme, but 5′-AMP and 5′-IMP did not. The enzyme deaminated the 5′-hydroxyl terminal adenosine residue in dinucleotides and trinucleotide, but not the 3′-hydroxyl terminal one in dinucleotides. The 5′-hydroxyl group of the ribose moiety was necessary for the substrate binding and catalytic activity of the squid enzyme. These results indicated that the recognition of ribose moiety in the substrate by the squid enzyme might be intermediate between those by adenosine deaminase and adenosine (phosphate) deaminase from microorganisms.     

Adenosine dialdehyde and neplanocin A: Potent inhibitors of S-adenosylhomocysteine hydrolase in neuroblastoma N2a cells

S-Adenosylhomocysteine hydrolase (AdoHcy hydrolase, E.C. 3.3.1.1) catalyzes the metabolism of S-adenosylhomocysteine (AdoHcy) to adenosine (Ado) and homocysteine (Hcy) in mouse neuroblastoma N2a cells. AdoHcy hydrolase in N2a cells can be inhibited completely by adenosine dialdehyde (Ado dialdehyde) or neplanocin A. The inhibitory effects of Ado dialdehyde (2.5 μM) and neplanocin A (1 μM) on cellular AdoHcy hydrolase were time-dependent, with total enzyme inhibition occurring after 30 min and 15 min of incubation, respectively. The inhibition of AdoHcy hydrolase produced by Ado dialdehyde and neplanocin A persisted for up to 72 h of incubation, and was paralleled by a time-dependent increase in endogenous AdoHcy levels reaching a maximum 4-fold elevation after 8 h of incubation with Ado dialdehyde and an 11-fold increase in the neplanocin A-treated cells. This increase in AdoHcy levels produced a subsequent inhibition of S-adenosylmethionine (AdoMet)-dependent cellular methylations (e.g. protein carboxylmethylation (PCM), lipid methylation). In addition, neplanocin A was metabolically converted to the corresponding AdoMet analog, S-neplanocylmethionine (NepMet), in neuroblastoma N2a cells. NepMet reached maximum levels after 8 h of incubation of the cells with neplanocin A.     

AdoHcy hydrolase of Trichomonas vaginalis: studies of the effects of 5'-modified adenosine analogues and related 6-N-cyclopropyl derivatives

Trypanosoma brucei and Trichomonas vaginalis are both parasitic protozoans that are known to share many similar biochemical pathways. Aristeromycin, as well as 5'-iodovinyl and 5'-oxime analogues of adenosine, are potent inhibitors of AdoHcy hydrolase in T. brucei, an enzyme that catalyses the hydrolysis of AdoHcy to adenosine and L-homocysteine. To help determine the role of this enzyme in T. vaginalis, we have tested a library of 5'-modified adenosine derivatives, including 5'-deoxy-5'-(iodomethylene)-adenosine and related 6-N-cyclopropyl analogues. Our results indicate that these inhibitors are effective at inhibiting the growth of T. vaginalis, by as much as 95%.     

Apparent suicide inactivation of human lymphoblast S-adenosylhomocysteine hydrolase by 2'-deoxyadenosine and adenine arabinoside. A basis for direct toxic effects of analogs of adenosine.

Adenosylhomocysteine hydrolase from human lymphoblasts binds 2'-deoxy[3H]adenosine tightly. Binding is associated with time-dependent, saturable, irreversible inactivation of catalytic activity which occurs with first order kinetics, suggesting "suicide" inactivation. Adenine arabinoside produces similar inactivation but is more potent. These results suggest a basis for a heretofore unrecognized mechanism of action for these and other analogs of adenosine, in which toxicity results from actions of the nucleosides themselves, rather than from nucleotides to which they may be converted.     

Reactions of adenosine 5′-phosphorimidazolide with adenosine analogs on a polyuridylic acid template: The uniqueness of the 2′-3′-unsubstituted β-ribosyl system

We have studied the reactions between adenosine 5′-phosphorimidazolide and various adenosine analogs on a poly(U) template. The nucleosides were adenosine (I), 2′-deoxyadenosine (II), 3′-deoxyadenosine (III), 2′-O-methyladenosine (IV), 3′-O-methyladenosine (V), 9-β-d-xylofuranosyladenine (VI), and 9-β-d-arabinofuranosyladenine (VII). We find that the various analogs form triple helices with poly(U) which are of comparable stability, but that only the β-riboside takes part in an efficient template-directed condensation.     

A study on the inhibition of adenosine deaminase

Adenosine deaminase (ADA, EC 3.5.4.4) catalyses the irreversible deamination of adenosine and 2′-deoxyadenosine to inosine and 2′-deoxyinosine, respectively. In this study the inhibition of ADA from bovine spleen by several molecules with structure related to that of the substrate or product has been quantified. The inhibitors adenine, purine, inosine, 2-aminopurine, 4-aminopyrimidine, 4-aminopyridine, 4-hydroxypyridine and phenylhydrazine are shown to be competitive inhibitors with K_I (mM) values of 0.17, 1.1, 0.35, 0.33, 1.3, 1.8, 1.4 and 0.25, respectively. Synergistic inhibition by various combinations of molecules that imitate the structure of the substrate has never been observed. Some general conclusions are: i) the enzyme ADA from bovine spleen we have used is appropriate for kinetic studies of inhibition and mechanistic studies; it can be a reference catalytic system for the homogeneous comparison of various inhibitors; ii) this enzyme presents very rigid requirements for binding the substrate: variations in the structure of adenosine imply the loss of important interactions.     

Overexpression, purification, and characterization of S-adenosylhomocysteine hydrolase from Leishmania donovani

The gene encoding S-adenosylhomocysteine (AdoHcy) hydrolase in Leishmania donovani was subcloned into an expression vector (pPROK-1) and expressed in Escherichia coli. Recombinant L. donovani AdoHcy hydrolase was then purified from cell-free extracts of E. coli using three chromatographic steps (DEAE-cellulose chromatofocusing, Sephacryl S-300 gel filtration, and Q-Sepharose ion exchange). The purified recombinant L. donovani enzyme exists as a tetramer with a molecular weight of approximately 48 kDa for each subunit. Unlike recombinant human AdoHcy hydrolase, the catalytic activity of the recombinant L. donovani enzyme was shown to be dependent on the concentration of NAD+ in the incubation medium. The dissociation constant (Kd) for NAD+ with the L. donovani enzyme was estimated to be 2.1 +/- 0.2 microM. The Km values for the natural substrates of the enzyme, AdoHcy, Ado, and Hcy, were determined to be 21 +/- 3, 8 +/- 2, and 82 +/- 5 microM, respectively. Several nucleosides and carbocyclic nucleosides were tested for their inhibitory effects on this parasitic enzyme, and the results suggested that L. donovani AdoHcy hydrolase has structural requirements for binding inhibitors different than those of the human enzyme. Thus, it may be possible to eventually exploit these differences to design specific inhibitors of this parasitic enzyme as potential antiparasitic agents.     

John Montgomery's Legacy: Carbocyclic Adenosine Analogues as Sah Hydrolase Inhibitors with Broad-Spectrum Antiviral Activity

Ever since the S-adenosylhomocysteine (AdoHcy, SAH) hydrolase was recognized as a pharmacological target for antiviral agents (J. A. Montgomery et al., J. Med. Chem. 25:626–629, 1982), an increasing number of adenosine, acyclic adenosine, and carbocyclic adenosine analogues have been described as potent SAH hydrolase inhibitors endowed with broad-spectrum antiviral activity. The antiviral activity spectrum of the SAH hydrolase inhibitors include pox-, rhabdo-, filo-, arena-, paramyxo-, reo-, and retroviruses. Among the most potent SAH hydrolase inhibitors and antiviral agents rank carbocyclic 3-deazaadenosine (C-c³Ado), neplanocin A, 3-deazaneplanocin A, the 5′-nor derivatives of carbocyclic adenosine (C-Ado, aristeromycin), and the 2-halo (i.e., 2-fluoro) and 6′-R-alkyl (i.e., 6′-R-methyl) derivatives of neplanocin A. These compounds are particularly active against poxviruses (i.e., vaccinia virus), and rhabdoviruses (i.e., vesicular stomatitis virus). The in vivo efficacy of C-c³Ado and 3-deazaneplanocin A has been established in mouse models for vaccinia virus, vesicular stomatitis virus, and Ebola virus. SAH hydrolase inhibitors such as C-c³Ado and 3-deazaneplanocin A should in the first place be considered for therapeutic (or prophylactic) use against poxvirus infections, including smallpox, and hemorrhagic fever virus infections such as Ebola.     

Adenosine-elicited accumulation of cyclic AMP in brain slices: potentiation by agents which inhibit uptake of adenosine

The uptake and incorporation of low concentrations of radioactive adenosine into guinea pig cerebral cortical slices is effectively inhibited by dipyridamole, hexobendine, papaverine, 6-($\text{p}$-nitrobenzylthio) guanosine, 5′-deoxy-adenosine and N⁶-phenyladenosine and ineffectively inhibited by other adenosine analogs such as 2-chloroadenosine, 3′-deoxyadenosine and tubercidin or by phosphodiesterase inhibitors such as theophylline, isobutylmethylxanthine, and N, 0-dibutyrylcyclic AMP. When uptake of 10–20

adenosine is inhibited 50–70% by dipyridamole, hexobendine, papaverine or 6-($\text{p}$-nitrobenzylthio)-guanosine, the adenosine-elicited accumulation of cyclic AMP is potentiated 2–3 fold. Potentiation of the effects of low concentrations of adenosine by various agents parallels more closely their efficacy as inhibitors of adenosine uptake rather than their potency as phosphodiesterase inhibitors. Amine-elicited accumulations of cyclic AMP are enhanced by hexobendine, dipyridamole, papaverine and 6-($\text{p}$-nitrobenzylthio) guanosine and this enhancement is blocked by an adenosine antagonist, theophylline. The stimulatory effects of the adenosine analogs, 5′-deoxyadenosine, 2-chloroadenosine and N⁶-phenyladenosine are blocked by theophylline and potentiated by hexobendine. The results are compatible with the hypothesis that the specific inhibition of uptake of adenosine potentiates adenosine or amine-elicited accumulations of cyclic AMP by increasing the effective extracellular concentration of adenosine within the slice. The inhibition or stimulation of cyclic AMP accumulation by adenosine analogs is consonant with differential activities as agonist or antagonist at an extracellular adenosine receptor.     

Catalytic mechanism of S-adenosylhomocysteine hydrolase: roles of His 54, Asp130, Glu155, Lys185, and Aspl89

S-adenosylhomocysteine hydrolase (AdoHcyase) catalyzes the hydrolysis of S-adenosylhomocysteine (AdoHcy) to form adenosine and homocysteine. The crystal structure of the K185N mutated enzyme, which has weak catalytic activity (0.1%), has been determined at 2.8 A resolution and supports the previously predicted mechanism [Takata, Y., Yamada, T., Huang, Y., Komoto, J., Gomi, T., Ogawa, H., Fujioka, M., & Takusagawa, F. (2002). Catalytic mechanism of S-adenosylhomocysteine hydrolase. Site-directed mutagenesis of Asp-130, Lys-185, Asp-189, and Asn-190. J. Biol. Chem. 277, 22670-22676]. The mutated enzyme has an intermediate structure between the open and closed conformation, observed in the substrate-free enzyme and in the inhibitor complexes, respectively. H54, H300, and H352 were mutated to asparagine, respectively, to identify the roles of the histidine residues in catalysis. The kinetic data of H54N, H300N, and H354N mutated enzymes suggest that H54 is the amino acid residue that acts as a general acid/base to cleave the C5'-S(D) bond of AdoHcy. The E155Q mutated enzyme retained a large portion of the catalytic activity (31%), while the E155D mutated enzyme lost most of it (0.3%). The NADH accumulation measurements of the mutated enzymes indicated that the C3'-oxidation and the C4'-proton abstraction are a concerted event and the C5'-S(D) bond cleavage is an independent event. The C4'-proton exchange measurements indicate that the enzyme has an open conformation when AdoHcy is converted to 3'-keto-4', 5'-dehydro-Ado in the active site. With the results of this study and those of the previous studies, a detailed catalytic mechanism of AdoHcyase is described. K185 facilitates the C3'-oxidation, D130 abstracts the C4'-proton, D189, and E155 act as a communicator between the concerted C3'-oxidation and C4'-proton abstraction, and H54 plays as a general acid to cleave the C5'-S(D) bond of AdoHcy.     

Synthesis and evaluation of 4′-5′-dehydro-5′-fluoroaristeromycins as S-adenosyl-L-homocysteine (AdoHcy) hydrolase inhibitors

4′,5′-Dehydro-5′-fluoro analogs of aristeromycin were synthesized and shown to be potent inhibitors of recombinant rat liver AdoHcy hydrolase.