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.     

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-c3 Ado), 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-c3 Ado 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 thefirst place be considered for therapeutic (or prophylactic) use against poxvirus infections, including smallpox, and hemorrhagic fever virus infections such as Ebola.     

Antiviral potency of adenosine analogues: correlation with inhibition of S-adenosylhomocysteine hydrolase

For a series of adenosine analogues a close correlation (r = 0.986) was found between their antiviral potency (against vesicular stomatitis virus) and their inhibitory effects (Ki/Km) on S-adenosylhomocysteine (AdoHcy) hydrolase; thus, in order of increasing inhibitory potency for both virus replication and AdoHcy hydrolase activity: (S)-9-(2,3-dihydroxypropyl)adenine less than (RS)-3-adenin-9-yl-2-hydroxypropanoic acid (isobutyl ester) less than carbocyclic 3-deazaadenosine less than neplanocin A. Our findings point to AdoHcy hydrolase as the target for the broad-spectrum antiviral activity of these adenosine analogues.     

Synthesis and antiviral activities of 3-deaza-3-fluoroaristeromycin and its 5′ analogues

The naturally occurring adenine based carbocyclic nucleosides aristeromycin and neplanocin A and their 3-deaza analogues have found a prominent place in the search for diverse antiviral activity agent scaffolds because of their ability to inhibit S-adenosylhomocysteine (AdoHcy) hydrolase. Following the lead of these compounds, their 3-deaza-3-fluoroaristeromycin analogues have been synthesized and their effect on S-adenosylhomocysteine hydrolase and RNA and DNA viruses determined.     

Characterization of three rabbit liver lysosomal proteinases with fructose 1,6-bisphosphatase converting enzyme activity

A large number of nucleoside analogs have been found to inactivate S-adenosylhomocysteine (AdoHcy) hydrolase in a time-dependent irreversible manner. There are two classes of these irreversible inhibitors: (A) analogs that inactivate the enzyme in a pseudofirst-order process and are devoid of any side chain at the 5′-OH group; (B) analogs that inactivate the enzyme in a time-dependent but curvilinear process, and generally have a side chain at the 5′ position. Among the more potent irreversible inhibitors are 2-chloroadenosine, 9-β-d-arabinofuranosyladenine (Ara-A), and (±)aristeromycin. Release of adenine base from adenosine or Ara-A in the presence of AdoHcy hydrolase was observed, thus supporting the proposed catalytic mechanism of AdoHcy hydrolase, that entails the transient formation of 3′-ketoadenosine during enzymatic catalysis of either the formation or hydrolysis of AdoHcy. Both Ara-A and adenosine may exert their irreversible inactivation by a suicide mechanism, but nucleosides such as 5′-iodo-5′-deoxyadenosine and 3′-deoxyadenosine are probably strictly irreversible inhibitors per se in view of the catalytic mechanism proposed for AdoHcy hydrolase. Labeling of AdoHcy hydrolase, perhaps covalent in nature, by radioactive Ara-A and adenosine was demonstrated by gel electrophoresis.     

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).     

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.     

New Neplanocin Analogues. V. A Potent Adenosylhomocysteine Hydrolase Inhibitor Lacking Antiviral Activity. Synthesis And Antiviral Activity Of 6″-Carboxylic Acid Derivatives Of Neplanocin A 1

Abstract

The 6′-carboxylic acid derivative of neplanocin A 3 was synthesized from NPA, and was converted to the corresponding methyl ester 4 and amides 5 and 6. These were evaluated for their anti-RNA-virus activities. Of the derivatives synthesized, only 5 was active against RNA viruses within the concentration range of 0.14-4.88 μg/mL. Compounds 3 and 5 showed a potent inhibitory effect on S-adenosylhomocysteine (AdoHcy) hydrolase from rabbit erythrocytes. Although a close correlation between the inhibitory effect of adenosine analogues on AdoHcy hydrolase and their antiviral potency has been demonstrated, 3 did not show any anti-RNA-virus activities.

     

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.     

Inhibition of S-adenosylhomocysteine hydrolase by purine nucleoside analogues

To find out potent inhibitors of S-adenosylhomocysteine hydrolase (SAHase), several deazaadenosine analogues synthesized in this laboratory and some naturally occurring nucleoside analogues were examined with SAHases from yellow lupin seeds and rabbit liver. Neplanocin A, an antibiotic, inhibited both enzymes more potently than aristeromycin which was also an antibiotic and known as one of the most potent inhibitors of SAHase. The 3-deazaadenine derivatives (2'-deoxy, arabinosyl, xylosyl) inactivated lupin SAHase as potent as 3-deazaadenosine. Whereas, inhibitory activities of 1-deazaadenosine, its derivatives, and 7-deazaadenosine (tubercidin) were very weak.     

Open-Chain Carbocyclic Analogs of Adenosine with Dihalovinyl Unit as Potential Inhibitors of S-Adenosyl-L-homocysteine Hydrolase

Abstract

Vinylogously extended deoxyeritadenine derivatives were synthesized as acyclic/carbocyclic analogues of the 6′-halo(homovinyl)adenosines, which are known to be potent inhibitors of S-adenosyl-L-homocysteine hydrolase. Swern oxidation of 9-[3-(t-butyldimethylsilyloxy)-4-hydroxybutyl]adenine (4) followed by Wittig olefination and desilylation gave access to ethyl 6-(adenin-9-yl)-4-hydroxy-2(E)-hexenoate (7) and 5-(adenin-9-yl)-1,1-dibromo-1-penten-3-ol (9). No inhibition of AdoHcy Hydrolase was observed with 7 and 9.     

The enantiomers of the 1′,6′-isomer of neplanocin A: Synthesis and antiviral properties

Both enantiomers of 1′,6′-isoneplanocin have been prepared from a common substituted cyclopentane epoxide in 7 steps. Both compounds were subjected to DNA and RNA viral assessments with moderate to high activity found for both towards human cytomegalovirus, measles, Ebola, norovirus, and dengue. The D-like congener also showed vaccinia and HBV effectiveness. In many of the other antiviral assays both compounds showed cytotoxicity making, in some cases, an EC₅₀ determination not possible. The S-adenosylhomocysteine hydrolase inhibitory effects showed the D-like target to be equal that of neplanocin itself and better than 3-deazaneplanocin whereas the L-like analogue was 13 to 30 times less inhibitory than 3-deazaneplanocin and neplanocin, respectively.     

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.     

Activation of collagen IV gene expression in F9 teratocarcinoma cells by 3-deazaadenosine analogs. Indirect inhibitors of methylation.

3-Deazaadenosine analogs can function as inhibitors and also as alternative substrates of S-adenosylhomocysteine (AdoHcy) hydrolase. In cells treated with the analogs, AdoHcy invariably accumulates, leading to inhibition of cellular methylation. F9 teratocarcinoma cells, stably transfected with two collagen (IV) promoter-enhancer-CAT constructs and treated with 10 microM 3-deazaadenosine, 3-deaza-(+-)-aristeromycin or 3-deazaneplanocin, showed a strong induction of CAT activities without affecting differentiation. In comparison, the same 3-deaza analogs did not affect the CAT activity in F9 cells transfected with the beta-actin promoter-CAT construct. Furthermore, Northern blot analysis of endogenous mRNA from wild-type F9 cells treated with the 3-deaza nucleosides all showed an induction of the collagen alpha 1(IV) chain mRNA. Thus, the 3-deaza analogs most likely affect DNA methylation because their results are consistent with the previous observation that the integrated collagen alpha 1(IV) promoter-enhancer constructs were activated with 5-azacytidine.     

Inhibitory Effects of 1-Deazaadenosine Analogues on HIV Replication and Adenosine Deaminase

Abstract

A series of 2′,3′-dideoxy-N⁶-(cyclo)alkyl-1-deazaadenosine derivatives were synthesized starting from 2,6-dichloro-1-deazapurine (9). The new nucleosides proved to be good inhibitors of HIV-1 replication, the most active being the 2′,3′-dideoxy-2-chloro-N⁶-cycloctyl-1-deazaadenosine (14h, ED₅₀ = 0.4 μ).     

A Convenient Synthesis of Acyclic Adenosines with an Unsaturated Side Chain by Modification of 9-(2,3-O-Isopropylidene-D-Ribityl)Adenine

Abstract

In expectation of discovering their antiviral activity, acyclic adenosine derivatives 7, 11, 12, and 16 were designed as analogs of neplanocin A (NPA) and L-eritadenine which are strong inhibitors of S-adenosyl-L-homocysteine hydrolase. The 1′,5′-seco-analog of 4′-deoxymethyl-NPA (DHCA) 7 was synthesized by dideoxygenation of 9-(2,3-O-isopropylidene-D-ribityl)adenine (2). Acyclic DHCA analogs 11 and 16 were obtained by Wittig reaction of the aldehyde 3 with Ph₃P=CHCO₂Et and Ph₃P=CHCN, respectively. Hydrolysis of the ester 11 afforded a vinylog of L-eritadenine 12. The synthesized acyclic nucleosides 7, 10, and 11 were evaluated for antiviral activity, however, none of them showed any significant antiviral activity.     

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 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.     

Synthesis of 5′-Substituted Analogues of Carbocyclic 3-Deazaadenosine as Potential Antivirals

Abstract

Various 5′-substituted analogues of carbocyclic 3-deazaadenosine (la), a potent antiviral agent, have been prepared and tested against nine viruses.