Calf intestine adenosine deaminase. Substrate specificity

Simon, Lionel N.; Bauer, Randy J.; Tolman, Richard L.; Robins, Roland K.

doi:10.1021/bi00805a018

Cited by 69 publications

(29 citation statements)

References 10 publications

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“…While we have previously demonstrated (Marquez and colleagues) that this enzyme efficiently deaminates nucleoside analogs in the north conformation (43)(44)(45)(46), the kinetic analysis of ADA that we report here shows that the presence of a substituent at the 2 position of the adenine base makes these derivatives poor substrates for ADA. These results agree with previous studies reporting that 2-halo-substituted compounds are not efficiently deaminated by ADA (29,(31)(32)(33). The mechanism by which 2-halo substitutions affect ADA activity does not appear to involve steric interactions in the ADA binding pocket (Fig.…”

Section: Discussionsupporting

confidence: 92%

“…The enzyme adenosine deaminase (ADA), which is present at high concentrations in human serum, is involved in the catabolism of dA and its analogs (27,28). ADA is responsible for the deamination of adenosine or deoxyadenosine analogs to inosine-or deoxyinosine-based products (27,(29)(30)(31)(32)(33) and can therefore affect the activation pathway and intracellular concentration of dA-based compounds. For example, ADA converts dideoxyadenosine (ddA) to ddI, leading to a different and indirect activation pathway for ddA (28,34,35).…”

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confidence: 99%

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Effects of Substitutions at the 4′ and 2 Positions on the Bioactivity of 4′-Ethynyl-2-Fluoro-2′-Deoxyadenosine

Kirby

Michailidis

Fetterly

et al. 2013

Antimicrob Agents Chemother

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i Nucleos(t)ide reverse transcriptase inhibitors (NRTIs) form the backbone of most anti-HIV therapies. We have shown that 4=-ethynyl-2-fluoro-2=-deoxyadenosine (EFdA) is a highly effective NRTI; however, the reasons for the potent antiviral activity of EFdA are not well understood. Here, we use a combination of structural, computational, and biochemical approaches to examine how substitutions in the sugar or adenine rings affect the incorporation of dA-based NRTIs like EFdA into DNA by HIV RT and their susceptibility to deamination by adenosine deaminase (ADA). Nuclear magnetic resonance (NMR) spectroscopy studies of 4=-substituted NRTIs show that ethynyl or cyano groups stabilize the sugar ring in the C-2=-exo/C-3=-endo (north) conformation. Steady-state kinetic analysis of the incorporation of 4=-substituted NRTIs by RT reveals a correlation between the north conformation of the NRTI sugar ring and efficiency of incorporation into the nascent DNA strand. Structural analysis and the kinetics of deamination by ADA demonstrate that 4=-ethynyl and cyano substitutions decrease the susceptibility of adenosinebased compounds to ADA through steric interactions at the active site. However, the major determinant for decreased susceptibility to ADA is the 2-halo substitution, which alters the pK a of N1 on the adenine base. These results provide insight into how NRTI structural attributes affect their antiviral activities through their interactions with the RT and ADA active sites. There are 10 nucleos(t)ide reverse transcriptase inhibitors (NRTIs) that are currently approved for the treatment of human immunodeficiency virus type 1 (HIV-1) infections (1-5). Several more nucleoside analog drugs are approved or being studied for the treatment of viruses, such as herpes simplex virus (HSV), hepatitis C virus (HCV), and hepatitis B virus (HBV), or as anticancer agents (6-10). NRTIs are among the most effective anti-HIV drugs. All approved anti-HIV NRTIs lack a 3=-hydroxyl moiety and, thus, act as chain terminators following their incorporation by the viral reverse transcriptase (RT) into the nascent DNA chain. However, the absence of a 3=-OH, while essential for the inhibition of DNA synthesis, also imparts detrimental properties to these inhibitors, including reduced intracellular phosphorylation to the active triphosphate form and reduced RT binding affinity (11). Prolonged exposure to NRTI-based treatments causes mitochondrial toxicity (12-14) and leads to the development of NRTI resistance mutations (15-18), giving rise to complications in the treatment of HIV-infected patients.Ideally, an NRTI should have a strong binding affinity for the RT target, a high barrier for the development of resistance, and low toxicity. We have reported that a series of 4=-substituted nucleosides in which the 3=-OH is retained has exceptional inhibitory activity against HIV-1 RT (19). Among these compounds, 4=-ethynyl-2-fluoro-2=-deoxyadenosine (EFdA) is a highly active RT inhibitor that prevents translocation of the nucleic acid from the n...

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Section: Discussionsupporting

confidence: 92%

mentioning

confidence: 99%

Effects of Substitutions at the 4′ and 2 Positions on the Bioactivity of 4′-Ethynyl-2-Fluoro-2′-Deoxyadenosine

Kirby

Michailidis

Fetterly

et al. 2013

Antimicrob Agents Chemother

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“…Furthermore, 2ClAdo is a low potency inhibitor of adenosine deaminase (19), an enzyme that regulates intracellular nucleotide concentrations. However, the effects of 2ClAdo could not be mimicked by N-ethylcarboxamidoadenosine, a purinergic P 1 agonist with similar receptor potencies to 2ClAdo, nor by reversible (erythro-9-(2-hydroxy-3-nonyl)adenine) or irreversible (deoxycoformycin) adenosine deaminase inhibitors ( Fig.…”

Section: Resultsmentioning

confidence: 99%

Interruption of Escherichia coli Heat-stable Enterotoxin-induced Guanylyl Cyclase Signaling and Associated Chloride Current in Human Intestinal Cells by 2-Chloroadenosine

Parkinson

Alekseev

Gómez

et al. 1997

Journal of Biological Chemistry

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Diarrhea induced by current, demonstrating that inhibition of Cl؊ flux reflected selective disruption of ligand stimulation of GCC rather than the chloride channel itself. Thus, the components required for adenine nucleotide inhibition of GCC signaling are present in intact mammalian cells, establishing the utility of this pathway to elucidate the mechanisms regulating ST-dependent guanylyl cyclase signaling and intestinal fluid homeostasis. In addition, these data suggest that the adenine nucleotide inhibitory pathway may be a novel target to develop antisecretory therapy for enterotoxigenic diarrhea.Guanylyl cyclase C (GCC), 1 the receptor for Escherichia coli heat-stable enterotoxin (ST a ) expressed in intestinal mucosa cells, is a member of the receptor guanylyl cyclase family that possesses receptor and catalytic domains on a single transmembrane protein (1, 2). Occupancy by ST a of the extracellular receptor domain induces catalytic conversion of intracellular GTP to cyclic GMP (cGMP), resulting in sequential alterations in epithelial cell chloride flux, electrolyte and fluid secretion, and diarrhea (3-7). Interventions that specifically interrupt the ST a -induced GCC-mediated signal sequence have not been defined. In cell-free systems, GCC is allosterically inhibited by 2-substituted adenine nucleotides (8, 9). Yet, the impermeance of intact cells to phosphorylated nucleotides and the absence of endogenous 2-substituted nucleotides has precluded the disruption of ST a -induced signaling in intestinal cells through this inhibitory pathway. However, intestinal cells express transporters, which carry 2-substituted nucleosides into the cytosol, and adenosine kinase, which catalyzes conversion of 2-substituted nucleosides into 2-substituted nucleotides (10). The present studies examine whether that mechanism can be exploited to interrupt transmembrane signaling and alterations in chloride flux induced by ST a in intact intestinal epithelial cells. EXPERIMENTAL PROCEDURESCyclic GMP Accumulation in Intact Cells-Caco 2 cells, well differentiated human colon carcinoma cells, were seeded in 24-well plates, allowed to reach confluence, and grown for an additional 14 -21 days to ensure differentiation of these cells into colonic enterocytes. HEK293 cells, human embryonic kidney cells expressing recombinant GCC, were seeded in 24-well plates, allowed to reach confluence, and used for assays at least 5 days after seeding (1, 11). Cells were incubated in OPTI-MEM serum-free media (Life Technologies, Inc.) (0.5 ml/well) containing indicated concentrations of the test substances for the given period of time. Cells were washed three times with OPTI-MEM, then incubated in OPTI-MEM (0.2 ml/well) containing 0.12 mM isobutylmethylxanthine to inhibit endogenous phosphodiesterases for 10 min. ST a was added to a final concentration of 0.5 M for 10 min. Trichloroacetic acid (0.2 ml of 12% solution) was added to the wells to lyse the cells and terminate the reaction. Well contents were collected and centrifuged 15 min in a micro...

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“…However, it is known that 3-deazaadenosine is neither a substrate nor an inhibitor of adenosine deaminase (10), that 3-deazaadenosine nucleotideis a poorer substrate for polymerase than is the parent compound (11), and that 3-deaza-6-methylthiopurine ribonucleotide is 1/1000 as inhibitory as its parent compound (2). These properties of the 3-deaza analogs parallel somewhat those of the 8-substituted nucleoside and it is natural to consider the same explanations for the inactivity of the 3-deaza analogs already given for the 8-substituted derivatives (15)(16)(17)(18)(19), i.e., that substrates for certain enzymes must be present in the anti conformation. In this regard the 3-deaza analogs may prove to be useful models for assessing the hypothesis of an optimized rotamer for enzymic recognition once there is assurance that N3, per se, is not used to bind the nucleoside to the enzyme.…”

mentioning

confidence: 84%

Comparative study by circular dichroism of the conformation of deazapurine nucleosides and that of common purine nucleosides.

Miles¹,

Townsend²,

Redington³

et al. 1976

Proc. Natl. Acad. Sci. U.S.A.

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Purine nucleoside analogs modified by replacement of the nitrogen atom at the 3 position by a CH group give a characteristic circular dichroism curve that is not substantially modified by chemical substitution at the 8 position.Since it is rather well established that 8-substituted purine nucleosides are predominantly in the syn conformation in aqueous solution, it follows that the 3-deazapurine nucleosides, whether substituted at position 8 or not, also favor the syn conformation. These data are in sharp contrast to the circular dichroism data obtained on 8-halogenated and 8-alkylated derivatives of adenosine and guanosine, which give circular dichroism profiles substantially different from those obtained on the parent compounds. Certain purine-nucleoside-utilizing enzymes fail to interact effectively with either the unsubstituted 3-deaza analogs or the 8-substituted derivatives of adenosine and guanosine. The hypothesis recently given that the inactivity of the 8-substituted derivatives springs from their syn-conformational preference is tentatively accepted to explain the inactivity of the 3-deaza analogs. Included in the search for purine analogs active against bacteria, tumor cells, and viruses have been nucleosides and nucleotides modified by replacement of the nitrogen atom at the 3 position by a CH group (1-9). This modification has, however, rarely led to recognized biological activity, giving rise to speculation over the biological significance of N3 (2,3,(8)(9)(10)(11). It has been suggested several times (12)(13)(14), and as early as 1962, that one role of N3 is to stabilize an optimal glycosyl conformation at the enzyme surface by internally hydrogen bonding with the ribose ring. Direct interaction between N3 and the enzymes has also been proposed (2).Unfortunately the "active" conformation of a substrate molecule and its energy are at present beyond the scrutiny of any experimental technique. In aqueous solution intermolecular hydrogen bonding prevails; nevertheless the discovery of the preferred solution conformation is relevant to either the internal or the external hydrogen bonding role of N3, for the energy of the "active" conformation relative to the stable solution conformation must influence the overall kinetics and energetics of complex formation. A comparative study of the solution conformations of purine nucleosides and their 3-deaza analogs has not been made despite the obvious importance of understanding the conformational consequences of replacing nitrogen by carbon. This paper presents a preliminary report on a comparative study of the solution conformation of purine nucleosides and their 3-deaza analogs. An analysis of the circular dichroism data obtained so far shows rather conclusively that 1,3-dideazapurine nucleoside and 3-deaza purine nucleoside have strong if not exclusive preferences for the syn conformation in aqueous solution, in sharp contrast to the substantial anti population of the parent compounds. Moreover, gas phase conformations from molecular orbital calcula...

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Calf intestine adenosine deaminase. Substrate specificity

Cited by 69 publications

References 10 publications

Effects of Substitutions at the 4′ and 2 Positions on the Bioactivity of 4′-Ethynyl-2-Fluoro-2′-Deoxyadenosine

Effects of Substitutions at the 4′ and 2 Positions on the Bioactivity of 4′-Ethynyl-2-Fluoro-2′-Deoxyadenosine

Interruption of Escherichia coli Heat-stable Enterotoxin-induced Guanylyl Cyclase Signaling and Associated Chloride Current in Human Intestinal Cells by 2-Chloroadenosine

Comparative study by circular dichroism of the conformation of deazapurine nucleosides and that of common purine nucleosides.

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