Adenylyl cyclase (IC50 = 3 µM)[1]
2',5'-Dideoxyadenosine targets adenylyl cyclase (adenylate cyclase), the enzyme responsible for the conversion of ATP to cyclic AMP (cAMP). It binds to the intracellular P-site of the enzyme in a non-competitive manner, meaning it does not compete with the substrate ATP for binding. The P-site is an allosteric regulatory site located on the cytoplasmic domain of adenylyl cyclase. By binding to this site, 2',5'-Dideoxyadenosine inhibits the enzyme's catalytic activity and reduces cAMP production. This inhibition is specific to certain isoforms of adenylyl cyclase and is dependent on the presence of adenine nucleotides.

2',5'-Dideoxyadenosine (10 μM, 30 minutes) inhibits the phosphorylation of GluA1 at Ser845 caused by carbachol (CCh) and decreases the generation of cAMP [3]. In 30 minutes, 10 μM 2',5'-Dideoxyadenosine phosphorylates Akt and disconnects Ser2448 phosphorylation produced by CCh [3]. The inotropic and chronotropic effects of isopropanol (8-54 pmol) β-hexanosine are substantially and reversibly blocked by 2',5'-Dideoxyadenosine (20-150 mM), just like adenosine does. up to 50% and 70%, in that order [2].
2',5'-Dideoxyadenosine exhibits potent in vitro inhibitory activity against adenylyl cyclase. It inhibits forskolin-induced activation of a cAMP-dependent reporter gene in HEK293 cells with an IC50 value of 33 µM. The compound binds to the P-site of adenylyl cyclase with an IC50 of 3 µM. As a non-competitive inhibitor, it does not interfere with the binding of the substrate ATP but instead reduces the enzyme's catalytic efficiency. The compound's activity is specific to adenylyl cyclase and does not affect other components of the cAMP signaling pathway directly. These in vitro results demonstrate that 2',5'-Dideoxyadenosine is a valuable tool for studying the regulation of cAMP production.

In this study the effects of 2',5'-dideoxyadenosine (DDA), an agonist of the intracellular adenosine binding site (P-site), on myocardial contractility, coronary resistance and cAMP-metabolism in the isolated guinea-pig heart were compared with those of adenosine. DDA (20-150 microM), like adenosine, dose dependently and reversibly inhibited the positive inotropic and chronotropic effect of beta-adrenergic stimulation with isoproterenol (8-54 pmol) up to 70% and 50%, respectively. In contrast to the known vasodilatory action of adenosine, however, basal coronary resistance remained unchanged with DDA. The antiadrenergic action of DDA was parallelled by changes in cAMP release from heart: stimulation with isoproterenol (16 pmol) increased cAMP release from 1.5 +/- 0.14 pmol cAMP/min under basal conditions to 5.2 +/- 0.45 pmol/min (mean +/- SE; n = 4). This increase was inhibited by 49% in presence of DDA (90 microM). Theophylline (50 microM), a well known antagonist of extracellular adenosine receptors, did not alter the potency of DDA. Our findings demonstrate, that DDA does not alter basal coronary flow but exerts a potent antiadrenergic action in heart which is P-site mediated[2].
In vivo activity data for 2',5'-Dideoxyadenosine are limited, as the compound is primarily used as a research tool in cell-based assays. Its cell-permeable nature suggests that it may be able to cross biological membranes and inhibit adenylyl cyclase activity in intact cells. However, comprehensive in vivo studies evaluating its pharmacokinetic properties, tissue distribution, and efficacy in animal models have not been extensively reported. The compound's short half-life and rapid metabolism may limit its utility in vivo. Further research is needed to assess its potential for in vivo applications.

M1 muscarinic acetylcholine receptors are highly expressed in key areas that control cognition, such as the cortex and hippocampus, representing one potential therapeutic target for cognitive dysfunctions of Alzheimer's disease and schizophrenia. We have reported that M1 receptors facilitate cognition by promoting membrane insertion of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor AMPA receptor subunit 1 (GluA1) through phosphorylation at Ser845. However, the signaling pathway is still unclear. Here we showed that adenylyl cyclase inhibitor 2',5'-dideoxyadenosine and PKA inhibitor KT5720 inhibited enhancement of phosphorylation of Ser845 and membrane insertion of GluA1 induced by M1 receptor activation. Furthermore, PI3K inhibitor LY294002 and protein kinase B (Akt) inhibitor IV blocked the effects of M1 receptors as well. Remarkably, the increase of the activity of PI3K-Akt signaling induced by M1 receptor activation could be abolished by cAMP-PKA inhibitors. Moreover, inhibiting the mammalian target of rapamycin (mTOR) complex 1, an important downstream effector of PI3K-Akt, by short-term application of rapamycin attenuated the effects of M1 receptors on GluA1. Furthermore, such effect was unrelated to possible protein synthesis promoted by mTOR. Taken together, these data demonstrate that M1 receptor activation induces membrane insertion of GluA1 via a signaling linking cAMP-PKA and PI3K-Akt-mTOR pathways but is irrelevant to protein synthesis.-Zhao, L.-X., Ge, Y.-H., Li, J.-B., Xiong, C.-H., Law, P.-Y., Xu, J.-R., Qiu, Y., Chen, H.-Z. M1 muscarinic receptors regulate the phosphorylation of AMPA receptor subunit GluA1 via a signaling pathway linking cAMP-PKA and PI3K-Akt[3].
The in vitro enzyme assay for 2',5'-Dideoxyadenosine involves measuring its inhibitory activity against adenylyl cyclase in a cell-free system. The assay is typically performed by incubating the compound with purified adenylyl cyclase enzyme in the presence of ATP and Mg²⁺. The production of cAMP is quantified using a radioactive or fluorescence-based detection method. Dose-response curves are generated by incubating the enzyme with increasing concentrations of the compound to calculate the IC50 value. The assay conditions are optimized to ensure that the compound's solubility is maintained, and appropriate controls are included to validate the specificity of the inhibition. Binding to the P-site is confirmed by the non-competitive nature of the inhibition.

Western Blot Analysis [3]
Cell Types: Primary hippocampal neurons
Tested Concentrations: 10 μM
Incubation Duration: 30 minutes
Experimental Results: Reduce cAMP production and block GluA1 Ser845 phosphorylation induced by carbachol (CCh).
The in vitro cellular assay for 2',5'-Dideoxyadenosine is conducted using cell lines such as HEK293 cells that express a cAMP-dependent reporter gene. Cells are seeded in multi-well plates and treated with forskolin to stimulate adenylyl cyclase activity and increase cAMP levels. The compound is added at various concentrations, and the inhibition of forskolin-induced reporter gene activation is measured. The IC50 value is calculated from the dose-response curve. Cytotoxicity is assessed in parallel using cell viability assays to ensure that the observed inhibition is not due to cell death. The compound's cell-permeable nature allows it to enter cells and inhibit adenylyl cyclase activity.

Animal/Disease Models: Male Wistar rats (3-4 months old) [3] 2',5'-dideoxyadenosine (0.1 mg/kg; intraperitoneal (ip) injection; sham for 15 minutes) completely inhibits Fr?EtOAc in suspension [4 ].
Doses: 0.1 mg/kg
Route of Administration: IP; pretreatment for 15 minutes.
Experimental Results: Completely inhibited the diuretic, natriuretic and K+ and Cl- preserving effects of Fr•EtOAc on rats.
In vivo animal experiments for 2',5'-Dideoxyadenosine would typically involve administration of the compound to rodents via intraperitoneal or intravenous injection. Tissues would be collected to measure cAMP levels as a readout of adenylyl cyclase inhibition. However, comprehensive in vivo studies have not been extensively reported for this compound. The compound's rapid metabolism and short half-life may limit its utility in animal models. Further research is needed to evaluate its pharmacokinetic properties and efficacy in vivo. Researchers using this compound in animal studies should consider its stability and dosing regimen.

Pharmacokinetic properties of 2',5'-Dideoxyadenosine have not been extensively characterized. As a nucleoside analog with a molecular weight of 235.24, the compound is expected to have moderate water solubility and may be able to cross biological membranes. Its cell-permeable nature suggests that it can enter cells. However, detailed studies on its absorption, distribution, metabolism, and excretion are limited. The compound's stability in plasma and its half-life have not been well defined. Further pharmacokinetic studies would be required to assess its potential for in vivo applications.

Toxicological data for 2',5'-Dideoxyadenosine are limited. As a nucleoside analog, it may have potential cytotoxic effects at high concentrations. In vitro cytotoxicity assays are typically performed alongside activity assays to ensure that the observed inhibition of adenylyl cyclase is not due to cell death. Comprehensive in vivo toxicology studies, including acute and chronic toxicity, have not been reported. Researchers handling this compound should follow standard safety protocols for nucleoside analogs, including the use of personal protective equipment and working in a well-ventilated area.

[1]. Tissue levels, source, and regulation of 3'-AMP: an intracellular inhibitor of adenylyl cyclases. Mol Pharmacol. 1990 Dec;38(6):848-53.

[2]. Isoproterenol antagonistic effect of 2',5'-dideoxyadenosine in the isolated perfused guinea-pig heart. J Mol Cell Cardiol. 1993 Mar;25(3):331-8.

[3]. M1 muscarinic receptors regulate the phosphorylation of AMPA receptor subunit GluA1 via a signaling pathway linking cAMP-PKA and PI3K-Akt. FASEB J. 2019 May;33(5):6622-6631.

[4]. Role of prostaglandin/cAMP pathway in the diuretic and hypotensive effects of purified fraction of Maytenus ilicifolia Mart ex Reissek (Celastraceae). J Ethnopharmacol. 2013 Oct 28;150(1):154-61.

2',5'-Dideoxyadenosine is a deoxyribonucleoside.
2',5'-Dideoxyadenosine is a research compound that has not entered clinical trials or received regulatory approval for therapeutic use. It is primarily used as a tool compound in biochemical research to study adenylyl cyclase and cAMP signaling pathways. The compound's ability to inhibit adenylyl cyclase in a non-competitive manner makes it valuable for investigating the regulation of cAMP production and its role in various cellular processes. Its P-site binding mechanism provides insights into the allosteric regulation of adenylyl cyclase. The compound is not intended for human therapeutic use.

C₁₀H₁₃N₅O₂

235.24

235.107

6698-26-6

65166

Typically exists as White to off-white solids at room temperature

1.77 g/cm3

547ºC at 760 mmHg

284.6ºC

1.825

0.658

2

6

1

17

292

3

C[C@H]1O[C@H](C[C@@H]1O)N1C=NC2=C1N=CN=C2N

FFHPXOJTVQDVMO-DSYKOEDSSA-N

InChI=1S/C10H13N5O2/c1-5-6(16)2-7(17-5)15-4-14-8-9(11)12-3-13-10(8)15/h3-7,16H,2H2,1H3,(H2,11,12,13)/t5-,6+,7-/m1/s1

(2R,3S,5R)-5-(6-aminopurin-9-yl)-2-methyloxolan-3-ol

2',5'Dideoxyadenosine; 2',5'-Dideoxyadenosine; 6698-26-6; (2R,3S,5R)-5-(6-Amino-9H-purin-9-yl)-2-methyltetrahydrofuran-3-ol; Adenosine, 2',5'-dideoxy-; 988H339Z1L; Adenosine,2',5'-dideoxy-; 5-(6-AMINOPURIN-9-YL)-2-METHYLTETRAHYDROFURAN-3-OL; NSC-95943; 2',5' Dideoxyadenosine

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~125 mg/mL (~531.37 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (10.63 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (8.84 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (8.84 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)