ADAR1 (Adenosine Deaminase Acting on Double-Stranded RNA 1); A-to-I RNA editing. 8-Azaadenosine is a potent inhibitor of ADAR1 and A-to-I editing activity. It binds to the active site of ADAR1, preventing the deamination of adenosine to inosine in double-stranded RNA. This blocks the RNA editing process that is often dysregulated in cancer. By inhibiting ADAR1, 8-Azaadenosine restores the expression of tumor suppressor microRNAs such as let-7, which are normally suppressed by A-to-I editing in cancer cells. The compound does not affect A-to-I editing of double-stranded mRNA, indicating specificity for ADAR1 over other RNA editing enzymes. It also serves as a substrate for adenosine kinase but does not inhibit purine biosynthesis.

8-Azaadenosine does not alter the levels of ADAR1 mRNA; instead, it solely targets the activity of ADAR (adenosine deaminases acting on double-stranded RNA)[1][2]. After two weeks in stromal co-culture, 8-adenosine (10–25 nM) restores let-7 miRNA biogenesis in proportion to a decrease in ADAR1 expression, RNA editing activity, and LIN28B expression in JAK2/BCR-ABL1 transduced progenitors[1]. Based on qRT-PCR and Western blot analysis of p-CRKL and p-STAT5a, 8-azaadenosine (10, 100 nM) has no effect on BCR-ABL and JAK2 signaling[1]. In TPC1 and Cal62 cells, 8-azaadenosine (0.1, 0.5, 1, 2 µM; 5 days) reduces cell viability/proliferation in a dose-dependent manner[2]. 8-Azaadenosine (1, 2 µM; 16 hours) inhibits TPC1 and Cal62 cell invasion and migration[2]. In TPC1 and Cal62 cells, 8-azaadenosine (1, 2 µM) suppresses the editing activity. In contrast, both cell lines' levels of ADAR1 mRNA stay constant[2].

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8-Azaadenosine (1-2 microM, 16 hours) inhibits TPC1 and Cal62 thyroid cancer cell invasion and migration. It blocks RNA editing and inhibits proliferation, 3D growth, invasion, and migration in thyroid cancer cells. In leukemia cell lines, 8-Azaadenosine reduces A-to-I editing activity, restores let-7 expression, and inhibits leukemia stem cell self-renewal. The compound is a potent purine biosynthesis inhibitor? Actually, 8-Azaadenosine is a potent inhibitor of purine biosynthesis, but 8-Aza-HR (8-azaadenosine? There is some confusion in the literature). In H.Ep.2 cells, 8-aza-HR (8-azaadenosine? Or a related compound?) blocks the conversion of uridylic acid to uridine monophosphate and leads to uracil accumulation. However, exogenous uracil does not reverse inhibition of cell growth, suggesting that inhibition of pyrimidine biosynthesis is not the primary cause of cytotoxicity. 8-Azaadenosine (8-aza-AR) is a potent purine biosynthesis inhibitor, but 8-aza-HR (8-azaadenosine?) does not inhibit this process even at concentrations far exceeding inhibitory concentrations of 8-aza-AR. This suggests that 8-Azaadenosine may have multiple mechanisms of action depending on the context. Further clarification is needed; however, the primary reported mechanism is ADAR1 inhibition. The compound shows activity in various cancer cell lines.

In vivo studies have not been extensively reported for 8-Azaadenosine. However, based on its in vitro activity as a potent ADAR1 inhibitor and its ability to inhibit cancer cell proliferation, invasion, and migration in thyroid cancer cells, the compound may have potential for in vivo efficacy in cancer models. ADAR1 is a promising therapeutic target in cancer, as its overexpression is associated with poor prognosis in many cancer types. Studies in leukemia stem cells suggest that ADAR1 inhibition may target cancer stem cells, which are often resistant to conventional therapies. Further in vivo studies in xenograft models and genetically engineered mouse models would be valuable to determine the therapeutic potential of 8-Azaadenosine.

In vitro ADAR1 inhibition assay: The activity of ADAR1 can be assessed using an A-to-I editing assay. A target RNA substrate containing a known editing site (e.g., the GluR2 Q/R site or a synthetic double-stranded RNA substrate) is incubated with recombinant ADAR1 enzyme in the presence of varying concentrations of 8-Azaadenosine (0.1-100 microM). The reaction is carried out at 37degC for 1-4 hours. RNA is extracted and subjected to reverse transcription and PCR amplification. The extent of A-to-I editing is quantified by Sanger sequencing (comparing peak heights of A and G) or by restriction enzyme digestion (since inosine is recognized as guanosine by many restriction enzymes). Alternatively, the RNA is digested with RNase T1, which cleaves at inosine but not at adenosine, and products are analyzed by denaturing PAGE or LC-MS/MS. IC₅0 values for ADAR1 inhibition are calculated from dose-response curves. The compound's effect on A-to-I editing in double-stranded mRNA (not ADAR1's main target) should be examined to confirm specificity.

Cell proliferation and invasion assays: Thyroid cancer cells (TPC1, Cal62) or leukemia cell lines are seeded in appropriate culture plates. Cells are treated with 8-Azaadenosine (0.5-10 microM) for 16-72 hours. Proliferation is measured by MTT, CCK-8, or cell counting. 3D growth is assessed by culturing cells in Matrigel or soft agar for 7-14 days, followed by colony counting. Invasion and migration are assessed using Transwell chambers with or without Matrigel coating. Cells (1-5 × 10⁴) are seeded in the upper chamber in serum-free medium containing 8-Azaadenosine (1-2 microM). The lower chamber contains medium with 10% FBS as a chemoattractant. After 16-24 hours, cells that have invaded/migrated to the lower side of the membrane are fixed, stained with crystal violet, and counted in 5-10 random fields per well. A-to-I editing activity in cells is assessed by RT-PCR and sequencing of editing sites such as the FLNA, COPA, or AZIN1 transcripts. let-7 microRNA expression is measured by qRT-PCR. All assays are performed in triplicate.

Animal cancer models: For potential in vivo studies, immunodeficient mice can be subcutaneously injected with thyroid cancer cells (e.g., TPC1 or Cal62 cells) or leukemia cells. When tumors reach an appropriate size, mice are treated with 8-Azaadenosine (dose and route to be determined, likely 10-100 mg/kg, oral or intraperitoneal, daily or every other day). Tumor volume is measured every 2-3 days. At the end of the study, tumors are excised and analyzed for ADAR1 expression, A-to-I editing levels, let-7 expression, proliferation (Ki-67), and apoptosis (TUNEL). For leukemia models, peripheral blood, bone marrow, and spleen are analyzed for leukemia burden by flow cytometry. Body weight and toxicity are monitored. These studies would be necessary to establish in vivo efficacy. Detailed protocols would need to be optimized based on the specific cancer model and compound pharmacokinetics.

8-Azaadenosine (MW 268.23, formula C₉H12N₆O4). Solubility: soluble in DMSO (245 mg/mL, 913.4 mM). For in vivo studies, formulations using 10% DMSO + 40% PEG300 + 5% Tween 80 + 45% Saline are recommended (working concentration up to 5 mg/mL). Storage: Powder at -20degC (stable for 3 years). In solution at -80degC (stable for 1 year). Pharmacokinetic properties (half-life, Cmax, AUC, bioavailability) have not been extensively reported in the literature for this compound. As a nucleoside analog, 8-Azaadenosine is likely subject to rapid metabolism by adenosine deaminase and other nucleoside-metabolizing enzymes. The compound is stable in DMSO solution when stored at -80degC. Oral and injection formulations have been validated by commercial suppliers, indicating feasibility for in vivo administration. Oral administration: CMC-Na suspension (≥5 mg/mL). Injection: 5% DMSO + 40% PEG300 + 5% Tween 80 + 50% ddH2O (clear solution, 2.7 mg/mL) or 5% DMSO + 95% corn oil (clear solution, 0.15 mg/mL).

Based on in vitro studies using cancer cell lines at concentrations up to 10 microM, 8-Azaadenosine appears to have selective toxicity for cancer cells over normal cells in some contexts. However, comprehensive toxicological studies have not been extensively performed for this compound. As a nucleoside analog, there is potential for off-target effects on normal cellular metabolism and DNA/RNA synthesis. The compound is a substrate for adenosine kinase, which could lead to accumulation of phosphorylated metabolites with potential cytotoxic effects. Standard safety precautions should be followed when handling 8-Azaadenosine. The compound is intended for research use only and is not approved for human therapeutic use. Long-term toxicity, genotoxicity, and reproductive toxicity studies would be required for clinical development.

[1]. ADAR1 Activation Drives Leukemia Stem Cell Self-Renewal by Impairing Let-7 Biogenesis. Cell Stem Cell. 2016 Aug 4;19(2):177-191.

[2]. ADAR1-mediated RNA editing is a novel oncogenic process in thyroid cancer and regulates miR-200 activity. Oncogene. 2020 Apr;39(18):3738-3753.

8-azaadenosine is an N-glycoside compound.

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8-Azaadenosine is a research-use-only compound that is not approved for human therapeutic use. It is available as a high-purity biochemical (typically >98% purity) for laboratory research applications. The compound has been studied as a potential therapeutic agent for cancer, particularly for cancers characterized by dysregulated RNA editing or ADAR1 overexpression. Studies have shown activity in thyroid cancer and leukemia models. 8-Azaadenosine is also a valuable tool compound for studying the biological functions of ADAR1 and A-to-I RNA editing in normal physiology and disease. The compound's ability to inhibit ADAR1 and restore tumor suppressor microRNA expression makes it a promising lead for cancer drug development. Additional research is needed to optimize its pharmacokinetic properties, evaluate in vivo efficacy, and assess safety. The compound should be stored at appropriate conditions to maintain stability. The inhibitory effects of Cap on survivin, MMP-2, MMP-9, and STAT3 activation may account for its anti-invasive effects.

C9H12N6O4

268.23

268.092

10299-44-2

96410

White to off-white solid powder

2.29 g/cm3

702.6ºC at 760 mmHg

378.7ºC

1.01E-20mmHg at 25°C

2

-2.2

4

9

2

19

336

4

C1=NC(=C2C(=N1)N(N=N2)[C@H]3[C@@H]([C@@H]([C@H](O3)CO)O)O)N

OAUKGFJQZRGECT-UUOKFMHZSA-N

InChI=1S/C9H12N6O4/c10-7-4-8(12-2-11-7)15(14-13-4)9-6(18)5(17)3(1-16)19-9/h2-3,5-6,9,16-18H,1H2,(H2,10,11,12)/t3-,5-,6-,9-/m1/s1

(2R,3R,4S,5R)-2-(7-aminotriazolo[4,5-d]pyrimidin-3-yl)-5-(hydroxymethyl)oxolane-3,4-diol

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture.

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

DMSO: 250 mg/mL (932.04 mM)
H2O: 3.33 mg/mL (12.41 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (7.75 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 2: ≥ 2.08 mg/mL (7.75 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 20.8 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 3: ≥ 2.08 mg/mL (7.75 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.)