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Decoyinine

Alias: Decoyinine; Angustmycin A; decoyinine; 2004-04-8; Decoyinin; Antibiotic A 14; (2R,3R,4S)-2-(6-aminopurin-9-yl)-2-(hydroxymethyl)-5-methylideneoxolane-3,4-diol; AI3-50816; DTXSID50173859; Antibiotic A 14
Cat No.:V3339 Purity: ≥98%
Decoyinine (also known asAngustmycin A) is a potent and selective inhibitor of GMP synthetase (GMPS,guanosine monophosphate synthase).
Decoyinine
Decoyinine Chemical Structure CAS No.: 2004-04-8
Product category: Others 2
This product is for research use only, not for human use. We do not sell to patients.
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Purity & Quality Control Documentation

Purity: ≥98%

Product Description

Decoyinine (also known as Angustmycin A) is a potent and selective inhibitor of GMP synthetase (GMPS, guanosine monophosphate synthase). Decoyinine allows sporulation in Bacillus subtilis to initiate and proceed under otherwise catabolite-repressing conditions. Decoyinine did not overcome catabolite repression of alpha-amylase synthesis in a wild-type strain of B. subtilis but did cause premature and enhanced synthesis in a mutant strain specifically blocked in catabolite repression of alpha-amylase synthesis. Decoyinine had no effect on alpha-amylase enzymatic activity. Thus, it appears that the catabolite control mechanisms governing alpha-amylase synthesis and sporulation in B. subtilis differ in their responses to decoyinine and hence must consist at least partially of separate components.

Biological Activity I Assay Protocols (From Reference)
Targets
GMP synthetase (GMPS)
ln Vitro
Decoyinine, an adenosine analogue, inhibits GMP synthase, resulting in a drop in intracellular GTP levels. Decoyinine only stimulates the synthesis of oa amylase in strains whose gra-10 mutations have rendered downstream regulatory components of α-amylase control unworkable. Decoyinine's apparent enhancement of a-amylase activity in WLN-11 is not a result of variations in the enzyme's activity level. Purine nucleotides, particularly GMP, have been shown to have strong inhibitory effects on B's activity. amylase frontis F-2. At 1.07 mM, the concentration utilized in the research, Decoyinine did not affect B's activity. α-amylase from subtilis WLN-11 in vitro. GMP slightly inhibits α-amylase activity at the same concentration [2].
Pharmacological targeting of GMPS by Decoyinine/angustmycin A decreases invasion [1]
Angustmycin A is a selective GMPS inhibitor that was tested in mice for non-cancer-related properties. Thus, we were interested in whether GMPS inhibition via angustmycin A may inhibit melanoma cells invasion. Treatment of SK-Mel-28 and SK-Mel103 cells with 2 mM of Angustmycin A (maximum concentration not affecting proliferation of studied cells) reduced their invasion by ~30% compared with vehicle-treated cells (Figure 4a), mirroring the phenotype caused by GMPS depletion (Figure 1b). Furthermore, as observed with GMPS depletion, supplementation with 100 μM of guanosine negated the effect of angustmycin A (Figure 4a). Thus, pharmacological inhibition of GMPS phenocopies the effects of GMPS genetic inhibition on melanoma cells invasive ability and its guanosine dependence.
Decoyinine, an inhibitor of GMP synthetase, allows sporulation in Bacillus subtilis to initiate and proceed under otherwise catabolite-repressing conditions. The effect of Decoyinine on alpha-amylase synthesis in B. subtilis, an event which exhibits regulatory features resembling sporulation initiation, was examined. Decoyinine did not overcome catabolite repression of alpha-amylase synthesis in a wild-type strain of B. subtilis but did cause premature and enhanced synthesis in a mutant strain specifically blocked in catabolite repression of alpha-amylase synthesis. Decoyinine had no effect on alpha-amylase enzymatic activity. Thus, it appears that the catabolite control mechanisms governing alpha-amylase synthesis and sporulation in B. subtilis differ in their responses to decoyinine and hence must consist at least partially of separate components [2].
ln Vivo
Decoyinine also inhibits the growth of xenografts made from cells with NRASQ61R (SK-Mel-103) or BRAFV600E (SK-Mel-28) mutations, suggesting that its effects, like those of GMPS activity, do not appear to be subtype potential [1].
Angustmycin A/Decoyinine suppresses growth of melanoma cell xenografts in SCID mice [1]
Based on the above results, we were interested in the anti-melanoma efficacy of angustmycin A/Decoyinine in a preclinical mouse model. Previously, the only in vivo study of angustmycin A/Decoyinine was performed in mice to investigate immunological responses to skin allografts.31 Using data from this paper, we determined the repetitive maximum tolerated dose (rMTD) of angustmycin A/Decoyinine in SCID mice as of 120 mg/kg.
IMPDH1 and IMPDH2 are rate-limiting enzymes of de novo guanylate biosynthesis (Figure 1a) and their expression is elevated in tumor cells.7, 33, 34 A specific inhibitor of IMPDH enzymes, MPA has been used as an immunosuppressant during organ transplant. A better bioavailable form of MPA is its prodrug MMF (over 200% improvement in bioavailability), which has also been proposed as anti-cancer therapy. Therefore, for the purpose of comparison, we evaluated angustmycin A head-to-head with MMF. Since angustmycin A/Decoyinine was delivered i.p. we chose the same route of administration for MMF. We selected a therapeutically active dose of MMF (30 mg/kg) based on previous studies and information on bioavailability.
SK-Mel-103 and SK-Mel-28 cells were inoculated subcutaneously in both flanks of SCID mice (18 mice/cell line). Once tumors volume reached approximately 100 mm3, mice were randomly assigned to one of four groups and treated with daily i.p. injections of angustmycin A/Decoyinine (120 mg/kg), MMF (30 mg/kg), or with respective vehicles. Tumor size was measured every other day with a caliper and mice were killed once tumor volume reached 1000 mm3 or the animals showed signs of morbidity. In SK-Mel-103 cells, angustmycin A treatment resulted in a 36% reduction of xenografts volume while MMF caused only 19% volume reduction compared with respective vehicle controls, (Figure 4b). In SK-Mel-28 cells, the xenograft volume reduction caused by angustmycin A and MMF were of 62% and 30%, respectively (Figure 4b).
Cell Assay
Immunoblotting [1]
Whole cell extracts were prepared in NP-40 buffer (20 mM Tris pH7.4, 150 mM NaCl; 1% NP-40, 20 mM NaF) supplemented with 1 mM Na-orthovanadate, 1 mM PMSF, and proteases inhibitors (aprotinin 1 μg/ml, pepstatin A 1 μg/ml, and leupeptin 2 μg/ml). Samples (20–80 μg/lane) were resolved on denaturing polyacrylamides gel and transferred onto nitrocellulose membranes. Membranes were incubated o/n at 4 °C with primary antibodies, followed by 1 h incubation at RT with the appropriate HRP-conjugated secondary antibody. Signals were visualized using the Pierce ECL western blotting substrate and X-ray films.
Immunohistochemistry [1]
Formalin-fixed and paraffin-embedded human melanocytic cells, cutaneous and metastatic melanoma tissues were processed at the Pathology Core Facility. Positive and negative control slides were supplied by the Pathology Core Facility and were included with every immunochemistry run. The GMPS antibody was visualized with the Novocastra PowerVision kit, followed by Fast Red The slides were manually counterstained with hematoxylin. Human tissue specimens were scored for intensity of staining by a board-certified pathologist as described in Wawrzyniak et al.
Matrigel-based invasion assay [1]
Invasion assay was performed using the BioCoat Matrigel invasion chambers according to the manufacturer's instructions, as described in Wawrzyniak et al.8 Experiments were performed in duplicates and repeated at least twice.
Combined gelatin degradation assay [1]
Coverslips were coated with warm Oregon Green 488-conjugated gelatin as described in Wawrzyniak et al. Melanoma cells (7.5 × 104) were seeded on the coverslips and after 16-h incubation at 37 °C they were fixed in 4% paraformaldehyde in PBS. After permeabilization in 0.05% Triton X-100 in PBS, cells were stained with rhodamine-conjugated phalloidin and hoechst. Coverslips were mounted onto glass slides with aqua-mount media. Fluorescent images were captured using a Nikon TE2000-E inverted microscope equipped with Roper CoolSnap HQ CCD camera and MetaVue software.
The isogenic strains WLN-4 (sacA321 amyRJ-amyE+) and WLN-11 (sacA321 gra-10-amyE+) are inoculated into minimal S7 medium containing 2% (wt/vol) glucose, using washed exponential-phase seed cultures grown in the same medium.
At mid-logarithmic growth phase, each culture is evenly divided into two flasks:
One flask receives 1/10 volume of fresh S7 medium
The other receives 1/10 volume of filter-sterilized S7 medium containing 2.5 mg/mL Decoyinine (resulting in a final Decoyinine concentration of 250 μg/mL)
Sampling occurs at regular intervals:
Before Decoyinine addition
After Decoyinine addition
Culture supernatants are assayed for α-amylase activity using previously described methods.
At 16 hours post-Decoyinine addition, the frequency of heat-resistant spores in each culture is determined [2].
Animal Protocol
Animal studies using a subcutaneous xenograft model [1]
SK-Mel-103 or SK-Mel-28 cells expressing control vector or shRNA to GMPS were inoculated subcutaneously in both flanks of 4- to 6-week-old female SCID mice, which are bred and maintained by the in-house transgenic mouse facility at RPCI (1.0x106 cells/flank, and 5.0 × 106 cells/flank, respectively). For all cohorts, the time of the appearance of tumor ≥2 mm in at least one dimension was recorded and tumors were measured thereafter every other day. For drug treatment studies, uninfected SK-Mel-103 or SK-Mel-28 cells were inoculated as described above. When tumors reached 100 mm3 in size, mice were randomly assigned to different treatment groups and treated with daily i.p. injection of Decoyinine (120 mg/kg in a solution of 10% DMSO in PBS), MMF (30 mg/kg in a suspension of 0.9% benzyl alcohol, 0.9% sodium chloride, 0.5% carboxymethylcellulose, and 0.4% polysorbate 80 in water), or the correspondent vehicle control.
Toxicity/Toxicokinetics
121578 mouse LD50 oral >2500 mg/kg Compounds Available for Fundamental Research, Volume II-6, Antibiotics, A Program of Upjohn Company Research Laboratory., 2(6)(-), 1971
121578 mouse LD50 intraperitoneal >1 gm/kg Compounds Available for Fundamental Research, Volume II-6, Antibiotics, A Program of Upjohn Company Research Laboratory., 2(6)(-), 1971
121578 mouse LD50 subcutaneous 2500 mg/kg CRC Handbook of Antibiotic Compounds, Vols.1- , Berdy, J., Boca Raton, FL, CRC Press, 1980, 5(294), 1981
References

[1]. Pharmacological targeting of guanosine monophosphate synthase suppresses melanoma cell invasion and tumorigenicity. Cell Death Differ. 2015 Nov; 22(11): 1858–1864.

[2]. Effect of Decoyinine on the regulation of alpha-amylase synthesis in Bacillus subtilis. J Bacteriol. 1987 Dec; 169(12): 5867–5869.

Additional Infomation
Decoyinine is a member of 6-aminopurines.
Decoyinine has been reported in Streptomyces angustmyceticus with data available.
Malignant melanoma possesses one of the highest metastatic potentials among human cancers. Acquisition of invasive phenotypes is a prerequisite for melanoma metastases. Elucidation of the molecular mechanisms underlying melanoma invasion will greatly enhance the design of novel agents for melanoma therapeutic intervention. Here, we report that guanosine monophosphate synthase (GMPS), an enzyme required for the de novo biosynthesis of GMP, has a major role in invasion and tumorigenicity of cells derived from either BRAF(V600E) or NRAS(Q61R) human metastatic melanomas. Moreover, GMPS levels are increased in metastatic human melanoma specimens compared with primary melanomas arguing that GMPS is an attractive candidate for anti-melanoma therapy. Accordingly, for the first time we demonstrate that Decoyinine/angustmycin A, a nucleoside-analog inhibitor of GMPS produced by Streptomyces hygroscopius efficiently suppresses melanoma cell invasion in vitro and tumorigenicity in immunocompromised mice. Our data identify GMPS as a powerful driver of melanoma cell invasion and warrant further investigation of angustmycin A as a novel anti-melanoma agent.[1]
The isolation of the gra-JO mutation (8) afforded a unique opportunity to examine the effect of Decoyinine addition on the regulation of a postexponentially expressed gene whose catabolite repression component had been inactivated. The finding that a-amylase synthesis could be stimulated by decoyinine only when the catabolite repression me'chanism had been inactivated by the gra-10 mutation suggests that the drop in the GTP level associated with the end of exponential growth may play a role in temporally regulated gene activation and not necessarily in the relief of catabolite repression. In addition to our findings with ca-amylase synthesis, decoyinine has been shown to elicit the expression of spoOH(2), spoVG(18), and citB(1). Each of these genes is subject to temporal regulation. It is intriguing to speculate that mechanisms regulating the expression of these genes may share a common feature with the mechanism that regulates the temporal expression of a-amylase synthesis even though one of the genes, spoVG, is transcribed by a different form of RNA polymerase.[2]
These protocols are for reference only. InvivoChem does not independently validate these methods.
Physicochemical Properties
Molecular Formula
C11H13N5O4
Molecular Weight
279.25202
Exact Mass
279.096
Elemental Analysis
C, 47.31; H, 4.69; N, 25.08; O, 22.92
CAS #
2004-04-8
Related CAS #
2004-04-8
PubChem CID
121578
Appearance
Typically exists as solid at room temperature
Density
1.9±0.1 g/cm3
Boiling Point
580.3±60.0 °C at 760 mmHg
Flash Point
304.7±32.9 °C
Vapour Pressure
0.0±1.7 mmHg at 25°C
Index of Refraction
1.837
LogP
-1.25
Hydrogen Bond Donor Count
4
Hydrogen Bond Acceptor Count
8
Rotatable Bond Count
2
Heavy Atom Count
20
Complexity
410
Defined Atom Stereocenter Count
3
SMILES
C=C(O1)[C@@H](O)[C@@H](O)[C@]1(CO)N2C=NC3=C(N)N=CN=C32
InChi Key
UZSSGAOAYPICBZ-SOCHQFKDSA-N
InChi Code
InChI=1S/C11H13N5O4/c1-5-7(18)8(19)11(2-17,20-5)16-4-15-6-9(12)13-3-14-10(6)16/h3-4,7-8,17-19H,1-2H2,(H2,12,13,14)/t7-,8-,11-/m1/s1
Chemical Name
Adenine, 9-(tetrahydro-3,4-dihydroxy-2-(hydroxymethyl)-5-methylene-2-furyl)-
Synonyms
Decoyinine; Angustmycin A; decoyinine; 2004-04-8; Decoyinin; Antibiotic A 14; (2R,3R,4S)-2-(6-aminopurin-9-yl)-2-(hydroxymethyl)-5-methylideneoxolane-3,4-diol; AI3-50816; DTXSID50173859; Antibiotic A 14
HS Tariff Code
2934.99.9001
Storage

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Shipping Condition
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
Solubility Data
Solubility (In Vitro)
DMSO: ~50 mg/mL (~180 mM);
Water: ~6.7 mg/mL (~24 mM)
Ethanol:N/A
Solubility (In Vivo)
Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

Injection Formulations
(e.g. IP/IV/IM/SC)
Injection Formulation 1: DMSO : Tween 80: Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution 50 μL Tween 80 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO 400 μLPEG300 50 μL Tween 80 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).
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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO 100 μLPEG300 200 μL castor oil 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10: 10 : 80 (i.e. 100 μL Ethanol 100 μL Cremophor 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH 400 μLPEG300 50 μL Tween 80 450 μL Saline)


Oral Formulations
Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).
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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders


Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

 (Please use freshly prepared in vivo formulations for optimal results.)
Preparing Stock Solutions 1 mg 5 mg 10 mg
1 mM 3.5810 mL 17.9051 mL 35.8102 mL
5 mM 0.7162 mL 3.5810 mL 7.1620 mL
10 mM 0.3581 mL 1.7905 mL 3.5810 mL

*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.

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In vivo Formulation Calculator (Clear solution)
Step 1: Enter information below (Recommended: An additional animal to make allowance for loss during the experiment)
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Working concentration mg/mL;

Method for preparing DMSO stock solution mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.

Method for preparing in vivo formulation:Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O,mix and clarify.

(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
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Biological Data
  • Decoyinine

    GMPS is upregulated during melanoma progression.2015 Nov;22(11):1858-64.

  • Decoyinine

    Angustmycin A treatment affects melanoma invasionin vitroand xenograft growthin vivo.2015 Nov;22(11):1858-64.

  • Decoyinine

    GMPS contributes to the invasive capability of melanoma cells.2015 Nov;22(11):1858-64.

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