Adenosine kinase (IC50 = 26 nM)

5-Iodotubercidin (NSC 113939) has IC50 values of 0.4, 3.5, 5-10, 5-10, 10.9, and 27.7 μM, respectively, and inhibits CK1, insulin receptor tyrosine kinase, phosphorylase kinase, PKA, CK2, and PKC[1]. 5-A significant drop in ATP concentration and a corresponding, minor increase in AMP concentration are caused by iodotubercidin (20 μM). 5. Iodotubercidin lowers the rates of fatty acid and cholesterol synthesis as well as the activity of ACC. The intracellular concentration of malonyl-CoA is significantly reduced by 5-iodotubercidin, which is consistent with the iodotubercidin-mediated Inhibition of ACC[4].

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SAR of Enzyme Inhibition. [2]
Compounds listed in Table 1 were evaluated as inhibitors of the recombinant human AK, and the IC50s were determined by a radiochemical assay by a slightly modified literature method.34 The purpose of the enzyme inhibition study was to evaluate the effect of modifications at the C4-, C5-, and C5‘-positions of tubercidin on the AKI activity. Initial efforts were focused on the C5-position since substitution with an iodo group converts a modest AK substrate tubercidin (KM = 10 μM) into a potent AK inhibitor (1a/5-Iodotubercidin, IC50 = 0.026 μM). The results indicate that a halogen atom such as Cl, Br, or I at the C5-position of pyrrolopyrimidine nucleosides is essential for potent AKI activity. Moreover, the inhibitory potency increases with increasing size and electron density of the halogen atom. The large atomic size of I appears to provide a hydrophobic interaction with the active site of the enzyme. To examine the effect of hydrophobic and electron-donating groups on the AKI activity, groups such as CH3 and SCH3 were introduced at the C5-position. These compounds (11a,b), however, were found to be poor inhibitors of the enzyme. The loss of AKI potency shown by 11a may be attributed to the smaller size of the CH3 group capable of providing only weak hydrophobic interactions compared to any of the halogen atoms. Although SCH3 is an electron-rich group, the decreased potency of 11b appears to be due to poor fitting of the group in the active site. Therefore, the potent AKI activity exhibited by 1a/5-Iodotubercidin,c,d appears to due to the combined effect of lipophilicity and electronegativity of the C5-substituent.

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Addition of micromolar concentrations of the adenosine derivative 5-Iodotubercidin (Itu) initiates glycogen synthesis in isolated hepatocytes by causing inactivation of phosphorylase and activation of glycogen synthase [Flückiger-Isler and Walter (1993) Biochem. J. 292, 85-91]. We report here that Itu also antagonizes the effects of saturating concentrations of glucagon and vasopressin on these enzymes. The Itu-induced activation of glycogen synthase could not be explained by the removal of phosphorylase a (a potent inhibitor of the glycogen-associated synthase phosphatase). When tested on purified enzymes, Itu did not affect the activities of the major Ser/Thr-specific protein phosphatases (PP-1, PP-2A, PP-2B and PP-2C), but it inhibited various Ser/Thr-specific protein kinases as well as the tyrosine kinase activity of the insulin receptor (IC50 between 0.4 and 28 microM at 10-15 microM ATP). Tubercidin, which did not affect glycogen synthase or phosphorylase in liver cells, was 300 times less potent as a protein kinase inhibitor. Kinetic analysis of the inhibition of casein kinase-1 and protein kinase A showed that Itu acts as a competitive inhibitor with respect to ATP, and as a mixed-type inhibitor with respect to the protein substrate. We propose that Itu inactivates phosphorylase and activates glycogen synthase by inhibiting phosphorylase kinase and various glycogen synthase kinases. Consistent with the broad specificity of Itu in vitro, this compound decreased the phosphorylation level of numerous phosphopolypeptides in intact liver cells. Our data suggest that at least some of the biological effects of Itu can be explained by an inhibition of protein kinases.[1]

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Diverse mechanisms of action have been proposed for 5-Iodotubercidin, although it is widely used as an adenosine kinase inhibitor that consequently interferes with the metabolism of adenosine and adenine nucleotides. Incubation of rat hepatocytes with iodotubercidin produced important effects on lipid metabolism. (i) Both acetyl-CoA carboxylase and fatty acid synthesis de novo were inhibited in parallel by 5-Iodotubercidin, with no change in the activity of fatty acid synthase. The inhibition of both activities showed a comparable dependence on iodotubercidin concentration and was accompanied by a similar decrease (about 60%) in the intracellular malonyl-CoA concentration. (ii) Iodotubercidin stimulated palmitate oxidation, although octanoate oxidation was unaffected. However, this effect can be attributed to the decrease of malonyl-CoA concentration and the concomitant relief of the inhibition of carnitine palmitoyltransferase I, because the activity of this enzyme was found unaltered when determined in cells permeabilized with digitonin. (iii) Iodotubercidin also inhibited cholesterol synthesis de novo. Results, thus, indicate that iodotubercidin increases fatty acid oxidation activity of the liver at the expense of lipogenesis, and we suggest that these effects on fatty acid metabolism are mediated by the inhibition of acetyl-CoA carboxylase, probably due to a more than twice increase in the AMP/ATP ratio and the concomitant stimulation of the AMP-activated protein kinase [3].

5-Iodotubercidin (1 mL/kg, ip) has been shown to be effective against bicuculline-induced seizures after AKI was administered locally to the prepiriform cortex[2].

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SAR of Antiseizure Activity. [2]
Several AKIs were evaluated in the standard maximum electroshock seizure (MES) model (Table 2). The anticonvulsant effect observed with 5-Iodotubercidin (1a) was in agreement with activity observed against bicuculline-induced seizures following local administration of the AKI into the prepiriform cortex.20 The most potent compound, 1b, had an ED50 of 0.3 mg/kg, whereas the others showed potencies within the range of 4.2−7.1 mg/kg. The rank order of anticonvulsant activity in vivo did not strongly correlate with the order of potency in the enzyme inhibition assay in vitro, suggesting that variations in the molecular substituents result in substantial changes in pharmacokinetic properties such as clearance rate, brain penetration, or accessibility to the intracellular enzyme. For example, compounds 15a,b, C5‘-amino analogues of 1b,c, showed lower potencies in vivo despite greater potencies against the enzyme; this may be a result of decreased blood−brain barrier penetration by the C5‘-amino analogues which are less hydrophobic than their corresponding 5‘-deoxy analogues. It is also possible that these compounds may act by additional mechanisms that may influence in vivo activity. Such mechanisms would likely be related to adenosine, since the in vivo activities of these compounds are antagonized by administration of adenosine receptor antagonists.39 The involvement of direct agonist activity on A1 or A2 adenosine receptors has been ruled out as these compounds show little affinity for the receptors.15 Similarly the compounds show no detectable activity on adenosine deaminase (ADA) or on adenosine monophosphate deaminase (AMPDA) and little or no affinity to the nitrobenzylthioinosine binding site associated with a transport mechanism.15 However undefined transport systems could be modulated by the compounds, perhaps influencing the accumulation of extracellular adenosine.

Enzyme Assay. [2]
AK activity was measured in a radiochemical assay similar to the procedure of Yamada et al., with minor modifications. The final reaction volume was 100 μL and contained 70 mM Tris-maleate (pH 7.0), 0.1% (w/v) bovine serum albumin, 1.0 mM MgCl2, 1.0 mM ATP, 1.0 μM [U-14C]adenosine (400−600 mCi/mmol; Moravek Biochemicals, Inc.) and various inhibitor concentrations. Inhibitors were prepared as 10 mM stock solutions in DMSO. The final DMSO concentration in the assay was 5% (v/v). Eleven different concentration of the test solutions ranging from 0.001 to 10.0 μM were utilized to determine a dose response curve of the inhibition of the enzyme. Reactions were started by adding the appropriate amount of purified human recombinant AK and incubated for 20 min at 37 °C. The reactions were terminated by addition of the potent AKI GP3269.40 A 30-μL aliquot of each reaction was spotted on DEAE cellulose filter paper (cut in squares of ∼1 × 1 cm) and air-dried for 30 min. The dry filters were then washed for 3 min in deionized water to remove residual [U-14C]adenosine, rinsed with ethanol and dried at 90 °C for 20 min. The filter papers were counted in 5.5 mL of Ready Safe liquid scintillation cocktail using a Beckman LS3801 scintillation counter. Control AK activity was determined from the amount of [14C]AMP formed in the presence of 5% DMSO. The concentration of inhibitor required to inhibit 50% of the AK activity (IC50) was determined graphically from plots of inhibitor concentration versus percent (%) control enzyme activity. The results are shown in Table 1.

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Biochemical assays [3]
ACC activity was determined in digitonin-permeabilized hepatocytes in an assay coupled to the fatty acid synthase reaction. To measure enzyme activity, 100 μL of hepatocyte suspension was added to 100 μL of prewarmed digitonin-containing assay medium, and the reaction was carried out for 2 min, exactly as described. Fatty acid synthase (FAS) activity was monitored in digitonin-permeabilized hepatocytes as described previously. Hepatocyte suspension (100 μL) was added to 100 μL of digitonin-containing assay medium, and the reaction was carried out for 4 min. CPT-I activity was determined in digitonin-permeabilized hepatocytes as the tetradecylglycidate (TDGA)-sensitive incorporation of radiolabeled l-carnitine into palmitoylcarnitine. In brief, hepatocytes were preincubated for 20 min in the absence or presence of 5 μM TDGA, a potent and specific inhibitor of CPT-I. Aliquots (100 μL) of both sets of hepatocyte incubations were added to 100 μL of prewarmed digitonin-containing assay medium, and the reaction was carried out for 40 s. Intracellular levels of malonyl-CoA were determined in neutralized perchloric acid cell extracts by a radioenzymatic method. Rates of cholesterol synthesis de novo were determined as the incorporation of [1- ]acetate into non-saponifiable sterols extracted with light petroleum ether. Nucleotide levels were determined in neutralized perchloric acid cell extracts by HPLC exactly as described by Gualix et al. Protein was determined by the method of Lowry et al.

MES Seizure Assay. [2]
Male SA rats (100−150 ) were maintained on a 12:12 light:dark cycle in temperature-controlled facilities with free access to food and water. One hour prior to seizure testing, the animals were injected intraperitoneally (1 mL/kg) with DMSO vehicle or with test compound dissolved in DMSO. At the time of the test, an electrolyte solution (2% lidocaine in 0.9% sodium chloride) was applied to the eyes. Maximal electroshock seizures were induced by administering a 60-Hz current of 150 mA for 0.2 s via corneal electrodes, using a Wahlquist Model H stimulator.41 The endpoint measured was suppression of hindlimb tonic extension (HTE) and expressed as percentage of animals in which the response was inhibited. At this supramaximal stimulation level, virtually 100% of control (vehicle-treated) animals showed HTE. ED50 values were calculated from a dose−response curve using probit analysis.42 The N for the screening doses was 6−8; dose−response determinations were conducted with at least 5 animals/dose. [2]

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Dissolved in saline; 1, 2.5 and 5 mg/kg; i.p. injection

Male Mongolian gerbils

[1]. Identification of the glycogenic compound 5-iodotubercidin as a general protein kinase inhibitor. Biochem J. 1994 Apr 1;299 (Pt 1):123-8.

[2]. Adenosine kinase inhibitors. 1. Synthesis, enzyme inhibition, and antiseizure activity of 5-iodotubercidin analogues. J Med Chem. 2000 Jul 27;43(15):2883-93.

[3]. Effects of 5-iodotubercidin on hepatic fatty acid metabolism mediated by the inhibition of acetyl-CoA carboxylase. Biochem Pharmacol. 2002 Jun 1;63(11):1997-2000.

[4]. A small-molecule inhibitor of Haspin alters the kinetochore functions of Aurora B. J Cell Biol. 2012 Oct 15;199(2):269-84.

[5]. Adenosine Kinase Inhibition Protects against Cranial Radiation-Induced Cognitive Dysfunction. Front Mol Neurosci. 2016 Jun 3;9:42.

5-iodotubercidin is an organoiodine compound with functions similar to tuberculin. In summary, we investigated the effects of various substituents at the C4, C5, and C5' positions of tuberculin on AK inhibition and described several novel AK inhibitors with enhanced potency. We also demonstrated anticonvulsant activity against maximal electroshock (MES)-induced seizures in rats. These studies indicate that halogen atoms (such as Cl, Br, or I) at the C5 position and Cl, NH2, or SCH3 groups at the C4 position are crucial for the effective inhibition of AK by tuberculin analogs. The C5' position appears to accommodate both hydrophobic and hydrophilic groups, with an amino group at this position yielding the three most potent AK inhibitors reported to date (15a, 15b, and 16). In vivo, these compounds exhibit potency similar to 5-iodotuberculin and require further modification to enhance their in vivo properties. The ability of potent AK inhibitors to suppress electroconvulsive seizures suggests that these compounds are highly effective as anticonvulsants and suggests that AK inhibition is a viable purinergic method of seizure suppression by adenosine receptor agonists. [2]

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In summary, our results indicate that itu significantly affects hepatic fatty acid metabolism by stimulating mitochondrial fatty acid oxidation and inhibiting lipogenesis. We have identified acetyl-CoA carboxylase as the target enzyme for itu to exert these effects. Furthermore, these data provide indirect evidence that could suggest a mechanism based on an increased AMP/ATP ratio and corresponding activation of AMPK to explain this cellular response. Clearly, further research is needed to fully elucidate this mechanism of action of itu, and related studies are currently underway. In addition, these studies may contribute to a better understanding of this widely used adenosine kinase inhibitor. Interestingly, itu has been shown to inhibit partially purified AMPK, but only in the absence of AMP, and therefore is unlikely to function in isolated hepatocytes. [3]

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Haspin promotes the recruitment of the chromosome passenger complex (CPC) to the centromere during mitosis by phosphorylating the Thr3 site of histone H3. Aurora B kinase is a subunit of CPC that maintains bidirectional chromosome orientation and the spindle assembly checkpoint (SAC). In this study, we identified the small molecule 5-iodotuberculin (5-ITu) as a potent Haspin inhibitor. In vitro experiments showed that 5-ITu effectively inhibited Haspin but had no inhibitory effect on Aurora B. Consistent with this, 5-ITu antagonized the centromere localization of CPC in HeLa cells without affecting most of the activity of Aurora B. The mislocalization of Aurora B is associated with dephosphorylation of CENP-A and Hec1 and uncontrolled spindle assembly checkpoint (SAC) dysregulation caused by high concentrations of nocodazole. 5-ITu also impairs the recruitment of Bub1 and BubR1 kinases to the kinetochore, while inhibition of phosphatase activity reverses this effect. Under the influence of 5-ITu, forcing Aurora B to localize to the centromere can restore the localization of Bub1 and BubR1, but cannot salvage SAC out-of-control. This result suggests that the target of 5-ITu (possibly Haspin itself) may further promote SAC signaling downstream of Aurora B. [4] Clinical radiotherapy for central nervous system cancers can lead to unexpected cognitive impairment, which can cause serious cognitive damage. Radiation-induced cognitive impairment is prolonged; however, its underlying molecular and cellular mechanisms are not fully elucidated. Since ionizing radiation can activate microglia and astrocytes, we hypothesize that maladaptive changes in astrocyte function may be related to radiation-induced cognitive impairment. Astrocytes control adenosine availability by metabolizing and clearing adenosine (an endogenous neuroprotective and cognitive modulator) and other neurotransmitters through adenosine kinase (ADK). Adult rats receiving cranial irradiation (10 Gy) showed a significant decline in hippocampal-dependent cognitive tasks (including novel location recognition, novel object recognition, and situational fear conditioning) one month after receiving a clinically relevant dose of ionizing radiation. Irradiated rats spent less time exploring new locations or objects. Compared to the control group, rats in the cranial irradiation group also exhibited reduced rigidity in the situational fear conditioning task. Importantly, immunohistochemical analysis of the irradiated group's brain tissue revealed significantly increased ADK immunoreactivity in the hippocampus, which was associated with astrocyte proliferation and increased expression of glial fibrillary acidic protein (GFAP). Conversely, rats treated with the ADK inhibitor 5-iodotriacontin (5-ITU, 3.1 mg/kg, intraperitoneal injection, for 6 consecutive days) before cranial irradiation showed significant behavioral improvements in all cognitive tasks one month after irradiation. 5-ITU treatment alleviated radiation-induced astrocyte proliferation and reduced ADK immunoreactivity in the hippocampus. These results confirm an astrocyte-mediated mechanism in which the preservation of extracellular adenosine can play a neuroprotective role against radiation-induced pathological damage. These innovative findings link radiation-induced cognitive and central nervous system functional changes with changes in purine metabolism and astrocyte proliferation, thus revealing the importance of adenosine homeostasis in the brain for radiation damage. [5]

C11H13IN4O4

392.15

391.998

24386-93-4

97297

White to gray solid powder

2.5±0.1 g/cm3

701.5±60.0 °C at 760 mmHg

216-217ºC dec.

378.0±32.9 °C

0.0±2.3 mmHg at 25°C

1.919

0.91

4

7

2

20

365

4

IC1C2=C(N([H])[H])N=C([H])N=C2N(C=1[H])[C@@]1([H])[C@@]([H])([C@@]([H])([C@@]([H])(C([H])([H])O[H])O1)O[H])O[H]

WHSIXKUPQCKWBY-IOSLPCCCSA-N

InChI=1S/C11H13IN4O4/c12-4-1-16(10-6(4)9(13)14-3-15-10)11-8(19)7(18)5(2-17)20-11/h1,3,5,7-8,11,17-19H,2H2,(H2,13,14,15)/t5-,7-,8-,11-/m1/s1

(2R,3R,4S,5R)-2-(4-amino-5-iodopyrrolo[2,3-d]pyrimidin-7-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

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

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.38 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 25.0 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: ≥ 2.5 mg/mL (6.38 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 3: ≥ 2.5 mg/mL (6.38 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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: ≥ 2.5 mg/mL (6.38 mM) (saturation unknown) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
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 5: ≥ 2.5 mg/mL (6.38 mM) (saturation unknown) in 5% DMSO + 95% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
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 6: ≥ 0.5 mg/mL (1.28 mM) (saturation unknown) in 1% DMSO 99% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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