Award
Dr Larissa Balakireva, CEO & Founder of NovoCIB, was awarded with the Trophy of
"Femmes en Or 2011, Femme de l'Innovation"
in September 2011
.

PRECICE® ADK Assay Kit

Ref. #K0507-01
Size Price*
1 plate (96 assays)
€ 420.00
5 plates (5 x 96 assays)
€ 1,680.00

* Pricing updated November 20th, 2013.
Kit is provided in stable lyophilized form and shipped without dry ice.

To buy ADK Assay Kit click here or ask for Quotation


Download in vitro ADK asssay protocol  
(User Manual)

High Throughput Assay for Human Adenosine Kinase

PRECICE® ADK Assay Kit provides a convenient, high throughput compatible and non-radioactive assay for in vitro measurement of ADK activity and for Drug Discovery of novel ADK inhibitors. Novel tool for Drug Discovery, this kit allows HTS search of novel ADK inhibitors.

Principle :
Adenosine kinase uses ATP as the phosphate donor. It transfers the terminal phosphate group of ATP to a substrate, producing ADP as a by-product. The assay is based on the use of inosine as a surrogate ADK substrate and a coupled reaction involving a highly active IMPDH (Inosine Monophosphate Dehydrogenase) for a direct measurement of the inosine monophosphate (IMP) formed by ADK. In the presence of inosine and ATP, ADK catalyses the phosphorylation of inosine to form IMP and ADP. IMP is then immediately oxidized to XMP by a highly active IMPDH in the presence of NAD, leading to NADH2 formation.

Key Features:

Convenient

    Non-radioactive
    Homogenous
    Continuous absorbance monitoring at 340nm

Quantitative

    The IMP is immediately transformed into NADH2 in a quantitative manner allowing for accurate kinetic analysis

High-Throughput Format

    The assay is performed in standard multi-well microplates
    Utilizes commun plate reader fitted with 340nm filter
    Meets the requirements of a HTS assay (Z' factor 0.68)

Reliable

    Validated with known ADK inhibitor A-134974 and nucleoside analog ribavirin known to be phosphorylated by adenosine kinase


For rapid analysis of in vitro phosphorylation of novel nucleoside analogs for human adenosine kinase see our PRECICE® ADK Phosphorylation Assay Kit..

ADK role in human body

Adenosine kinase is a ubiquitous enzyme regulating intra- and extracellular concentrations of adenosine (AR), an important modulator of central nervous system functions and a signal molecule involved in hypoxia, inflammation and nociception. Together with adenosine demainase, adenosine kinase regulates intra- and extracellular cellular adenosine concentration. Inhibition of adenosine kinase results in selective increase of local adenosine concentrations and reduced seizure susceptibility and nociception in vivo(3). ADK dysfunction is involved in several pathologies, including diabetes(13), epilepsy(6), and cancer. Consequently, ADK emerges as a rational therapeutic target, and adenosine-regulating drugs have been extensively tested (1) as new analgesic and anti-inflammatory agents(4) to treat schizophrenia(7) or to limit brain injury after an ischemic stroke(8).

Validation

The use of PRECICE® ADK Screening Assay Kit for the screening of ADK inhibitors and IC50 measurements was validated with compound A-134974, a known ADK inhibitor (Sigma-Aldrich, under license from Abbott Laboratories). Z'-Factor was calculated from 44 negative (no ADK inhibition) and positive (inhibition of ADK with 500nM A-134974) and measured as 0.68, which is an excellent level of performance for a screening assay

ADK Inhibition curve with compound A-134974 at 12 different concentrations.
All data points represent the mean (+/-SD) of 8 wells (2 wells - duplicate - on 4 independent plates). IC50 was determined by fitting a 4-parameter non-linear curve to the data with least-square method. IMPDH activity assays were run to confirm that IMPDH is not inhibited by compound A-134974 (data not shown).

References
1. K. S. Toti, D. Osborne et al. (2016):South (S)- and North (N)-Methanocarba-7-Deazaadenosine Analogues as Inhibitors of Human Adenosine Kinase J. Med. Chem. 59 (14), pp 6860–6877
2. M. F. Jarvis et al. (2000): ABT-702 (4-amino-5-(3-bromophenyl)-7-(6-morpholinopyridin-3-yl)pyrido[2, 3-d]pyrimidine), a novel orally effective adenosine kinase inhibitor with analgesic and anti-inflammatory properties: I. In vitro characterization and acute antinociceptive effects in the mouse J. Pharmacol. Exp. Ther. 295(3), 1156-1164
3. S. McGaraughty et al. (2005): Anticonvulsant and antinociceptive actions of novel adenosine kinase inhibitors Curr. Top. Med. Chem. 5(1):43-58
4. D. E. Fedele et al. (2005): Astrogliosis in epilepsy leads to overexpression of adenosine kinase, resulting in seizure aggravation Brain 128(Pt 10), 2383-2395
5.D. Boison (2008): The adenosine kinase hypothesis of epileptogenesis Prog. Neurobiol. 84(3), 249-262
6. D. Boison et al. (2011): Adenosine hypothesis of schizophrenia – Opportunities for pharmacotherapy doi:10.1016/J.neuropharm.2011.01.048
7. H. Y. Shen et al. (2011): Adenosine kinase determines the degree of brain injury after ischemic stroke in mice J. Cereb. Blood Flow Metab. 31(7): 1648-1659
8. J. Annes et al. (2012): Adenosine kinase inhibition selectively promotes rodent and porcine islet β-cell replication Proc. Natl. Acad. Sci. USA Vol: 109:3915-3920
9. R. C. Willis et al. (1978): Adenosine kinase initiates the major route of ribavirin activation in a cultured human cell Proc. Natl. Acad. Sci. USA 75(7), 3042-3044
10. R. L. Miller et al. (1979): Adenosine kinase from rabbit liver. II. Substrate and inhibitor specificity J. Biol. Chem., 254(7), 2346–2352
11. P. P. Saunders et al. (1990): Tiazofurin is phosphorylated by three enzymes from Chinese hamster ovary cells Cancer Res. 50(17), 5269-5274
12. Yamada Y, Goto H, Ogasawara N. (1980) Purification and properties of adenosine kinase from rat brain. Biochim Biophys Acta. 616(2):199-207.




Related Links
ADK enzyme
ADK Phosphorylation Assay Kit
dCK Assay Kit
dCK enzyme
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