• 
AK Inhibition - Contract Research Service for In vitro Screening
Once released outside the cell, adenosine, a naturally occurring ribonucleoside, reveals strong neuroprotective and anti-inflammatory properties. One of the strategies to increase extracellular adenosine consists in inhibiting adenosine kinase (AK). Thus, adenosine kinase is an attractive and experimentally validated target for the development of new analgesic and anti-inflammatory agents(1). In addition, AK has recently emerged as a novel target to predict and to prevent epileptogenesis(2,3). The X-ray crystallographic structure of human AK has been described(4) and provides structural basis for rational design and optimisation of new AK inhibitors.
Our Contract Research Service for AK Inhibition screening is performed on Human recombinant Adenosine Kinase (Ref. E-Nov5), produced and purified by NOVOCIB. The assay was validated with a known AK inhibitor (A-134974, IC50=20nM) and meets the requirements of a convenient and reliable HTS assay (microplate format, “add-and measure”, spectrophotometric continuous readout, Z'-Factor = 0.68).
Once released outside the cell, adenosine, a naturally occurring ribonucleoside, reveals strong neuroprotective and anti-inflammatory properties. One of the strategies to increase extracellular adenosine consists in inhibiting adenosine kinase (AK). Thus, adenosine kinase is an attractive and experimentally validated target for the development of new analgesic and anti-inflammatory agents(1). In addition, AK has recently emerged as a novel target to predict and to prevent epileptogenesis(2,3). The X-ray crystallographic structure of human AK has been described(4) and provides structural basis for rational design and optimisation of new AK inhibitors.
Our Contract Research Service for AK Inhibition screening is performed on Human recombinant Adenosine Kinase (Ref. E-Nov5), produced and purified by NOVOCIB. The assay was validated with a known AK inhibitor (A-134974, IC50=20nM) and meets the requirements of a convenient and reliable HTS assay (microplate format, “add-and measure”, spectrophotometric continuous readout, Z'-Factor = 0.68).
Aim: Screening of compounds for their abilities to inhibit human Adenosine Kinase (AK, ADK) in vitro. Determination of the inhibition kinetics of a given compound on human recombinant AK and measurement of its IC50 value.
Performance of the Screening Assay: Z'-Factor was calculated from a series of 40 positive (ADK inhibition) and negative (no ADK inhibition) control assays run on a 96-well microplate. Z'-Factor value was 0.68, which indicates an excellent assay performance.
Description: Assays are run using a human recombinant Adenosine Kinase, cloned, produced and purified by NOVOCIB. In vitro screening for AK inhibition is based on a spectrophotometric measurement of Adenosine Kinase activity using a coupling reaction with IMPDH. For further details, please refer to our "ADK Screening Assay Kit".
The assays are performed on 96-well microplate, at 37°C, in 200µl of reaction mixture. Compounds A-134974 (Sigma-Aldrich, under licence from Abbott Laboratories), is used as positive control for AK inhibition. Other positive control can be used if available. Both negative and positive controls are done in duplicate.
- For Screening Assays, positive control consists in inhbiting AK with A-134974 at final concentrations of 500nM. It is run in duplicate for every assay.
In case of positive signal during the screening, assays are run to control that IMPDH is not inhibited by the compound. Then, IC50 of the compound can be obtained in parallel to A-134974 inhibition curve. Additionally, specificity of potential ADK inhibitor can also be checked through our dCK Inhbition assay.
- For Inhibition Kinetics (IC50), the inhibition curve used as a positive control is obtained with A-134974 at 8 or 12 different concentrations, equally spaced by 3- or 2-fold dilutions respectively, to cover a 3.3-log wide range.
 • half of the data points +/- 1 above the IC50 value or half +/- 1 below • well-defined top and bottom plateau values, at least within a 15% margin of theoretical values. * Abiding by these constraints depends on the availability of information about the compound before starting the assay. When the results of the assay do not meet two of these three constraints, whereas AK inhibition by the compound is demonstrated, an additional assay can be performed with ad hoc alterations of the procedure (e.g. inhibitor concentration range, additional points…) |
Ask for Quotation