challenges of dating a man in a wheelchair - Validating aurora b as an anticancer drug target

The assay has to be very robust with a high Z-factor [18] and easy to scale up.

validating aurora b as an anticancer drug target-36

In a first trial, we developed an ELISA assay in 96-well plates to follow the phosphorylation of histone H3, a well-known substrate of aurora kinases.

Unfortunately, the black plastic microplates required for fluorescent reading on the robot were not suitable for coating the basic histone.

Several of these ATP-competitive inhibitors are currently in clinical development.

Most of them including benzo[e]pyridoindoles were identified by high throughput screening (HTS) [14].

Finally we decided to measure the consumption of ATP by kinase-Glo Figure 1. (A) Description of the kinase assay: recombinant histone H3 was phosphorylated by the catalytic domain of aurora A under a non-saturating ATP concentration.

Remaining ATP was measured by the addition of kinase Glo; (B) Representation of a typical HTS result.

assays, we performed a High Throughput Screening (HTS) to identify kinase inhibitors among 10,000 small chemical compounds.

In this didactic paper, we describe step-by-step the approach to validate the hits as well as the major pitfalls encountered in the development of active molecules.

When the kinase is inactive the ATP concentration is high.

Each point represents a molecule, the yellow points are controls in the presence of staurosporine and blue points the full active kinase in the absence of molecule. We selected molecules that inhibit aurora kinase by more than 80% at 15 µM.

We selected molecules that inhibit aurora kinase by more than 80%.

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