Fluorescence-based Detection of Kinase Allostery for High-throughput Allosteric Inhibitor Discovery

FRET Technology Identifies Allosteric Structural Changes within Proteins

A new protein kinase biosensor uses Förster resonance energy transfer (FRET) technology to identify allosteric binders that trigger conformational changes within the activation loop of kinase proteins.  The method can identify allosteric small molecule inhibitors or peptide inhibitors using a high-throughput screening method. The unique components of this technology include:

• specific dye placement and labeling strategies,
• high-throughput measurement,
• specific design of the screening assay,
• analysis of steady-state and time-resolved fluorescence data
• FRET-based structural information on the allosteric effects of inhibitors
• applying the assay to identify new allosteric inhibitors of Aurora A and other biomedically important protein kinases

Highly Sensitive, Robust Method Detects Allosteric Inhibitors of Kinase Proteins

Protein kinases are major drug targets in oncology, but existing inhibitors are ATP-competitive and poorly selective for specific kinases. Most kinase inhibitor screening assays measure kinase inhibition or ligand binding and cannot distinguish between allosteric and conventional inhibitors. No robust assay technology currently exists that can directly identify allosteric kinase inhibitors and distinguish them from ATP-competitive inhibitors that bind without causing a conformational change. This new technology offers a screening assay for drugs that act as protein kinase inhibitors and identifies drugs that inhibit protein kinases by means other than inhibiting ATP binding in the enzyme active site. The method detects allosteric inhibitors of kinase proteins using a FRET based high-throughput assay that directly monitors structural changes in the kinase domain upon ligand binding. The assay measures an important structural parameter of the drug target, the position of the kinase activation loop, giving direct information on the nature of the induced structural change, and allowing it to distinguish between different subtypes of allosteric inhibitors. The assay is highly sensitive and robust and has considerable potential to accelerate allosteric kinase inhibitor discovery.

BENEFITS AND FEATURES:

• Determines allosteric inhibitors of kinase proteins using a FRET based high-throughput assay
• Identifies allosteric inhibitors of kinases
• Identifies completely new classes of drugs that inhibit protein kinases
• Additional features as needed

APPLICATIONS:

• Drug development for protein kinase inhibitors
• Identification of allosteric inhibitors of kinases, including Aurora A
• Drug screening assays for allosteric kinase inhibitor discovery
• Treating cancer and inflammatory and immunological diseases dictated by kinase activation
• Assay design
• Pharmaceuticals

Phase of Development - Proof of concept assay created.