Kinase Profiling Panels

Applications:

  • Rapidly screen test compounds or compound libraries at single concentration
  • Quantitate inhibition in dose response titrations, measure IC50s
  • Measure selectivity of an inhibitor against a kinase profiling panel
  • Measure the impact of full-length vs kinase domain constructs on test compounds

Cell-free KinaseSeeker™ Assays

Luceome offers two assay formats for evaluating kinase inhibitors. The Cell-free KinaseSeeker assay uses the proprietary three-hybrid split-luciferase technology, where the two luciferase fragment-fusion proteins are expressed in rabbit reticulocyte lysate and the reassembly of split-luciferase, mediated by a CID-probe, takes place in this cell-free system. Competitive displacement of the CID-probe by a kinase inhibitor is measured by a change in luminescence signal. The assay is a homogeneous binding assay, which enables rapid kinase profiling in a high-throughput format.

Cell-free-KinaseSeeker-assay-2

Increasing examples have shown that regulatory domains outside of the kinase domain can greatly influence the activation state of the kinase and consequently impact inhibitor binding. The KinaseSeeker assay platform is highly amenable to full-length/intracellular domain testing. These full-length and intracellular-domain containing kinases provide for numerous opportunities to investigate autoinhibited states, conformation-selective and allosteric inhibitors that otherwise would not be available in a standard kinase panel. Luceome has over 90 such constructs (across multiple kinase families) available for profiling in the cell-free format.

A Type II inhibitor PDGFRa

Type II inhibitor PDGFRA

B Type I inhibitor LCK

Type I inhibitor LCK

(A) PDGFRa binding to a Type II inhibitor. Type II inhibitor binds the autoinhibited, juxtamembrane containing PDGFRa-intracellular module with reduced affinity (IC50 > 10 µM), compared to the PDGFRa-kinase domain (IC50 = 44 nM). (B) LCK binding to a Type I inhibitor. A dramatic increase in binding potency is observed with the LCK-kinase domain (IC50 = 0.14 nM) over the SH2-SH3 domain containing LCK-full length construct (IC50 = 32 nM).

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