How it Works

KINOMEscan Assay Overview

The KINOMEscan screening platform employs a novel and proprietary active site-directed competition binding assay to quantitatively measure interactions between test compounds and more than 450  kinase assays and disease relevant mutant variants. This robust and reliable assay technology affords investigators the ability to extensively annotate compounds with accurate, precise and reproducible data. KINOMEscan assays do not require ATP and thereby report true thermodynamic interaction affinities, as opposed to IC50 values, which can depend on the ATP concentration.

Assay Process

  1. 1.  Assemble Assay Components

      E. coli or mammalian cell-expressed kinase labeled with DNA tag for qPCR readout
  1.       Known active site binding ligand immobilized on a solid support
  2.       Test compound or DMSO control

  3. 2.  Equilibrate

  4. 3.  Wash solid support to remove unbound kinase

  5. 4.  Quantify kinase captured on solid support (qPCR)

  6. 5.  Compare captured kinase levels in test compound and DMSO control samples


Assay Principle

Compounds that bind the kinase active site and directly (sterically) or indirectly (allosterically) prevent kinase binding to the immobilized ligand, will reduce the amount of kinase captured on the solid support (Panels A & B). Conversely, test molecules that do not bind the kinase have no effect on the amount of kinase captured on the solid support (Panel C). Screening “hits” are identified by measuring the amount of kinase captured in test versus control samples by using a quantitative, precise and ultra-sensitive qPCR method that detects the associated DNA label (Panel D). In a similar manner, dissociation constants (Kds) for test compound-kinase interactions are calculated by measuring the amount of kinase captured on the solid support as a function of the test compound concentration.

          Panel A                  Panel B                  Panel C                       Panel D

How it works technology schematic
How it works technology schematic binding readout

Platform Benefits & Features

  • Rapid turnaround time
  • Largest commercially available kinase assay panel
  • Flexible: screen any number of compounds against any number of kinases
  • Custom panel capabilities to support ongoing drug discovery programs
  • High throughput: >4000 compounds per month screened across full panel (>1.7 million interactions)
  • Accurate affinity (Kd) measurements from <100 pM to >10 uM
  • ATP not required: delivers true thermodynamic affinity data as opposed to IC50s
  • Includes clinically relevant catalytically inactive pseudokinases (e.g. ERBB3)
  • Includes activation state-specific assays that provide inhibitor binding mode information
  • No interference from compound fluorescence
  • All assays performed in parallel under standardized conditions as a single experiment
  • Accurate, precise, reproducible data


Performance Metrics & Data Quality

Assay Quality: Z’ values

  • Z’ values measure assay noise and predictive value
  • High Z’ values reproducibly measured across entire assay panel
  • High Z’ values ensure low false positive/negative rates

Assay Quality
Z' Factor Analysis - one metric of assessing assay quality
Average Z' values and standard deviations were calculated for each kinase based on fourteen control wells per experiment in over 135 independent experiments spanning a period of sixteen months. Average Z' = 0.71 [click graph to enlarge].
  

Assay Quality: Primary screen reproducibility

  • Outstanding screen to screen reproducibility
  • Enables data to be interpreted with confidence


Primary Screen Reproducibility & Data Consistency
Correlational values for four compounds over time
Profiling of the indicated compounds at 10uM in fourteen independent experiments against 442 kinases over a one year period. Correlation analysis was performed in a pair-wise comparison to calculate the correlation coefficient. The correlation coefficients range from 0.91 to 0.97 with an average of 0.95 [click graph to enlarge].



Primary screen data interpretation

  • Association between primary screen data and affinity (Kd)
  • Facilitates primary screen data analysis
  • Provides guideline to choose screening hits for Kd follow-up

Relationship Between Primary Data & Binding Constants (Kds)
Data correlation between primary and binding constants
Association between primary screening data (10uM concentration) and binding affinity (Kd values). Binding constants are compared with primary screening results, where lower POC values are associated with lower Kd values (higher affinity interactions) [click graph to enlarge].




Dynamic range for accurate affinity measurements

  • Accurate affinity measurements across >5 logs Kd values
  • Distinguish pM inhibitors from nM inhibitors

Assay Sensitivity
Assay Sensitivity and Range

Binding constant (Kd) determinations for the indicated compounds against LOK demonstrate the broad range (> 5 logs) of interaction affinities quantitatively measured using the KINOMEscan assay platform. Assays are performed at low kinase concentrations (<0.1 nM), which enables the measurement of accurate Kd values in the pM range [click graph to enlarge].