Study Reports & Data Analysis

Study report interpretation and analysis: getting the most out of your KINOMEscan study report

Study Report Contents

Primary Study Data Reports

Primary Study results are delivered as three separate data files and include:

1. Study Report - A PDF file containing a project summary, description of the assay protocol, color coded data matrix which provides a view of compound potency against the tested kinases, and TREEspot™ compound/kinase interaction maps for each compound tested (view primary sample report).

2. Data Report - A spreadsheet file containing average values for both replicates (reported as Percent of Control, or %Ctrl) of all compound/kinase interactions, which can be easily imported into most data management and visualization tools (including KINOMEscan TREEspot Profile Visualization Tool).

3. S-Scores Report - A spreadsheet containing S-Score values for each compound
(view primary sample report).

     Sample Primary Study Report Data Matrix

 

KdELECT Study Data Reports (11-point dose response curves)

KdELECT Study reports are delivered as three separate data files and include:

1. Study Report - A PDF file containing a project summary, description of the assay protocol and algorithm used to fit curves, color coded data matrix which provides a Kd values for each compound against the tested kinases and curve images which display the data points, and the best fit curve for each Kd determination (view Kd sample report).

2. Data Report - A spreadsheet file containing the average Kd value reported from duplicate Kd determinations (reported in nM), which can be easily imported into most data management and visualization tools (including TREEspot Profile Visualization Tool).

3. Raw Data Report - A spreadsheet containing data values obtained for each replicate (reported as relative signal) for each compound at each tested concentration.

     Sample Curve Images

 

Data Analysis & Interpretation

Definitions

Percent of Control (%Ctrl)

The results for single concentration (primary screen) binding interactions for tested compound(s) are reported in your study report and spreadsheets as '%Ctrl' and is calculated in the following manner:

    test compound = client supplied compound
    negative control = DMSO (100% control)
    positive control = control compound (0% control)

Binding Constant (Kd)

The results for an 11-point dose response curve compound/kinase interactions are reported in your study report and spreadsheets as Kd, which are values derived using the Hill equation:

The Hill Slope is set to -1. Curves are fitted using a non-linear least square fit with the Levenberg-Marquardt algorithm.

Selectivity Score (S-Scores)

Selectivity Score or S-score is a quantitative measure of compound selectivity. It is calculated by dividing the number of kinases that compounds bind to by the total number of distinct kinases tested, excluding mutant variants.


This value can be calculated using %Ctrl as a potency threshold (below) and provides a quantitative method of describing compound selectivity to facilitate comparison of different compounds.

     S(35) = (number of non-mutant kinases with %Ctrl <35)/(number of non-mutant kinases tested)
     S(10) = (number of non-mutant kinases with %Ctrl <10)/(number of non-mutant kinases tested)
       S(1) = (number of non-mutant kinases with %Ctrl <1)/(number of non-mutant kinases tested)

Using S-Score Data to Quantitate Selectivity

Selectivity Profile for 38 Small Molecule Kinase Inhibitors

 

TREEspot Kinase Dendrograms: A Visual Representation of Compound Selectivity

TREEspot is an innovative compound profile visualization tool for visualizing screening data. Kinases found to bind are marked with red circles, where larger circles indicate higher-affinity binding. 

RSK Inhibitor                  

 

TREEspot Compound Profile Visualization Tool

TREEspot is an innovative, secure access, web-based, compound profile visualization tool for analysis of KINOMEscan screening data. TREEspot is an ideal companion tool for existing data analysis applications and facilitates compound profile visualization through its simple yet powerful user interface.

Gain new perspectives of kinase profile data in a visual environment and distill essential knowledge to drive your discovery programs.  TREEspot is provided as a complimentary tool to our clients. To learn more about how you can visualize your data using TREEspot or to request access credentials, please contact KINOMEscan.

• Easy-to-use
• Facilitates evaluation and analysis of profiling data
• Generates publication quality of TREEspot images
• Provides global visualization of profile data
• Visualize your data in a whole new way

Additional Follow Up Services

KdELECT

An ideal follow-on service for Primary Screening. Generate a quantitative Kd value (binding constants) of compound/kinase interactions observed in initial KINOMEscan study data. KdELECT is performed in an 11-point dose-response curve (in duplicate) and can be requested for any number of kinases making it an ideal tool for follow up primary study follow up.

• 11-point dose-response curve performed in duplicate
• Consistent & reproducible results
• No minimum number of kinases or compounds required
• Can be performed against any of our 451 kinase assays

 

Investigative Tools & MOA Services

Learn more about your kinase inhibitor’s biochemical mechanism of action.  KINOMEscan offers a suite of investigative tools that provides a detailed biochemical characterization of the interaction between inhibitors and their targets.  The thermodynamic, kinetic, and structural information provided by these tools enables a detailed comparison of inhibitors from common or distinct lead series and facilitates the interpretation of data from downstream cellular and in vivo pharmacology models.

 

Frequently Asked Questions

Is there a correlation between the data obtained in single concentration primary screens and Kd determinations?

Yes. Based on screening data from thousands of profiled compounds, a proportional relationship between primary screening results and corresponding affinities may be described. Evident in the correlation graph below is a range of binding constants (Kd values) for the indicated ranges of POC values with tighter binding (higher affinity) interactions associated with lower POC values and weaker binding (lower affinity) associated with higher POC values. This distribution of binding constants is characteristic of single concentration primary screens and underscores the importance of following up observed ‘hits’ or apparent high affinity interactions with quantitative binding constant determinations. For more information on Kd determinations, please visit our KdELECT service page.

Relationship between Binding Constant Distribution (Kds)& Single Concentration 10uM Primary Screen Values

Can %Ctrl be converted to Percent Inhibition?

Although data derived from competition binding assays is fundamentally different from that obtained in activity assays, there is generally good agreement between the data sets. This allows data comparisons and assessments to be made between KINOMEscan binding data (POC) and inhibition data derived from other sources. If desired, POC values may be converted to percent competition by taking the inverse of the POC value:

     (100 – POC) = Percent Competition
     Eg: 30 POC = (100- 30) or 70 Percent Competition

What is the concentration of ATP in your assays?

ATP is not required for the KINOMEscan competition binding assay platform. Therefore, no ATP is added to the assay which avoids artifacts and variations that can be associated with performing assays in the presence of different ATP concentrations.

 

Where can I learn about the technical specifications of KINOMEscan assays?

Full technical details for each assay including expression, reference compound information, and other technical details can be readily accessed on individual Technical Data Sheets.

 

What is the importance of profiling against mutant kinases?  What diseases are associated with mutant kinases that my compound inhibits?

Mutated kinases have been implicated as causative agents in a diverse range of diseases, including many forms of cancers. These same mechanisms can also confer resistance to therapeutic agents thereby rendering them ineffective or lead to relapse after initial positive response. The availability of disease relevant mutant kinases affords investigators an important tool to elucidate the mechanisms of action for their compounds, reveal novel therapeutic opportunities, or as starting points for next generation drugs.  For a list of mutant kinases available in our panel and associated disease-relevance, please visit our Mutant Kinase Reference page.

 

Does the affinity of the ligand for a respective kinase used in the assays affect affinity measurements of my compounds?

No. Binding assays are performed under conditions in which binding constants measured for the interaction between kinases and test compounds are independent of the affinity of the immobilized ligand for the kinase. This has been experimentally verified by measuring binding constants for several kinase/test compound combinations using different immobilized ligands (for some of the kinases, more than one immobilized ligand can be used to build an assay). Test compound binding affinity did not change with the immobilized ligand used and confirms that results are independent of the affinity of the immobilized ligand for the kinase. Additional details regarding compound binding constant relative to affinity of bait can be found on p.335, of Fabian, M. et al. (2005) and supplementary details of Wodicka, L. et al. (2010)

 

Can you provide a list of known liability or safety kinases associated with off-target toxicities that my compound should not hit?

Although off-target liabilities can be program and target specific, there are kinases which have been described in literature as being associated with varying degrees of toxicity.  For example, Olaharski, A. et al. PLoS Comput. Biol. 5, 1-10 (2009) reported on a panel of kinases found to be predictive of a positive micronucleus test result, a sign of potential chromosomal damage, while Cheng, H. et al. Prog. Cardiovasc. Dis. 53, 114–120 (2010), describe a number of kinases thought to be at least partially responsible for cardiotoxicity observed in existing therapeutics.

 

What is the standard immobilized ligand used in your competition binding assays?

We utilize over 30 different small molecule active-site directed ligands, each extensively tested and validated to deliver high quality, consistent data.  A few of the inhibitors we use include Staurosporine and Purvalanol B.

 

What is the naming convention used your assays? For example, what does EGFR(L858R) mean?

This naming convention is frequently used to denote the presence of an amino acid mutation or deletion in the sequence and its location.  For example, EGFR(L858R) is kinase EGFR where Leucine (L) at position 858 was replaced with an Arginine (R).  Similarly, EGFR(E746-A750del) is a deletion of amino acids starting with Glutamic acid (E) at position 746 to Alanine (A) at position 750.