PathHunter Primary Study Data Reports
Primary Study results are delivered as two separate data files and include:
1.
Study Report - A PDF file containing a project summary, description of the assay protocol, control agonist dose response curves for each target (where applicable), and data matrix (
view primary sample report).
2. Data Report – An Excel spreadsheet file containing duplicate raw data values, average % activity or % inhibition of all compound/target interactions and summary tables.
Sample Primary Study Report Data Matrix
PathHunter EC50/IC50 Determination Study Reports (10-point dose response curves)
PathHunter EC50/IC50 Determination Study reports are delivered as two separate data files and include:
1. Study Report - A PDF file containing a project summary, description of the assay protocol, control agonist dose response curves for each target (where applicable), data matrix which provides EC50/IC50 values for each compound against the tested targets, curve images which display the data points and the best fit curve for each EC50/IC50 determination (
view PathHunter EC50/IC50 sample report).
2.
Data Report - A spreadsheet file containing the raw data values reported from duplicate EC50/IC50 determinations, which can be easily imported into most data management and visualization tools.
Sample Curve Images
Data Analysis & Interpretation
Definitions
Percent Activity/Inhibition
The results for single concentration (primary screen) for tested compound(s) are reported in your study report and spreadsheets as '% Activity' or '% Inhibition' and are calculated in the following manner:
% Activity(Agonist Mode) =
100% x
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[
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Mean RLU (test sample)- Mean RLU(vehicle control)
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]
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Mean MAX RLU (control ligand)- Mean RLU (vehicle control)
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test sample = client supplied compound
vehicle control = DMSO (0% activity)
control ligand = control compound (100% activity)
% Inhibition (Antagonist mode) = 100% x
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[
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1-
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Mean RLU (test sample)- Mean RLU(vehicle control)
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]
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|
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Mean MAX RLU (control ligand)- Mean RLU (vehicle control)
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test sample = client supplied compound
vehicle control = DMSO (0% activity)
EC80 control = control compound (80% activity)
Dose Response Curve (EC50/IC50)
The results for 10-point dose response curve compound/target interactions are reported in the study report and spreadsheets as EC50/IC50, which are values derived using the Hill equation:
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 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.
Frequently Asked Questions
How does EFC work?
Enzyme Fragment Complementation (EFC) is a non-radioactive, mix-and-read detection system based on two genetically engineered fragments of β-galactosidase: a large protein fragment, called Enzyme Acceptor or EA and a small peptide fragment called Enzyme Donor or ED. Separately, the β-galactosidase fragments are inactive. But in solution, the fragments rapidly recombine to form active enzyme that is capable of hydrolyzing substrate and producing a chemiluminescent or fluorescent signal.
How do PathHunter Arrestin GPCR cell-based assays work?
PathHunter® β-Arrestin GPCR assays are whole cell functional assays that directly measure GPCR activity by detecting the interaction of β-Arrestin with the activated GPCR. In this system, the GPCR is fused in frame with the small, 42 amino acid fragment of β-gal called ProLink™ and co-expressed in cells stably expressing a fusion protein of β-Arrestin and the larger, N-terminal deletion mutant of β-gal (called enzyme acceptor or EA). Activation of the GPCR stimulates binding of β-Arrestin to the ProLink-tagged GPCR and forces complementation of the two enzyme fragments, resulting in the formation of active β-gal enzyme. This action leads to an increase in enzyme activity that can be measured using chemiluminescent PathHunter® Detection Reagents. Because Arrestin recruitment is independent of G-protein signaling, these assays offer a powerful and universal screening and profiling platform that can be used for virtually any Gi-, Gs, or Gq-coupled receptor.
How do PathHunter Internalization cell-based assays work?
PathHunter® GPCR Internalization assays are whole cell functional assays that measure GPCR receptor recycling and desensitization. These non-imaging, non-antibody-based chemiluminescent assays, provide a direct and quantitative measurement of internalized GPCR localized to intracellular endosomes and allow the fate of unlabelled, activated GPCRs to be monitored in live cells without the need for expensive microscopy.
PathHunter Activated GPCR Internalization Assays detect Arrestin-mediated GPCR internalization. In this system, the small enzyme fragment of β-galactosidase called ProLink™ (PK) is localized to intracellular endosomes and the larger complementing enzyme fragment termed Enzyme Acceptor or EA is fused to β-Arrestin. Stimulation of the receptor results in Arrestin binding to the activated GPCR, internalization of the receptor and trafficking to cellular endosomes. In the PathHunter Total GPCR Internalization System, the large portion of β-galactosidase called Enzyme Acceptor or EA is localized exclusively to intracellular endosomes and PK is fused to the GPCR of interest. GPCR activation results in internalization of the receptor and trafficking to endosomes.
How do PathHunter® Cell-Based Kinase assays work?
PathHunter Receptor Tyrosine Kinase cell lines express a full length receptor tyrosine kinase (RTK) protein fused in frame with a small epitope tag called ProLink™ on the intracellular C-terminus of the receptor. These cells co-express a fusion protein of the SH2 domain protein and the larger, N-terminal deletion mutant of β-gal (called enzyme acceptor or EA). Activation of receptor results in phosphorylation of RTK dimers and subsequent interaction with the SH2 protein which forces complementation of the two enzyme fragments, resulting in the formation of active β-gal enzyme. This action leads to an increase in enzyme activity that can be measured using chemiluminescent PathHunter® Detection Reagents.
How do Hit Hunter Cell-Based cAMP assays work?
DiscoverX have developed a panel of cell lines stably expressing non-tagged GPCRs that signal through cAMP. The Hit Hunter cAMP Hunter assay monitors the activation of a GPCR via Gi and Gs secondary messenger signaling in a homogenous, non-imaging assay format using a technology developed by DiscoverX called complementation. This utilizes an enzyme fragment complementation (EFC) assay with β-galactosidase (β-Gal) as the functional reporter. The enzyme is split into two complementary portions. ProLabel donor peptide is fused to cAMP and in the assay competes with cAMP generated by cells for binding to a cAMP-specific antibody. Active β-Gal is formed by complementation with EA to any unbound ED-cAMP. The active enzyme can convert a chemiluminescent substrate to generate an output signal detectable on a standard microplate reader.
How do Hit Hunter Cell-Based Calcium mobilization assays work?
The Calcium No Wash assay monitors the activation of a GPCR via Gq secondary messenger signaling in a live cell, non-imaging assay format. In this assay, a calcium sensitive dye is loaded into cells to allow for the real time detection of intracellular calcium mobilization. Activation of a GPCR by injection of a compound results in the release of calcium from intracellular stores and an increase in dye fluorescence that is measured in real time. PathHunter cell lines stability expressing GPCRs that are coupled to Gq can be readily used to measure intracellular calcium flux.
What are the benefits of PathHunter® Screening and Profiling Services?
- Largest commercially available portfolio of drug discovery targets
- Accurate pharmacology from rigorously validated cell lines
- Single assay format for detection in agonist, antagonist, inverse agonist, allosteric modulator modes
- Broad applications ideal for small molecule and biologics screening or profiling
- Competitive pricing and fast turn around times to expedite your discovery programs
How are PathHunter® and HitHunter® cell-based assays validated?
- Passage stability tested
- Control ligand dose response curves run on every plate, compared to historical values
- Growth media and assay media is optimized for every cell line
Can allosteric modulators be detected using the technology?
Yes. Both positive (PAM) and negative (NAM) allosteric modulators can be detected using DiscoverX PathHunter and HitHunter technologies
How is % Activity or % Inhibition calculated?
The results for single concentration (primary screen) for tested compound(s) are reported in your study report and spreadsheets as '% Activity' or '% Inhibition' and are calculated in the following manner:
test sample = client supplied compound
vehicle control = DMSO (0% activity)
control ligand = control compound (100% activity)
test sample = client supplied compound
vehicle control = DMSO (0% activity)
EC80 control = control compound (80% activity)
Can you determine the EC50/IC50 of my compound?
Yes. DiscoverX routinely performs ten-point dose response curves to determine EC50 and IC50s as part of our profiling services.
How are data reported?
PathHunter® Primary Study Data Reports are delivered as two separate data files:
1. Study Report (PDF file) that contains a project summary, description of the assay protocol, control agonist dose response curves for each target (where applicable), and data matrix.
View a sample report.
2. Data Report (excel file) that contains duplicate raw data values, average % activity or % inhibition of all compound/target interactions and summary tables.
View a sample report.
PathHunter® EC50/IC50 Determination Study Reports are delivered as two separate data files:
1. Study Report (PDF file) that contains a project summary, description of the assay protocol, control agonist dose response curves for each target (where applicable), data matrix which provides EC50/IC50 values for each compound against the tested targets and curve images which display the data points, and the best fit curve for each EC
50/IC
50 determination.
View a sample report.
2. Data Report (excel file) that contains the raw data values reported from duplicate EC50/IC50 determinations, which can be easily imported into most data management and visualization tools.
View a sample report.
What controls do we use for the assays?
Controls are used to define MIN and MAX activity for the assay. We also run agonist dose response curves.
How much DMSO can your assays tolerate?
We do not typically profile compounds that contain greater than 1% DMSO. Please
contact us for more information about this requirement
How do I order?
Visit our
Request a Quote page to select assays and request a no obligation quote.
How do I send my compounds?
Before shipping your compounds to DiscoverX, please carefully review our
Compound Preparation & Shipping Instructions. This documentation is included with each quote request and is available under the "Related documents" link on this page. Prepare and ship compounds using the included compound submission guidelines. You will receive an email confirmation from our compound management team upon receipt of compound(s).
How do I supply compounds, and how much do you need?
Compound may be provided as a stock DMSO solution or dry powder. For single concentration (primary) screens, we will need 20 ul of 1000x the desired screening concentration for the first target and an additional 2 ul for each target thereafter or an equivalent amount of dry powder such that when reconstituted, will yield the required volume.