In Vitro Safety Pharmacology Profiling

Setups: Standard panel testing consists of single concentration testing in duplicates. Alternate testing formats may be available upon request.  

Controls:
Binding assays: Total binding (DMSO vehicle) and non-specific binding (containing saturating concentration of reference compound) and a full concentration-response curve with an appropriate reference compound are conducted with each target.
Enzymatic activity assays: No inhibitor control (DMSO vehicle) and a full concentration-response curve with an appropriate reference compound are conducted with each target.

Turnaround time: 10 business days for standard projects. Expedited scheduling and data delivery can be arranged prior to the commencement of the studies. 

Report: The raw data, % inhibition, and control compound IC50 values will be reported in Excel format.  Assay conditions, target, and substrate information are available upon request. Requirements for this information should be noted prior to the commencement of the study.

Screening facility: This assay is performed at our screening facility in Malvern, PA, US.

Compound requirements: In brief, for a standard project with a final test concentration of 10 µM we require 100 µl of a 10 mM stock solution in DMSO or 5 mg powder. Less material per test is needed for large-scale screening. Please refer to our FAQs for information regarding compound preparation and shipping.

Radioligand Binding Assay 

A. Commercially sourced membrane preparations and prepared rat brain membranes expressing the target receptors, respectively, are placed in multi-well plates. B. ³H-labelled ligands incubate with the membrane preparations in the presence and absence of test compounds for 90 minutes. C. The solution is pulled through a filter trapping the membranes and bound ligands. Unbound ligands are not retained on the filter. The radioactivity in the filter is measured via scintillation counting as a readout of ligand-target receptor binding. 

Fluorescence Polarization

The fluorescence polarization (FP) assays are binding assays where a ligand conjugated to a fluorophore binds to the target receptor.
A. The FP assay is performed with either soluble protein or (in the case of hERG) membrane preparations. B. The client compound and the fluorescent ligand incubate with the target. C. Ligand binding is quantified by measuring the amount of polarized light emitted by the fluorophores in the well. 

Principle: Light waves, passed through certain crystalline materials (polarizers), have their electrical vectors oriented in a single plane and are thus polarized. The fluorophores linked to the ligand absorb the light and emit fluorescence. Soluble, small fluorescent ligands have a high rotational movement (Brownian movement) that is shorter than their fluorescence decay time resulting in their emitted fluorescence light being depolarized. Fluorescent ligands bound to the larger receptor have a rotational movement that is slower than their fluorescence decay time thus, their emitted light remains polarized. 
 

Cell-based transcriptional regulation

Receptor-specific reporter cells express the luciferase reporter gene functionally linked to a bona fide receptor-responsive promoter. Thus, quantifying changes in luciferase expression in the treated reporter cells provides a sensitive surrogate measure of the changes in receptor activity. This assay format is suitable to investigate agonistic and antagonistic responses of the target receptor.
 

Enzymatic activity

Assays for testing the activity of an enzymatic target measure modifications to the target substrate. More information can be found for cytochrome P450, phosphodiesterases, or protease targets. 

Testing of the InVEST Panel is performed on a monthly basis. Please reach out to learn about upcoming screening dates. 

Fluorescence Polarization Assay with a Nuclear Receptor

Progesterone Receptor Fluorescence Polarization: Results of three independent experiments of progesterone binding are shown. 95% CV shown as dashed lines. Sigmoid fit (solid line) parameters: IC50=36.9 nM, Hill slope=−2.13. 95% confidence intervals: EC50: 30.4 to 44.8 nM, Hillslope: ‑3.0 to ‑1.2.

Fluorescence Polarization Assay with an Ion Channel

hERG Receptor Fluorescence Polarization: Results of six independent experiments of E-4031 binding to hERG containing membranes are shown.Average of 6 independent experiments. 95% CV shown as dashed lines. Competing drug: E-4031. Sigmoid fit (solid line) parameters: IC₅₀=20.9 nM, Hill slope=−1.46. 95% confidence intervals: IC₅₀: 14.7 to 29.7 nM, Hillslope: ‑-2.08 to ‑0.84.

Radioligand Assay with a GPCR

Histamine H1 Receptor Radioligand Binding Assay: Results of one experiment performed in duplicates of binding of reference compound pyrilamine to Histamine H1 receptor are shown.

Sigmoid fit parameters: EC50=1.25 nM, Hill slope=−0.99.

Enzymatic Activity Assay with a PDE

Three reference compounds IBMX, methoxyquinazoline, and Rolipram were tested against the activity of cAMP-specific cyclic phosphodiesterase 4A (PDE4A). Concentration-response curves are shown with semi-log concentrations in singlicates with the following parameters:

IBMX: IC₅₀= 1.4e-05, hillslope= -0.72
Methoxyquinazoline: IC₅₀= 7.82e-07, hillslope= -0.86
Rolipram: IC₅₀= 1.1e-06, hillslope= -0.86

Enzymatic Activity Assay with a Protease

Reference inhibitor gabexate mesylate (GM) was tested against thrombin for IC50 value determination with semi-log concentrations in singlicate. The fluorescent substrate Pefafluor TH containing AMC was used as a substrate. Parameters: IC₅₀= 5.9e-07, hillslope= -0.94