In Vitro Safety Pharmacology Profiling

Reaction Biology's in vitro safety assays allows for off-target screening of new drug candidates for early safety pharmacology assessment. Inhibition of unwanted targets is often associated with adverse drug reactions in animal models and clinical studies. In vitro safety pharmacology profiling involves the screening of compounds against a broad range of targets that may cause adverse drug reactions in humans including receptors, transporters, enzymes, and ion channels.

Predicting potential safety liabilities early in the drug discovery process is integral for lead compound selection. In vitro safety screening enables selectivity-focused structure-activity relationship studies to mitigate off-target effects while retaining or increasing the compound's potency at the primary target.

We have established in vitro safety assays for compound screening against an assortment of targets involved in toxicity issues in humans. Available assay formats include enzymatic assays, biochemical binding assays, and radioligand binding assays.

The targets include: 

  • GPCRs
  • Nuclear receptors
  • Cytochrome P450s 
  • Ion channels
  • Other targets

 

The InVEST Panel

Safety screening at Reaction Biology can be performed with our In Vitro Evaluation of Safety and Toxicity (InVEST) panel to investigate your compound's effects on a large selection of targets. Adding your compound to our monthly screening runs is an efficient and economical way to address your in vitro safety screening needs. 

  • The InVEST panel allows monthly screening runs of our panel.
  • Cost-efficient screening setup with single concentration testing in duplicates. 
  • Direct contact with our safety pharmacologists.
  • IC50 values of reference controls are included for each assay.

Our team of in vitro safety pharmacologists is available to discuss your research needs to ensure you perform the right assays at the right time during your drug discovery endeavor. Get in contact today.

The InVEST Panel

Early safety profiling with the In Vitro Evaluation of Safety and Toxicity (InVEST) panel includes 35 targets of 6 target families for broad coverage of potential adverse drug effects. 

All of the targets in the InVEST panel are clinically relevant. Their inhibition was shown to cause potentially serious health problems.  

In-vitro-safety-screening

Optional: Functional Ion Channel Panel

In addition to compound profiling with the InVEST panel, clients have the option to investigate the cardiac liability of new drug candidates against our ion channel panel. Performed with whole-cell patch clamp technique, the results reveal whether a compound inhibits cardiac ion channel function in live cells. Advanced cardiac safety assays including testing on the CiPA panel to comply with regulatory guidelines as well as testing on cardiac tissue or isolated hearts can be performed. Please read more on our cardiac safety assessment website.

Category Target name HGNC reference Synonyms Data
Calcium Ion Channel Cav1.2 Ion Channel CACNA1C Cav1.2, CACH2, CACN2, TS, LQT8, Voltage-dependent L-type calcium channel subunit alpha-1C Data sheet
Potassium Ion Channel hERG Ion Channel KCNH2 potassium voltage-gated channel subfamily H member 2, Ether-a-go-go-related gene potassium channel 1, Kv11.1 Data sheet
Sodium Ion Channel Nav1.5 Ion Channel SCN5A sodium voltage-gated channel alpha subunit 5, Nav1.5 LQT3, HB1, HBBD, PFHB1, IVF, HB2 HH1 SSS1 CDCD2, CMPD2, ICCD Data sheet

Additonal information to InVEST Panel Screening

Assay setup

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.

Assay formats

Radioligand Binding Assay 

safety-pharmacology-profiling

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

safety-pharmacology-profiling

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. 

Screening schedule

The next upcoming run starts on April 18th. The compound receiving deadline is April 13th. 

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

Example data

Fluorescence Polarization Assay with a Nuclear Receptor

safety-testing-progesterone

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

safety-testing-hERG

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

radioligand assay 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

safety-pharmacology-profilingThree 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

safety-pharmacology-profilingReference 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