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.

In Vitro Safety Screening Assay Formats

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.

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The InVEST Panel

Early safety profiling with the In Vitro Evaluation of Safety and Toxicity (InVEST) panel includes 53 targets of 7 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

List of the InVEST Panel (51 targets)

In Vitro Safety Panel
Target family	Target name	HGNC reference	Additional synonyms	Assay format	Species
Biogenic Amine Transporter	Serotonin Transporter	SLC6A4	Solute Carrier Family 6 (Neurotransmitter Transporter, Serotonin), Member 4, SET	Radioligand Filter Binding	Human
Cholinesterase	Acetylcholinesterase	ACHE	Acetylcholinesterase	Enzymatic Activity	Human
Cyclooxygenase	COX-1	PTGS1	PGHS-1, PTGHS, prostaglandin-endoperoxide synthase 1 (prostaglandin G/H synthase and cyclooxygenase)	Enzymatic Activity	Ovine
Cyclooxygenase	COX-2	PTGS2	prostaglandin-endoperoxide synthase 2 (prostaglandin G/H synthase and cyclooxygenase)	Enzymatic Activity	Human
Cytochrome P450	CYP1A2	CYP1A2	CYP 1A2	Enzymatic Activity	Human
Cytochrome P450	CYP2A6	CYP2A6	Cytochrome P450, family 2, subfamily A, polypeptide 6	Enzymatic Activity	Human
Cytochrome P450	CYP2B6	CYP2B6	CYP 2B6	Enzymatic Activity	Human
Cytochrome P450	CYP2C19	CYP2C19	CYP 2C19	Enzymatic Activity	Human
Cytochrome P450	CYP2C8	CYP2C8	Cytochrome P450, family 2, subfamily C, polypeptide 8	Enzymatic Activity	Human
Cytochrome P450	CYP2C9	CYP2C9	CYP 2C9	Enzymatic Activity	Human
Cytochrome P450	CYP2D6	CYP2D6	CYP 2D6	Enzymatic Activity	Human
Cytochrome P450	CYP2E1	CYP2E1	CYP 2E1, Cytochrome P450, family 2, subfamily E, polypeptide 1	Enzymatic Activity
Cytochrome P450	CYP2J2	CYP2J2	Cytochrome P450, family 2, subfamily J, member 2	Enzymatic Activity
Cytochrome P450	CYP3A4	CYP3A4	CYP 3A4	Enzymatic Activity	Human
Cytochrome P450	CYP3A5	CYP3A5	Cytochrome P450, subfamily IIIA, polypeptide 5	Enzymatic Activity
GPCR	Adenosine A1 Receptor	ADORA1	RDC7, Adenosine receptor A1	Radioligand Filter Binding	Human
GPCR	Adrenergic Alpha 1A Receptor	ADRA1A	Alpha-1A adrenergic receptor, adrenoceptor alpha 1A	Radioligand Filter Binding	Human
GPCR	Adrenergic Alpha 2A Receptor	ADRA2A	ADRAR, ADRA2, ADRA2R, Alpha-2A adrenergic receptor, adrenoceptor alpha 2A	Radioligand Filter Binding	Human
GPCR	Adrenergic Beta 1	ADRB1	Beta1 adrenergic receptor, adrenoceptor beta 1	Radioligand Filter Binding	Human
GPCR	Adrenergic Beta 2	ADRB2	Beta1 adrenergic receptor, adrenoceptor beta 2	Radioligand Filter Binding	Human
GPCR	CCK1 Receptor	CCL28	SCYA28, MEC, CC chemokine CCL28, C-C motif chemokine 28	Radioligand Filter Binding	Human
GPCR	Dopamine D1 Receptor	DRD1	D(1A) dopamine receptor	Radioligand Filter Binding	Human
GPCR	Dopamine D2S Receptor	DRD2	D(2) dopamine receptor	Radioligand Filter Binding	Human
GPCR	Dopamine D3 Receptor	DRD3	D(3) dopamine receptor	Radioligand Filter Binding	Human
GPCR	Histamine H1 Receptor	HRH1	Histamine H1 receptor, HH1R, H1R	Radioligand Filter Binding	Human
GPCR	Muscarinic M1 Receptor	CHRM1	Cholinergic receptor muscarinic 1, Muscarinic acetylcholine receptor M1	Radioligand Filter Binding	Human
GPCR	Muscarinic M2 Receptor	CHRM2	Cholinergic receptor muscarinic 2, Muscarinic acetylcholine receptor M2	Radioligand Filter Binding	Human
GPCR	Muscarinic M3 Receptor	CHRM3	Cholinergic receptor muscarinic 3, Muscarinic acetylcholine receptor M3	Radioligand Filter Binding	Human
GPCR	Muscarinic M4 Receptor	CHRM4	Cholinergic receptor muscarinic 3, Muscarinic acetylcholine receptor M3	Radioligand Filter Binding	Human
GPCR	Muscarinic M5 Receptor	CHRM5	Cholinergic receptor muscarinic 5, Muscarinic acetylcholine receptor M3	Radioligand Filter Binding	Human
GPCR	Opioid delta Receptor	OPRD1	opioid receptor, Delta-type opioid receptor delta 1	Radioligand Filter Binding	Human
GPCR	Opioid kappa Receptor	OPRK1	opioid receptor, Kappa-type opioid receptor kappa 1	Radioligand Filter Binding	Human
GPCR	Opioid mu Receptor	OPRM1	opioid receptor, Mu-type opioid receptor mu 1	Radioligand Filter Binding	Human
GPCR	Serotonin 5-HT1A Receptor	HTR1A	5-hydroxytryptamine receptor 1A, 5-HT1A, Serotonin receptor 1A	Radioligand Filter Binding	Human
GPCR	Serotonin 5-HT1B Receptor	HTR1B	5-hydroxytryptamine receptor 1B, 5-HT1B, Serotonin receptor 1B	Radioligand Filter Binding	Human
Ion Channel	GABA-A Receptor	GABRA1	gamma-aminobutyric acid type A receptor, GABA(A) receptor	Radioligand Filter Binding	Rat
Ion Channel	hERG	KCNH2	potassium voltage-gated channel subfamily H member 2, Ether-a-go-go-related gene potassium channel 1, Kv11.1	Fluorescence Polarization	Human
Monoamine oxidase	MAO-A	MAOA	MAO-A	Enzymatic Activity	Human
Monoamine oxidase	MAO-B	MAOB	MAO-B	Enzymatic Activity	Human
Nuclear Receptor	Androgen Receptor	AR	AIS, NR3C4, SMAX1. HUMARA, dihydrotestosterone receptor	Fluorescence Polarization	Human
Nuclear Receptor	Estrogen Receptor a	ESR1	ESR, NR3A1, Era, ER-alpha	Fluorescence Polarization	Human
Nuclear Receptor	Estrogen Receptor β	ESRRB	ERR-beta, ERRB2, Steroid hormone receptor ERR2, ERR beta-2, Estrogen receptor-like 2, NR3B2	Fluorescence Polarization	Human
Nuclear Receptor	Glucocorticoid Receptor	NR3C1	GR, GRL, nuclear receptor subfamily 3 group C member 1	Fluorescence Polarization	Human
Nuclear Receptor	PPARg Receptor	PPARG	PPARG1, PPARG2, NR1C3, PPARgamma, peroxisome proliferative activated receptor gamma	Fluorescence Polarization	Human
Nuclear Receptor	Progesterone Receptor	PGR	PR, NR3C3, Nuclear receptor subfamily 3 group C member 3	Fluorescence Polarization	Human
Phosphodiesterase	PDE3A	PDE3A	cGMP-inhibited 3′,5′-cyclic phosphodiesterase A	Enzymatic Activity	Human
Phosphodiesterase	PDE4A	PDE4A	cAMP-specific 3′,5′-cyclic phosphodiesterase 4A	Enzymatic Activity	Human
Phosphodiesterase	PDE4D	PDE4D	cAMP-specific 3′,5′-cyclic phosphodiesterase 4D	Enzymatic Activity	Human
Protease	ACE-1	ACE	Angiotensin-converting enzyme	Enzymatic Activity	Human
Protease	Cathepsin G	CTSG	CG	Enzymatic Activity	Human
Protease	Thrombin Alpha	F2	Coagulation factor II	Enzymatic Activity	Human

We consistently establish more targets for our InVEST Panel. Please reach out to inquire if the target of your interest is currently in development.

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.

Please see our FAQ page to access our assay condition form (US facility).

Assay formats

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.

Screening schedule

Testing of the InVEST Panel is performed on a monthly basis.

Please see the upcoming screening dates here.

Example data

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

In Vitro Safety Pharmacology Profiling

In Vitro Safety Screening Assay Formats

The InVEST Panel

The InVEST Panel

List of the InVEST Panel (51 targets)

Optional: Functional Ion Channel Panel

Additonal information to InVEST Panel Screening

Radioligand Binding Assay

Fluorescence Polarization

Cell-based transcriptional regulation

Enzymatic activity

Fluorescence Polarization Assay with a Nuclear Receptor

Fluorescence Polarization Assay with an Ion Channel

Radioligand Assay with a GPCR

Enzymatic Activity Assay with a PDE

Enzymatic Activity Assay with a Protease

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