Assay Principle, Validation& Applications

HitHunter® cAMP Assays forBiologics and Small Molecules

Split β-Galactosidase Enzyme

Enzyme Fragment Complementation (EFC)Technology

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Enzyme Fragment Complementation (EFC) technology consists of a β-galactosidase (β-gal) enzyme splitinto two inactive components, the enzyme donor peptide (ED) and enzyme acceptor (EA). Whenbrought together, ED complements with EA forming an active β-gal complex. With the addition ofsubstrate, the active β-gal catalyzes the formation of chemiluminescent products providing a highlyamplified signal.

Hydrolysis

Inactive Fragments

EFC-based cAMP Assay Principle

• At higher levels of cellular cAMP, the anti-cAMP antibody becomessaturated allowing the ED-cAMP complex to complement with the β-gal acceptor (EA) and form an active enzyme

• Following substrate addition, the active enzyme produces a signalthat is directly proportional to the cellular cAMP levels

Key Features and Benefits

• Flexible – Use with small molecules or biologics• Simple – Easy-to-follow protocol• Validated – In 400+ peer reviewed publications• Qualified – Unmatched performance for a variety of

applications• Robust – Exquisite sensitivity with large assay windows• Reliable – Reproducible lot-after-lot• Universal – Detect with any standard plate reader

Drug Discovery Applications forSmall Molecules and Biologics

Basic Research and AssayDevelopmentIdentify and fully characterizemolecules for correctpharmacology

Basic Research and AssayDevelopmentIdentify and fully characterizemolecules for correctpharmacology

ScreeningPerform functional cAMPresponse screening

ScreeningPerform functional cAMPresponse screening

Lead OptimizationProfiling molecules (rank order;EC50/IC50); obtain expectedpharmacology

Lead OptimizationProfiling molecules (rank order;EC50/IC50); obtain expectedpharmacology

Clinical StudiesMeasure drug efficacy andpotency in clinical samples

Clinical StudiesMeasure drug efficacy andpotency in clinical samples

QC BioassayRun quality control assays(potency assays for lotrelease and stability testing)

QC BioassayRun quality control assays(potency assays for lotrelease and stability testing)

Immunogenicity StudiesDetect neutralizingantibodies

Immunogenicity StudiesDetect neutralizingantibodies

Additional biologics applications

Rank Molecular Potency(EC50 or IC50 Comparison)

Profiling experiments of ranked ligands (agonists orantagonists) show assays can obtain the expectedpharmacology as indicated in the literature

Formeterol Isoproterenol Clenbuterol Salmeterol Salbutamol

EC50 1.7x10-10 1.7x10-9 2.7x10-9 2.8x10-9 1.6x10-7

S/B 14.5 11.6 9.1 13.1 12.3

Haloperidol Risperidon Chlorpromazine Clozapine

EC50 1.8x10-8 2.6x10-8 3.9x10-7 3.8x10-6

S/B 15.8 10.1 13.1 12.4

Measure Complex Assay Formats(Antagonists; Gαi-coupled Receptors)

Large assay windowsallow easier antagonistidentification andrescue of many difficultGαi-coupled receptors

Antagonist 1 Antagonist 2

EC50 2.4x10-8 8.0x10-9

S/B 4.2 6.2

Easily Identify Allosteric Modulators(PAMs and NAMs)

Quantify allosteric modulator activity by performing adose response curve study to determine the EC50and Schild analysis to show curve shifting withincreasing modulator concentrations

PAM Study on GRM4 (Glutamate Receptor) using cAMP Detection

Detect Inverse Agonism

Use assays to revealinverse agonists

Data from: Yao et.al; British Journal ofPharmacology (2006) 149, 145–154.

Inverse Agonist (SR144528) Studyon Cannabinoid Receptor CB2 using

cAMP Detection

Characterize Biologics

Characterization of theprotein SDF1α(stromal cell-derivedfactor 1α) using aCXCR4 Gαi-coupledchemokine receptorcell line

EC50 8.0x10-10

S/B 6.5

BiologicsExample

Obtain Excellent Reproducibility

Evaluate lot-to-lotconsistency forquality controlanalysis

Lot 1 Lot 2 Lot 3

EC50 8.7x10-11 7.4x10-11 7.6x10-11

S/B 10.2 12.0 12.6

BiologicsExample

Recognize Partial Agonists

• The large dynamic range of the assay helps to identify partialagonists

• Using the assay and validated cAMP Hunter™ cell lines showpartial agonism without difficulty

Data from: Yao et.al; British Journal ofPharmacology (2006) 149, 145–154.

BottomTopLogEC50HillSlopeEC50

Glucagon31.62514.8-7.9682.3401.076e-008

[des-His1,Glu9]-Glucagon26.65283.6-6.4671.9393.413e-007

Glucagon (des-His1, Glu9)-Glucagon

EC50 1.1x10-8 3.4x10-7

S/B 14.8 9.2

BiologicsExample

Partial Agonist (GW405,833) Studyon Cannabinoid Receptor CB2 using

cAMP Detection

Detect Neutralizing Antibodies

Performimmunogenicitystudies using serumand plasmasamples

BiologicsExample

Data from: Ryding et al. Journal of Immunological Methods (2013).

Neutralization Assay Study to select theMinimal Drug Concentration using the HumanFollicle-Stimulating Hormone (FSH) Receptor

Identify PDE Inhibitors

Study cAMP dependentphosphodiesterases(PDE) – enzymes thatregulate the cellularcAMP and cGMP levelsby controlling theirrates of degradation

0.04 μMcAMP

0.12 μMcAMP

0.37 μMcAMP

EC50 1.1x10-6 1.0x10-6 2.8x10-6

Phosphodiesterase Assay

BottomTopLogEC50HillSlopeEC50

0.37 uM cAMP-57.8398.030.4436-0.89212.777

0.12 uM cAMP-0.439397.230.01422-2.2381.033

0.04 uM cAMP5.45692.840.02689-2.4831.064

Quantify Low Levels of cAMP(Endogenous Receptor Studies)

Quantify endogenous(native) receptoractivity by monitoringlow levels of cAMP

EC50 1.8x10-7

S/B 12.3Best-fit valuesBottomTopLogEC50HillSlopeEC50

1321 N1 Gs PTGDR

86.25739.9-6.2621.6155.466e-007

Naked 1321 N1

47.09632.9-6.7360.95101.837e-007

Broad Range of Sample Compatibility

• Easily determine ligand activity in the presence of serum andplasma

• Assay works well with live cells or cell membrane fractions

2 μg/well

1 μg/well

0.5 μg/well

0.25 μg/well

EC50 7.5x10-7 8.4x10-7 1.2x10-6 2.5x10-6

BottomTopLogEC50HillSlopeEC50

2 g/well309.7496.7-6.1261.1917.475e-007

1 g/well308.6540.8-6.0750.90878.421e-007

0.5 g/well206.0474.8-5.9160.93331.213e-006

0.25 g/well132.8421.3-5.5960.75292.534e-006

0% FBS 5% FBS 10% FBS 25% FBS 50% FBS

EC50 1.70x10-8 1.57x10-8 1.59x10-8 1.90x10-8 2.28x10-8Best-fit valuesBottomTopLogEC50HillSlopeEC50

0% FBS

57.441813-7.7691.2031.703e-008

5% FBS

46.051997-7.8041.0491.571e-008

10% FBS

45.461954-7.7991.0521.588e-008

25% FBS

11.191878-7.7220.73121.898e-008

50% FBS

36.711525-7.6420.89322.281e-008

Simple Protocol

• Rapid, homogeneous protocol- Simply add cAMP assay reagents to stimulated cells,

incubate and read- Detection within 4 hours- Chemiluminescent readout using any standard plate reader

Scalable Assay(Miniaturization Compatible)

96-well 384-well 1536-well 3456-well

Reduce sample size and reagent costs

Weber M. (Merck), ELRIG 2006(384 and 3456 well format)

(384-well format)(1536-well format)

Validated Results

• Well-established, validated results since 2000- ~400 customer publications to date

HitHunter® cAMP Assay select citations

Simple User Manual

• Easy-to-follow, step-by-stepprocess

• Quick start guide• Helpful imagery, call-outs,

and schematics

Convenient Packaging

3 Sizes available for each applications

Bulk quantities are available upon request

Application Specific Kits

HitHunter cAMP Assay for Biologic HitHunter cAMP Assay for Small Molecules

Product No. Size Configuration Product No. Size Configuration

90-0075LM2 200/800 dp (96-well/384-well) 90-0075SM2 200/800 dp (96-well/384-well)

90-0075LM10 1,000/4,000 dp (96-well/384-well) 90-0075SM10 1,000/4,000 dp (96-well/384-well)

90-0075LM25 2,500/10,000 dp (96-well/384-well) 90-0075SM25 2,500/10,000 dp (96-well/384-well)

Complete Offering of Our EFC Technology

• Stable Cell Lines- Frozen cells (unlimited

culture)- Validated culture reagents

• Assay Ready Kits- Single-use frozen cells- Detection reagents- Cell plating media and

components

• LeadHunter™ Services- Leverage our expertise- Multiple flexible options- High throughput capabilities

• Custom Assay Development- Access to all technologies- Expertise from our R&D team- Flexible milestone structures- Experience with 300+ projects

cAMP Hunter™Stable Cell-lines

HitHunter® cAMP AssayDetection Reagents cAMP Hunter™ eXpress Kit

Assay Ready Kits withCells plus

Detection Reagents

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