PathHunter® Pharmacochaperone Trafficking Assays

PathHunter cell-based pharmacotrafficking assays provide a simple and quantitative tool for broad 
pharmacological characterization and interrogation of compound function in disease processes associated with protein trafficking and internalization due to protein misfolding. Eurofins DiscoverX PathHunter Pharmacochaperone assays provide a validated, cell-based chemiluminescent assay platform that enables identification and optimization of compounds which function as pharmacochaperones by promoting proper folding of mis-folded protein and assist in trafficking to their intended location.

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Learn more about the updated Cell Culture and Handling Procedure in the new Technical Bulletin.

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Assay Highlights

  • Functionally rescue disease relevant mutants
  • Correct assembly-related pathologies associated with ion channel/transporter proteins
  • Flexible — detect antagonists, agonists & allosterics
  • EFC-based chemiluminescent detection — uses standard plate reader
  • Small c-terminal peptide tag — no artifact or disruption of ligand binding
 

Key Resources

  • Knowledge-Based Video: Assays to identify pharmacological chaperones that rescue disease relevant mutant membrane proteins like GPCRs, ion channels and transporters.
  • Validation Slides: Identify compounds that rescue disease relevant mutant membrane proteins
  • Product Solution Guides and Product Lists for GPCRs and Ion Channels
  • Publications:
    • Review how customers used a pharmacochaperone-based high-throughput screening assay approach to discover chemical probes of orphan GPCRs (review abstract)
    • Use of pharmacotrafficking assays therapeutically beneficial for Type 2 Diabetes (review abstract)
 

Products


Parental Cell Lines

Cell Lines

 
Target Description Target Class Disease Relevance Readout Datasheet
ADRB2(W158A) Adrenergic receptor beta 2 GPCR   Endosome - EA View Datasheet
AVPR2(S167T) Vasopressin receptor 2 GPCR Nephrogenic diabetes insipidus Endosome - EA View Datasheet
CFTR-ΔF508 Cystic fibrosis transmembrane conductance regulator Ion Channel Cystic fibrosis Membrane - EA View Datasheet
KCNH2(G601S) Potassium voltage-gated channel, subfamily H (eag-related), member 2 Ion Channel Long QT syndrome (Cardiac arrhythmias) Membrane - EA View Datasheet
MC4R(T162I) Melanocortin 4 receptor GPCR Obesity Endosome - EA View Datasheet
mRHO(P23H) Rhodopsin GPCR Retinitis pigmentosa Endosome & Membrane - EA View Datasheet
SMO(W535L) Smoothened frizzled family receptor GPCR Basal skin cell carcinomas Membrane – EA View Datasheet

Not what you are looking for?  Learn more about our Custom Assay Development Services for custom solutions to meet your needs.

Assay Principle


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1. Cells lines co-express a ProLink™ (PK)-tagged transmembrane protein retained in the ER (due to protein misfolding), and an Enzyme Acceptor (EA) tag localized to the cell membrane (Membrane-EA format).  2. Cells lines co-express a ProLink™ (PK)-tagged trans-membrane protein retained in the ER (due to protein misfolding), and an Enzyme Acceptor (EA) tag localized to the early endosomes (Endosome-EA format).

In either system, binding of a chemical pharmacochaperone to the misfolded, PK-tagged protein stabilizes the protein in a conformation that enables its trafficking through the Golgi, then onward to the cell membrane. In the Membrane-EA format (1), complementation of the two β-galactosidase enzyme fragments (EA & PK) occurs at the membrane and in the Endosome-EA format (2), protein re-localized to the cell membrane subsequently internalizes (either passively or actively) into endosomes, forcing complementation of EA-PK.  The resulting functional complemented β-galactosidase enzyme hydrolyzes substrate to generate a chemiluminescent signal.

Create Your Own Trafficking Assays Using Engineered Parental Cell Lines

Study trafficking or translocation of proteins from one cellular compartment to another.
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Generate a PathHunter trafficking assay by first creating a plasmid vector with your target protein of choice tagged with enzyme donor [ED; ProLink (PK) or enhanced ProLabel® (ePL)]. Simply transfect this plasmid into a PathHunter EA parental cell line containing an EA-reporter protein (e.g. Endosome-EA [ENDO-EA] as shown in this example), and then perform a trafficking assay in the presence of a ligand. GPCR “total”-internalization from the cell membrane to the endosome is shown.

For creating a trafficking assay from the endoplasmic reticulum to the plasma membrane use the Membrane-EA [MEM-EA] PathHunter EA parental cell line (see customer publication).
 

 Discover Novel Pharmacochaperones

CFTR Assay Shows Expected Pharmacology and Localization

              

Localization of ADRB2(W158A)

PathHunter CFTR (ΔF508) cell line was treated with VX-661 and VX-809, known small molecules that repair the dysfunction of the mutant CFTR. Both VX compounds address the underlying cause of cystic fibrosis (CF), by helping the defective CF protein move to its proper place in the cell. Expected pharmacology and excellent S:B window makes this an ideal assay for screening of different modulators.
PathHunter ADRB2 (W158A) cell line was treated with various concentrations of Propranol indicating a dose dependent response of GPCR trafficking as measured by beta-gal complementation (bottom panel). Surface staining of U2OS cells expressing ADBR2(W158A) (red) after overnight incubation with ADRB2 antagonist Propranol (top left), and without Propranolol (top right) demonstrate that Propranol induces protein trafficking to the cell surface and proper localization of the GPCR.

Discover Pharmacochaperones That Rescue Disease-Associated Mutant Receptors

Identify pharmacologic chaperones (small molecule compounds) that function by promoting proper folding of misfolded GPCRs or ion channels and assist in trafficking from the endoplasmic reticulum (ER) to the plasma membrane. 

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Mutations in the potassium voltage-gated channel human ERG (KCNH2), can lead to reduced functional potassium current, long QT syndrome, and cardiac arrhythmias. A pharmacotrafficking assay was created using MEM-EA U2OS parental cells stably transfected with PK-tagged mutant KCNH2(G601S) (Cat. No. 93-1064C3). The pharmacochaperone clofilium was able to rescue the mutant ion channel by promoting proper folding resulting in successful trafficking from the ER to the membrane (EC50 = 564 nM; S/B = 3.5).
 

Early Investigate Multi-Subunit Ion Channel Protein Assembly

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A voltage-dependent calcium channel (Cav2.2) subunit assembly assay was created using MEM-EA parental cells stably transfected with PK-tagged (PK = EFC enzyme donor) subunit A1B (one of the three Cav2.2 subunits), along with neither, one, or both of the untagged Cav2.2 partner subunits 2D1 and B3. Low basal levels of EFC activity was obtained when only one or two subunits were present, indicating little to no trafficking from the ER to the plasma membrane. Only by adding all three subunits, A1B-PK/2D1/B3, did Cav2.2 form a proper ion channel subunit assembly and traffic properly to the cell membrane.