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. 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.
 
View Webinar View Validation Slides CFTR Assay Validation

 

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

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. View data validation »
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. View data validation »

Products


Cell Lines

Parental 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


 

1. 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 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.