Pharmacotrafficking Assays

Identify Pharmacochaperones that Rescue Disease Relevant Mutant Membrane Proteins

Small molecule pharmacochaperones function as protein chaperones to rescue and guide misfolded proteins from the endoplasmic reticulum (ER) to their intended cellular location. Mutated misfolded GPCRs, ion channels, or transporters trapped in the ER are unable to move to the plasma membrane and can lead to diseases such as cystic fibrosis, obesity, diabetes, cardiac arrhythmias, and more. Pharmacochaperones act as therapeutic agents to stabilize and correct the misfolded protein and allow for the proteins’ proper trafficking from the ER to the plasma membrane to prevent the disease.

 

Utilize PathHunter® cell-based pharmacotrafficking assays to identify, characterize, and optimize pharmacochaperones. These homogeneous cell-based assays are easy-to-use, quantitative, and provide a superior alternative to imaging or antibody-based methods



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Product Highlights

  • Extensive Portfolio – Cell lines, custom assays, and toolbox products applicable for disease relevant mutant GPCRs, ion channels, and transporters
  • Small Molecule Discovery – Assays to identify small molecule pharmacological chaperones that rescue or correct several disease relevant mutant membrane proteins
  • Scalable – Homogenous quantitative, easy-to-use assays amenable for low, medium, and high throughput screening (HTS) programs
  • Superior Readout — Antibody- and image-free assays with a sensitive chemiluminescent read-out

Key Resources

Products

Cell Lines and Assays

  • PathHunter Pharmacotrafficking Assays – Identify pharmacochaperones that rescue disease relevant mutant membrane proteins with easy-to-use, quantitative, and homogeneous cell-based assays based on the Enzyme Fragment Complementation technology.
    Target Class Target Protein Description Disease Relevance Readout Cell Line
    GPCR ADRB2(W158A) Adrenergic receptor beta 2   Endosome - EA 93-0986C3
    GPCR AVPR2(S167T) Vasopressin receptor 2 Nephrogenic diabetes insipidus Endosome - EA 93-0992C3
    Ion Channel CFTR-ΔF508 Cystic fibrosis transmembrane conductance regulator Cystic fibrosis Membrane - EA 93-0641C2 (CHO-K1)
    93-0987C3 (U2OS)
    Ion Channel KCNH2(G601S) Potassium voltage-gated channel, subfamily H (eag-related), member 2 Long QT syndrome (Cardiac arrhythmias) Membrane - EA 93-1064C3
    GPCR MC4R(T162I) Melanocortin 4 receptor Obesity Endosome - EA 93-1004C3
    GPCR mRHO(P23H) Rhodopsin Retinitis pigmentosa Endosome & Membrane - EA 93-0990C3 (ENDO-EA)
    93-1077C3 (MEM-EA)
    GPCR SMO(W535L) Smoothened frizzled family receptor Basal skin cell carcinomas Membrane – EA 93-0991C3

Custom and Toolbox Products

  • Custom Capabilities – Custom cell lines and assays capabilities optimized to fit your requirements
  • Parental Cell Lines – Engineered cells to generate your own pharmacotrafficking assays and investigate trafficking of your target protein to the plasma membrane, nucleus, or endosome.

Assay Principle

Assay Principle

 

Pharmacotrafficking assays involve the binding of a small molecule pharmacochaperone to a misfolded membrane protein. The pharmacochaperone stabilizes the membrane protein in a conformation that enables its trafficking from the ER through the Golgi and onward to the cell membrane and in some cases the endosome. There are two pharmacotrafficking assay formats that are both based on the Enzyme Fragment Complementation system involving a cell lines expressing a small enzyme donor ProLink™ (PK)-tagged membrane protein and an enzyme acceptor (EA) tagged protein localized to either the cell membrane or endosome. In the Membrane-EA format, complementation of the two β-galactosidase (β-gal) enzyme fragments (EA & PK) occurs at the membrane. In the Endosome-EA format, the membrane protein subsequently internalizes (either passively or actively) into endosomes with complementation of EA-PK within the endosome. The resulting functional β-gal enzyme hydrolyzes substrate to generate a chemiluminescent signal that can be detected on any standard luminometer.

Discover and Screen for Novel Pharmacochaperones that Rescue Disease-Associated Mutant Receptors

Discover and Screen for Novel Pharmacochaperones that Rescue Disease-Associated Mutant Receptors

 

Identify small molecule pharmacochaperones that function by stabilizing misfolded membrane proteins and assist in their trafficking from the endoplasmic reticulum (ER) to the plasma membrane. A. PathHunter CFTR (cystic fibrosis transmembrane conductance regulator) (ΔF508) Pharmacotrafficking cell line (membrane-EA format) was treated with VX-661 and VX-809, known small molecules that repair the dysfunction of the mutant CFTR. Both compounds address the underlying cause of cystic fibrosis by helping the defective CFTR protein move to its proper location (plasma membrane) in the cell. The expected pharmacology, high-throughput ability, and excellent signal-to-background (S:B) window makes this an ideal assay for screening of different modulators. B. 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 to the membrane (EC50 = 564 nM; S:B = 3.5).

 

Create Your Own Trafficking Assays Using Engineered Parental Cell Lines

Create Your Own Trafficking Assays Using Engineered Parental Cell Lines

 

Generate a PathHunter trafficking assay by first creating a plasmid vector with your target protein of choice tagged with the Enzyme Fragment Complementation (EFC) β-galactosidase (β-gal) enzyme donor (ED). Simply transfect a PathHunter EA parental cell line with the plasmid and perform a trafficking assay in the presence of a ligand. In this example, the Endosome-EA (ENDO-EA) PathHunter EA parental cell line is used, and the target protein is a GPCR. The assay detects the trafficking of the GPCR from the plasma membrane to the endosome upon ligand (pharmacochaperone) binding to GPCR. Once the GPCR moves to the endosome, the ED-tagged GPCR meets with the large EFC enzyme acceptor (EA) that bound to an endosomal reporter protein. Upon addition of the β-gal substrate and formation of the active enzyme (ED+EA), the enzyme hydrolyzes its substrate to produce a chemiluminescence signal.

 

For creating a trafficking assay from the ER to the plasma membrane, use the Membrane-EA (MEM-EA) PathHunter EA parental cell line (see an example of this in the following customer publication).