PHARMACOTRAFFICKING ASSAYS

Pharmacotrafficking assays involve the binding of a small molecule pharmacochaperone to a misfolded membrane protein, which is being retained in the ER. 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

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

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).