Servizio Comunitario di Informazione in materia di Ricerca e Sviluppo - CORDIS

A high throughput system to search for small molecules that would affect the function of CFTR

A high throughput system to search for small molecules that would affect the function of CFTR was developed. The screening process is performed routinely using an automate for rapid measurement of radiotracer flux allowing multiple tests of molecules on living cells cultured in multiwell plates. The rate of activation is determined after mathematical fitting, the EC50 (for an activator) and/or IC50 (for an inhibitor) are calculated using the software GraphPad Prism version 3.0 for Windows (Graphpad Software, San Diego, California, USA). Several protocols have been used. An adaptation to the robotic system was done in preliminary experiments during the first three month of the project. Then, four different protocols were designed.

Protocol #1 was designed to screen molecules on wild type CFTR activity expressed in CHO and Calu-3 cells. In the protocol 1a, drugs are tested alone in the absence of cAMP-promoting agent. In the protocol 1b, drugs are tested in the presence of low concentration of the cAMP-promoting agent forskolin used at 1 microM. The first protocol will tell us whether a drug is able to stimulate directly CFTR. The second one will determine whether the drug potentiates the response obtained with low concentration of forskolin.

Protocol #2 was designed to screen molecule on the class III CF mutation G551D-CFTR. Despite the fact that this mutant does not respond to forskolin at concentration as high as 10 microM, this mutant need high concentration of forskolin before further activation. In other words, the R-domain of CFTR need to be phosphorylated by protein kinase A (PKA). Drugs are tested on this mutant in the presence of 10 microM of forskolin. After incubation of cells with the selected molecule the activity of defective CFTR can be addressed and compared to that of wild-type CFTR.

Protocol #3 was designed to screen molecules on F508del-CFTR activity. The F508del homozygous human airway epithelial cell line CF15 are used for this protocol. Cells are cultured at 27°C for 24 to 48 hours before functional analysis.

Protocol #4 was designed to screen molecules on F508del-CFTR trafficking. The ability of molecules to activate F508del-CFTR is determined after incubation (2-4h, 37°C) to allow the localization of proteins toward the apical compartment. The rescue of F508del-CFTR protein at the cell surface is determined using a cAMP-promoting cocktail containing forskolin (10 microM) and genistein (30 microM). Protocol #3 is now used extensively not only to screen new agents able to rescue F508del from the ER to the plasma membrane but also to evaluate the effect of modulators of the biosynthetic pathway added in association with these agents. For example using the brefeldin A allow to demonstrate that F508del move from the ER to the golgi. With proteasome inhibitors or Ca-ATPase inhibitors or other agent we can potentiate the effect of the first agents. These items are now used in routine to further study the mechanism of action of any new agent that rescue F508del.

Informazioni correlate

Reported by

Université de Poitiers
Avenue du Recteur Pineau, 40
86022 Poitiers
France
See on map