Cystic Fibrosis is caused by loss of function mutations in the Cystic Fibrosis Transmembrane Conductance Regulator Gene: CFTR and is associated with severe airway disease. Loss of CFTR channel function on the surface of epithelial cells leads to mucus obstruction with recurrent infection. Drug discovery efforts have identified a “potentiator” drug (Ivacaftor or VX-770) that partially repairs the defective channel activity exhibited by the rare CF causing mutation: G551D and leads to improved lung function. Now, there is an urgent need to find a compound (or compounds) to rescue the defects caused by the major CF causing mutation: F508del present on at least one allele in 90% of CF patients. The F508del mutation causes the CFTR protein to misfold, leading to its retention in the endoplasmic reticulum and degradation. “Corrector” compounds have been identified that improve folding and trafficking of F508del-CFTR to the surface of cultured cells. However, the lead corrector: VX-809, failed to show efficacy in clinical trials of patients homozygous for this mutation. A recent press release reported promising, yet variable results for Phase 2a clinical trials of the combination: VX-809 plus VX-770. Most patients exhibited a relatively modest increase in FEV1 -highlighting the requirement for further drug development. The discovery of better modulators of mutant CFTR protein will be enabled by understanding the mechanism of action of these existing compounds. We hypothesize that these compounds bind directly to modulate CFTR folding and function and tested this hypothesis in novel in-vitro assays, using purified and reconstituted Wt and mutant CFTR. Our in-vitro studies show that the potentiator VX-770 directly modified the channel activity of purified and reconstituted purified CFTR proteins. Channel activity of purified protein was evaluated using both a proteoliposomal flux assay and in planar lipid bilayer studies. Regardless of the CFTR genotype, ie. Wt, F508del or G551D-CFTR, we observed an increase in channel activity of PKA phosphorylated CFTR protein in the presence of its natural activating ligand, ATP. Interesting, we also found that VX-770 enhanced channel activity of phosphorylated CFTR in the nominal absence of ATP, suggesting that it acts via a canonical pathway to modify the channel gate. Currently, we are employing chemical analogues of VX-770 to gain insight into the structural properties of the VX-770 binding site and the mechanism underlying ATP independent channel activity. Interestingly, we found that the two corrector compounds: VX-809 (in clinical trial) and C18 (an analog of VX-809) also bind directly to Wt-CFTR, F508del-CFTR and G551D-CFTR. The binding of C18 led to enhanced ATP-independent channel activity in all three of these genotypes, suggesting that it may share a similar binding site as the VX-770 or its binding leads to similar conformational changes. In the case of the purified mutant, F508del-CFTR, the binding of VX-809 or C18 also induces a more stable protein conformation, with a reduced propensity to aggregate. This latter observation, likely accounts for the partial “rescue” effect of these compounds on the misfolding of F508del-CFTR. As in our studies of VX-770, we are employing chemical analogues of C18 to probe the properties of its binding site on mutant CFTR. We anticipate that these binding studies will report the structural defects caused by the F508del-CFTR mutation and enable discovery of more efficacious corrector compounds. Together, these studies support the use of small chemical molecules in probing the structural basis for folding and function of CFTR.