Although some therapeutic progress has been achieved in developing small molecules that correct F508del-CFTR defects, the mechanism of action (MoA) of these compounds remain poorly elucidated. Here, we investigated the effects and MoA of MCG1516A, a newly developed F508del-CFTR corrector. MCG1516A effects on wild-type (WT) and F508del-CFTR were assessed by immunofluorescence microscopy, and biochemical and functional assays both in cell lines and in intestinal organoids. To shed light on the MoA of MCG1516A, we evaluated its additivity to the FDA-approved corrector VX-661, low temperature, genetic revertants of F508del-CFTR (G550E, R1070W, and 4RK), and the traffic-null variant DD/AA. Finally, we explored the ability of MCG1516A to rescue trafficking and function of other CF-causing mutations. We found that MCG1516A rescues F508del-CFTR with additive effects to VX-661. A similar behavior was observed for WT-CFTR. Under low temperature incubation, F508del-CFTR demonstrated an additivity in processing and function with VX-661, but not with MCG1516A. In contrast, both compounds promoted additional effects to low temperature to WT-CFTR. MCG1516A demonstrated additivity to genetic revertant R1070W, while VX-661 was additive to G550E and 4RK. Nevertheless, none of these compounds rescued DD/AA trafficking. Both MCG1516A and VX-661 rescued CFTR processing of L206W- and R334W-CFTR with greater effects when these compounds were combined. In summary, the absence of additivity of MCG1516A to genetic revertant G550E suggests a putative binding site for this compound on NBD1:NBD2 interface. Therefore, a combination of MCG1516A with compounds able to rescue DD/AA traffic, or mimicking the actions of revertant R1070W (e.g., VX-661), could enhance correction of F508del-CFTR defects.

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The R334W (c.1000C>T, p.Arg334Trp) is a rare cystic fibrosis (CF)-causing mutation for which no causal therapy is currently approved. This mutation leads to a significant reduction of CF transmembrane conductance regulator (CFTR) channel conductance that still allows for residual function. Potentiators are small molecules that interact with CFTR protein at the plasma membrane to enhance CFTR-dependent chloride secretion, representing thus pharmacotherapies targeting the root cause of the disease. Here, we generated a new CF bronchial epithelial (CFBE) cell line to screen a collection of compounds and identify novel potentiators for R334W-CFTR. The active compounds were then validated by electrophysiological assays and their additive effects in combination with VX-770, genistein, or VX-445 were exploited in this cell line and further confirmed in intestinal organoids. Four compounds (LSO-24, LSO-25, LSO-38, and LSO-77) were active in the functional primary screen and their ability to enhance R334W-CFTR-dependent chloride secretion was confirmed using electrophysiological measurements. In silico ADME analyses demonstrated that these compounds follow Lipinski’s rule of five and are thus suggested to be orally bioavailable. Dose–response relationships revealed nevertheless suboptimal efficacy and weak potency exerted by these compounds. VX-770 and genistein also displayed a small potentiation of R334W-CFTR function, while VX-445 demonstrated no potentiator activity for this mutation. In the R334W-expressing cell line, CFTR function was further enhanced by the combination of LSO-24, LSO-25, LSO-38, or LSO-77 with VX-770, but not with genistein. The efficacy of potentiator VX-770 combined with active LSO compounds was further confirmed in intestinal organoids (R334W/R334W genotype). Taken together, these molecules were demonstrated to potentiate R334W-CFTR function by a different mechanism than that of VX-770. They may provide a feasible starting point for the design of analogs with improved CFTR-potentiator activity.

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