Figure - available from: Scientific Reports
This content is subject to copyright. Terms and conditions apply.
Effect of chronic treatment with elexacaftor (VX-445) includes its actions as both a CFTR corrector and potentiator. (A) Representative It recordings of non-CF HNE treated for 24 h with DMSO or VX-445 (3 µM). (B–C) Changes in It after the additions of test compounds for the experiment presented in Panel A. The potentiating action of VX-445 accounts for the differences in post-amiloride It between DMSO and VX-445 treated cultures. (D) Representative It recordings of F508del-HNE treated for 24 h with tezacaftor (VX-661; 3 µM), ivacaftor (VX-770; 100 nM), and/or VX-445 (3 µM chronic; 100 nM acute). (E–F) Changes in It after the additions of test compounds for the experiment presented in (D). The triple combination of VX-661/77/445 significantly increased CFTR-mediated It. Acute action of VX-445 in stimulating It was only observed in VX-661/770 treated F508del/F508del nasal cells. See SI for additional experimental details and for supporting data. All data are presented as mean ± standard error. Bars with different letters (A, B, C…) are significantly different from each other (ANOVA; P < 0.05). Asterisks indicate specific P values: ***P < 0.001.
Source publication
Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), which lead to early death due to progressive lung disease. The development of small-molecule modulators that directly interact with CFTR to aid in protein folding (“correctors”) and/or increase channel function (“potentiators”) have proven...
Citations
... FIS levels in these PDIOs underestimated functional CFTR rescue, as has been observed before for organoids with SLA levels of over~40% 15 . Pre-swelling could be explained by the high efficacy of CFTR modulator VX-445 and its dual corrector and potentiator activity 32,33 . Previous drug-induced increase in lumen area was only observed upon combined 24 h corrector/potentiator incubation, likely by rendering the potentiated CFTR protein responsive to endogenous cAMP-PKA signalling 15 . ...
Epithelial ion and fluid transport studies in patient-derived organoids (PDOs) are increasingly being used for preclinical studies, drug development and precision medicine applications. Epithelial fluid transport properties in PDOs can be measured through visual changes in organoid (lumen) size. Such organoid phenotypes have been highly instrumental for the studying of diseases, including cystic fibrosis (CF), which is characterized by genetic mutations of the CF transmembrane conductance regulator (CFTR) ion channel. Here we present OrgaSegment, a MASK-RCNN based deep-learning segmentation model allowing for the segmentation of individual intestinal PDO structures from bright-field images. OrgaSegment recognizes spherical structures in addition to the oddly-shaped organoids that are a hallmark of CF organoids and can be used in organoid swelling assays, including the new drug-induced swelling assay that we show here. OrgaSegment enabled easy quantification of organoid swelling and could discriminate between organoids with different CFTR mutations, as well as measure responses to CFTR modulating drugs. The easy-to-apply label-free segmentation tool can help to study CFTR-based fluid secretion and possibly other epithelial ion transport mechanisms in organoids.
... Another example of long-range allostery in CFTR is the finding that point mutations at the cytosolic loops increase channel open probability (even in the absence of ATP) by shifting a pre-existing conformational equilibrium toward the ligand bound conducting conformation (Wang et al., 2010). Notably, allostery in CFTR has direct therapeutic relevance: The potentiator drug ivacaftor and the dual-function modulator elexacaftor do not bind in the proximity of the gating residues (Liu et al., 2019;Fiedorczuk and Chen, 2022a) yet increase the open probability (Po) of the channel (Eckford et al., 2012;Shaughnessy et al., 2021). These and other studies (Sorum et al., 2017;Scholl et al., 2021;Wei et al., 2014) significantly advanced our understanding of the function of CFTR and its allosteric control. ...
... There are currently four FDA-approved drugs that are used to treat CF: The type I correctors lumacaftor (VX-809) and tezacaftor (VX-661) stabilize mutant CFTR variants (predominantly the most common ΔF508 mutation) reducing premature protein degradation and improving surface presentation of the mature channel (Van Goor et al., 2011;Fiedorczuk and Chen, 2022b). The potentiator ivacaftor (VX-770) enhances the activity of conductance-defective mutants by increasing the open probability (Po) of the channel (Levring et al., 2023;Liu et al., 2019;Eckford et al., 2012), and the dual-function type III corrector elexacaftor (VX-445) stabilizes unstable variants and increases channel open probability (Shaughnessy et al., 2021;Veit et al., 2021). The recently published structures of WT and ΔF508CFTR bound to various drugs (Fiedorczuk and Chen, 2022a) enabled us to investigate the allosteric effects of the drugs. ...
Malfunction of the CFTR protein results in cystic fibrosis, one of the most common hereditary diseases. CFTR functions as an anion channel, the gating of which is controlled by long-range allosteric communications. Allostery also has direct bearings on CF treatment: the most effective CFTR drugs modulate its activity allosterically. Herein, we integrated Gaussian network model, transfer entropy, and anisotropic normal mode-Langevin dynamics and investigated the allosteric communications network of CFTR. The results are in remarkable agreement with experimental observations and mutational analysis and provide extensive novel insight. We identified residues that serve as pivotal allosteric sources and transducers, many of which correspond to disease-causing mutations. We find that in the ATP-free form, dynamic fluctuations of the residues that comprise the ATP-binding sites facilitate the initial binding of the nucleotide. Subsequent binding of ATP then brings to the fore and focuses on dynamic fluctuations that were present in a latent and diffuse form in the absence of ATP. We demonstrate that drugs that potentiate CFTR’s conductance do so not by directly acting on the gating residues, but rather by mimicking the allosteric signal sent by the ATP-binding sites. We have also uncovered a previously undiscovered allosteric ‘hotspot’ located proximal to the docking site of the phosphorylated regulatory (R) domain, thereby establishing a molecular foundation for its phosphorylation-dependent excitatory role. This study unveils the molecular underpinnings of allosteric connectivity within CFTR and highlights a novel allosteric ‘hotspot’ that could serve as a promising target for the development of novel therapeutic interventions.
... In 2019 (US) and 2021 (Australia), the first triple combination therapy involving elexacaftor-tezacaftor-ivacaftor was approved for patients aged over 12 years old. Elexacaftor is a next-generation modulator that is proposed to act as both corrector and potentiator (Shaughnessy et al., 2021). In phase 3 studies, clinical end points of FEV1 were superior to the tezacaftor-ivacaftor combination with fewer adverse events (Heijerman et al., 2019;Middleton et al., 2019). ...
Highly effective cystic fibrosis (CF) transmembrane conductance regulator (CFTR) modulator therapies (HEMT), including elexacaftor-tezacaftor-ivacaftor, correct the underlying molecular defect causing CF. HEMT decreases general symptom burden by improving clinical metrics and quality of life for most people with CF (PwCF) with eligible CFTR variants. This has resulted in more pregnancies in women living with CF. All HEMT are known to be able pass through the placenta and into breast milk in mothers who continue on this therapy while pregnant and breast feeding. Toxicity studies of HEMT in young rats demonstrated infant cataracts, and case reports have reported the presence of congenital cataracts in early life exposure to HEMT. This article reviews the evidence for how HEMT influences the dynamic and interdependent processes of healthy and abnormal lens development in the context of HEMT exposure during pregnancy and breastfeeding, and raises questions that remain unanswered.
... Of note, P67L CFTR FRT cells consistently displayed increased ion current with VX-445 treatment even though no significant increase in fully glycosylated CFTR could be observed (Figs. 1, D and E and S8C). This discrepancy between lack of band C correction and functional rescue is likely due to the potentiating activity of VX-445 (40,41), which may mask the small degree of functional rescue from the KDs. ...
... Prior work by Veit et al. similarly reported this discrepancy in response to VX-809 and VX-445 for P67L and L206W in CFBE cell lines (42), and a deep mutational scanning study reported this discrepancy in HEK293T cells (43). A mild increase in channel function in response to VX-445 in FRT cells stably expressing P67L CFTR as measured by Ussing chamber may also be explained in part by the potentiating activity of VX-445 in addition to a low-level correction (40,41). ...
... It is possible that fully glycosylated CFTR as observed by RPL37 KD were either nonfunctional or sequestered in post-Golgi compartments such as endosomes. VX-445 exhibits mild potentiating activity (40,41), which explains the increase in channel activation in the absence of trafficking correction (Fig. S8C). We did not see enhanced channel function upon VX-770 (potentiator) treatment with RPL37 KD, however, which indicates that the rescued post-Golgi CFTR proteins are either not trafficked to the cell surface or remain in nonfunctional conformations in FRT cells. ...
Cystic fibrosis (CF) is one of the most prevalent lethal genetic diseases with over 2000 identified mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Pharmacological chaperones such as Lumacaftor (VX-809), Tezacaftor (VX-661) and Elexacaftor (VX-445) treat mutation-induced defects by stabilizing CFTR and are called correctors. These correctors improve proper folding and thus facilitate processing and trafficking to increase the amount of functional CFTR on the cell surface. Yet, CFTR variants display differential responses to each corrector. Here, we report variants P67L and L206W respond similarly to VX-809 but divergently to VX-445 with P67L exhibiting little rescue when treated with VX-445. We investigate the underlying cellular mechanisms of how CFTR biogenesis is altered by correctors in these variants. Affinity purification-mass spectrometry (AP-MS) multiplexed with isobaric Tandem Mass Tags (TMT) was used to quantify CFTR protein-protein interaction changes between variants P67L and L206W. VX-445 facilitates unique proteostasis factor interactions especially in translation, folding, and degradation pathways in a CFTR variant-dependent manner. A number of these interacting proteins knocked down by siRNA, such as ribosomal subunit proteins, moderately rescued fully glycosylated P67L. Importantly, these knockdowns sensitize P67L to VX-445 and further enhance the trafficking correction of this variant. Partial inhibition of protein translation also mildly sensitizes P67L CFTR to VX-445 correction, supporting a role for translational dynamics in the rescue mechanism of VX-445. Our results provide a better understanding of VX-445 biological mechanism of action and reveal cellular targets that may sensitize unresponsive CFTR variants to known and available correctors.
... In particular, the functional YFP assay showed that the combination of VX445 with either VX809 or VX661 was particularly effective in primary bronchial epithelial cells derived from patients with CF [36][37][38][39]. Other studies proposed that VX445 acts as a potentiator [47] or plays a dual role as a potentiator and a corrector [41,48]. Finally, in a recent study, Fiedorczuk and Chen resolved the molecular structure of F508 CFTR bound to the three components of Kaftrio and identified the pocket formed by the transmembrane helices 10 and 11 and the lasso motif as the putative site of interaction of VX445 with F508 CFTR [42]. ...
Cystic fibrosis (CF) is one of the most frequent lethal autosomal recessive diseases affecting the Caucasian population. It is caused by loss of function variants of the cystic fibrosis transmembrane conductance regulator (CFTR), a membrane protein located on the apical side of epithelial cells. The most prevalent CF-causing mutation, the deletion of phenylalanine at position 508 (F508del), is characterized by folding and trafficking defects, resulting in the decreased functional expression of the protein on the plasma membrane. Two classes of small-molecule modulators, termed potentiators and correctors, respectively, have been developed to rescue either the gating or the cellular processing of defective F508del CFTR. Kaftrio, a next-generation triple-combination drug, consisting of the potentiator ivacaftor (VX770) and the two correctors tezacaftor (VX661) and elexacaftor (VX445), has been demonstrated to be a life-changing therapeutic modality for the majority of people with CF worldwide. While the mechanism of action of VX770 and VX661 is almost known, the precise mechanism of action and binding site of VX445 have not been conclusively determined. We investigated the activity of VX445 on mutant F508del to identify the protein domains whose expression is mostly affected by this corrector and to disclose its mechanisms of action. Our biochemical analyses revealed that VX445 specifically improves the expression and the maturation of MSD2, heterologously expressed in HEK 293 cells, and confirmed that its effect on the functional expression of defective F508del CFTR is additive either with type I or type II CFTR correctors. We are confident that our study will help to make a step forward in the comprehension of the etiopathology of the CF disease, as well as to give new information for the development and testing of combinations of even more effective correctors able to target mutation-specific defects of the CFTR protein.
... Responses were blocked by the CFTR inhibitor I-172 ( Figure S1F) and activity was lower in absence of IVA ( Figure S1E). Some CFTR correctors possess acute inhibitory (e.g., GLPG-2737 [25]), or co-potentiating effects (ELX, [21,26,27]) on CFTR currents. In HEK293 cells expressing the gating mutant G551D-CFTR, incubation (10 min) with IDOR-3 or IDOR-4 caused co-potentiation of IVA activity, similar to the effect of ELX, while a corrector from the company Galapagos (WO2017060874, example#331) displayed an acute inhibitory effect ( Figure S1H). ...
Three distinct pharmacological corrector types (I, II, III) with different binding sites and additive behaviour only partially rescue the F508del-CFTR folding and trafficking defect observed in cystic fibrosis. Here, we describe novel, uniquely effective, macrocyclic CFTR correctors that were additive to the known corrector types, thus exerting a new, complementary “type-IV” corrector mechanism. Macrocycles achieved wildtype-like folding efficiency of F508del-CFTR at the endoplasmic reticulum and normalized CFTR currents in reconstituted patient-derived bronchial epithelium. Using photo-activatable macrocycles, docking studies and site-directed mutagenesis a highly probable binding site and pose for type-IV correctors was identified in a cavity between lasso helix-1 (Lh1) and transmembrane helix-1 of membrane spanning domain-1 (MSD1), distinct from the known corrector binding sites. Since only F508del-CFTR fragments spanning from Lh1 until MSD2 responded to type-IV correctors, these likely promote co-translational assembly of Lh1, MSD1, and MSD2. Remarkably, previously corrector-resistant CFTR folding mutations were also robustly rescued, suggesting substantial therapeutic potential for this novel type-IV corrector mechanism.
Teaser
A novel type of macrocyclic CFTR corrector with new binding site, complementary mode of action and unique folding / trafficking efficacy is described.
... Another example of long-range allostery in CFTR is the finding that point mutations at the cytosolic loops increase channel open probability (even in the absence of ATP) by shifting a pre-existing conformational equilibrium towards the ligand bound conducting conformation (26). Notably, allostery in CFTR has direct therapeutic relevance: The potentiator drug ivacaftor and the dual-function modulator elexacaftor do not bind in the proximity of the gating residues (27,28) yet increase the open probability (Po) of the channel (29,30). These and other studies (31)(32)(33) significantly advanced our understanding of the function of CFTR and its allosteric control. ...
... There are currently four FDA-approved drugs that are used to treat CF: The type I correctors lumacaftor (VX-809) and tezacaftor (VX-661) stabilize mutant CFTR variants (predominantly the most common ΔF508 mutation) reducing premature protein degradation and improving surface presentation of the mature channel (54,55). The potentiator ivacaftor (VX-770) enhances the activity of conductance-defective mutants by increasing the open probability (Po) of the channel (24,27,29), and the dualfunction type III corrector elexacaftor (VX-445) both stabilizes unstable variants and increases channel open probability (30,56). The recently published structures of WT and ΔF508CFTR bound to various drugs (28) enabled to us to investigate the allosteric effects of the drugs. ...
Malfunction of the CFTR protein results in cystic fibrosis, one of the most common hereditary diseases. CFTR functions as an anion channel, the gating of which is controlled by long-range allosteric communications. Allostery also has direct bearings on CF treatment: CFTR drugs bind at the periphery of the protein yet affect the gating residues that lie at the center of it. Herein, we combined two computational approaches; Anisotropic Normal Mode-Langevin dynamics (ANM-LD) and Transfer Entropy (TE) and investigated the allosteric communications network of CFTR. The results are in excellent agreement with experimental observations and provide extensive novel insight. We identified residues that serve as pivotal allosteric sources and transducers, many of which correspond to disease causing mutations. We observe that the degenerate and catalytic ATP sites asymmetrically contribute to the allosteric communication, and that the catalytic site provides the greater allosteric input. We demonstrate that drugs that potentiate CFTR’s conductance do so not by directly acting on the gating residues, but rather by mimicking the allosteric signal sent by the ATP binding sites. We identify a hitherto unknown allosteric hotspot near the docking site of the phosphorylated R domain, providing a molecular basis for its phosphorylation dependent excitatory role. This study uncovers the molecular basis of allosteric connectivity in CFTR and reveals a novel allosteric hotspot that can serve as a target for the development of novel therapeutics.
... Another example of long-range allostery in CFTR is the finding that point mutations at the cytosolic loops increase channel open probability (even in the absence of ATP) by shifting a pre-existing conformational equilibrium toward the ligand bound conducting conformation (Wang et al., 2010). Notably, allostery in CFTR has direct therapeutic relevance: The potentiator drug ivacaftor and the dual-function modulator elexacaftor do not bind in the proximity of the gating residues (Liu et al., 2019;Fiedorczuk and Chen, 2022a) yet increase the open probability (Po) of the channel (Eckford et al., 2012;Shaughnessy et al., 2021). These and other studies (Sorum et al., 2017;Scholl et al., 2021;Wei et al., 2014) significantly advanced our understanding of the function of CFTR and its allosteric control. ...
... There are currently four FDA-approved drugs that are used to treat CF: The type I correctors lumacaftor (VX-809) and tezacaftor (VX-661) stabilize mutant CFTR variants (predominantly the most common ΔF508 mutation) reducing premature protein degradation and improving surface presentation of the mature channel (Van Goor et al., 2011;Fiedorczuk and Chen, 2022b). The potentiator ivacaftor (VX-770) enhances the activity of conductance-defective mutants by increasing the open probability (Po) of the channel (Levring et al., 2023;Liu et al., 2019;Eckford et al., 2012), and the dual-function type III corrector elexacaftor (VX-445) stabilizes unstable variants and increases channel open probability (Shaughnessy et al., 2021;Veit et al., 2021). The recently published structures of WT and ΔF508CFTR bound to various drugs (Fiedorczuk and Chen, 2022a) enabled us to investigate the allosteric effects of the drugs. ...
Malfunction of the CFTR protein results in cystic fibrosis, one of the most common hereditary diseases. CFTR functions as an anion channel, the gating of which is controlled by long-range allosteric communications. Allostery also has direct bearings on CF treatment: CFTR drugs bind at the periphery of the protein yet affect the gating residues that lie at the center of it. Herein, we combined two computational approaches; Anisotropic Normal Mode-Langevin dynamics (ANM-LD) and Transfer Entropy (TE) and investigated the allosteric communications network of CFTR. The results are in excellent agreement with experimental observations and provide extensive novel insight. We identified residues that serve as pivotal allosteric sources and transducers, many of which correspond to disease causing mutations. We observe that the degenerate and catalytic ATP sites asymmetrically contribute to the allosteric communication, and that the catalytic site provides the greater allosteric input. We demonstrate that drugs that potentiate CFTR’s conductance do so not by directly acting on the gating residues, but rather by mimicking the allosteric signal sent by the ATP binding sites. We identify a hitherto unknown allosteric hotspot near the docking site of the phosphorylated R domain, providing a molecular basis for its phosphorylation dependent excitatory role. This study uncovers the molecular basis of allosteric connectivity in CFTR and reveals a novel allosteric hotspot that can serve as a target for the development of novel therapeutics.
... Notably, allostery in CFTR has direct therapeutic relevance: The potentiator drug ivacaftor and the dual-function modulator elexacaftor do not bind in the proximity of the gating residues (27,28) yet increase the open probability (Po) of the channel (29,30). These and other studies (31)(32)(33) significantly advanced our understanding of the function of CFTR and its allosteric control. ...
Malfunction of the CFTR protein results in cystic fibrosis, one of the most common hereditary diseases. CFTR functions as an anion channel, the gating of which is controlled by long-range allosteric communications. Allostery also has direct bearings on CF treatment: CFTR drugs bind at the periphery of the protein yet affect the gating residues that lie at the center of it. Herein, we combined two computational approaches; Anisotropic Normal Mode-Langevin dynamics (ANM-LD) and Transfer Entropy (TE) and investigated the allosteric communications network of CFTR. The results are in excellent agreement with experimental observations and provide extensive novel insight. We identified residues that serve as pivotal allosteric sources and transducers, many of which correspond to disease causing mutations. We observe that the degenerate and catalytic ATP sites asymmetrically contribute to the allosteric communication, and that the catalytic site provides the greater allosteric input. We demonstrate that drugs that potentiate CFTR's conductance do so not by directly acting on the gating residues, but rather by mimicking the allosteric signal sent by the ATP binding sites. We identify a hitherto unknown allosteric hotspot near the docking site of the phosphorylated R domain, providing a molecular basis for its phosphorylation dependent excitatory role. This study uncovers the molecular basis of allosteric connectivity in CFTR and reveals a novel allosteric hotspot that can serve as a target for the development of novel therapeutics.
... ELX and TEZ function to correct CFTR misfolding and facilitate trafficking to the cell surface, increasing the amount of CFTR available at the cell surface (Veit et al., 2020). In addition, ELX has been described to have potentiator activity (Shaughnessy et al., 2021;Veit et al., 2021). In F508del/F508del differentiated hNECs, ELX/TEZ dual therapy significantly increased CFTR activity 14.06fold (p < 0.0001), reaching 18.80 μA/cm 2 above baseline ( Figure 4G; total I sc-Fsk : 20.24 ± 2.78 μA/cm 2 , Supplementary Figure S1D; Supplementary Table S5). ...
... This enhancement is expected to be largely contributed to by restoration of the F508del allele folding defect, since the G551D mutation impairs CFTR channel opening but does not cause a folding defect (Sermet-Gaudelus, 2013). However, it is possible that the G551D allele is potentiated by ELX which has recently been shown to exhibit synergy with IVA in potentiating the G551D mutation in human airway epithelia (Shaughnessy et al., 2021;Veit et al., 2021). In contrast, ELX/TEZ dual therapy in Q1291H/F508del differentiated hNECs only increased CFTR activity by 1.74-fold, reaching only 2.55 μA/cm 2 above baseline (total I sc-Fsk : 6.00 ± 0.80 μA/cm 2 ). ...
Background: Cystic fibrosis (CF) is caused by a wide spectrum of mutations in the
CF transmembrane conductance regulator (CFTR) gene, with some leading to
non-classical clinical presentations. We present an integrated in vivo, in silico and
in vitro investigation of an individual with CF carrying the rare Q1291H-CFTR allele
and the common F508del allele. At age 56 years, the participant had obstructive
lung disease and bronchiectasis, qualifying for Elexacaftor/Tezacaftor/Ivacaftor
(ETI) CFTR modulator treatment due to their F508del allele. Q1291H CFTR incurs a
splicing defect, producing both a normally spliced but mutant mRNA isoform and
a misspliced isoform with a premature termination codon, causing nonsense
mediated decay. The effectiveness of ETI in restoring Q1291H-CFTR is largely
unknown.
Methods: We collected clinical endpoint measurements, including forced
expiratory volume in 1 s percent predicted (FEV1pp) and body mass index
(BMI), and examined medical history. In silico simulations of the Q1291H-CFTR
were compared to Q1291R, G551D, and wild-type (WT)-CFTR. We quantified
relative Q1291H CFTR mRNA isoform abundance in patient-derived nasal
epithelial cells. Differentiated pseudostratified airway epithelial cell models at
air liquid interface were created and ETI treatment impact on CFTR was
assessed by electrophysiology assays and Western blot.
Results: The participant ceased ETI treatment after 3 months due to adverse
events and no improvement in FEV1pp or BMI. In silico simulations of Q1291HCFTR
identified impairment of ATP binding similar to known gating mutants
Q1291R and G551D-CFTR. Q1291H and F508del mRNA transcripts composed 32.91% and 67.09% of total mRNA respectively, indicating 50.94% of Q1291H mRNA
was misspliced and degraded. Mature Q1291H-CFTR protein expression was
reduced (3.18% ± 0.60% of WT/WT) and remained unchanged with ETI. Baseline
CFTR activity was minimal (3.45 ± 0.25 μA/cm2) and not enhanced with ETI (5.73 ±
0.48 μA/cm2), aligning with the individual’s clinical evaluation as a non-responder
to ETI.
Conclusion: The combination of in silico simulations and in vitro theratyping in
patient-derived cell models can effectively assess CFTR modulator efficacy for
individuals with non-classical CF manifestations or rare CFTR mutations, guiding
personalized treatment strategies and optimizing clinical outcomes.
