To date, however, the binding site for VX remains unsettled, with several studies proposing different locations, including 1 the interface between the lipid bilayer and TMDs Jih and Hwang, ; Yeh et al. In short, the apparent affinity of a potentiator is determined by how tight it binds to both the open and closed channel conformations, whereas the efficacy is determined by the differences in free energy of binding between the open and closed states, regardless of the absolute value of the free energy of binding in each state.
This idea of state-dependent binding demands that the structure of the binding site for a potentiator in an open state should differ from that in a closed state. Thus, in theory, one can manipulate the efficacy and affinity of the potentiator by altering its chemical interactions with the binding sites. However, to realize this ambition of structure-based drug design, one has to first identify the binding sites.
Taking advantage of the detailed molecular pictures of CFTR and the extensively studied mechanism of action for CFTR potentiators, we envision the structure-based drug design a real possibility once the molecular targets for CFTR potentiators are identified.
The comparative contribution of these two sites to the binding of CFTR potentiators and the implication of our results for structure-based drug design will be discussed. Transfected cells grown on a glass chip were transferred to a chamber filled with standard inside-out perfusate on the stage of an inverted microscope IX51; Olympus at room temperature.
VX was provided by R. All chemicals were diluted with the standard inside-out perfusate, and the pH was adjusted to 7. While this limiting concentration worked well for most of our constructs, some mutations may have resulted in a more drastic shift of the dose—response relationship, and therefore, a complete dose—response curve was not obtained e.
Igor Pro 7 Wave-Metrics was used to measure the steady-state mean current amplitude, estimate the relaxation time constant, and perform Hill equation fitting with built-in functions to determine the dose—response relationships. The normalized response y axis was plotted against the corresponding concentrations x axis , and Hill equation fitting was performed to estimate the half-effective concentration EC The base of the Hill equation was held at zero, and the maximal response predicted by the Hill equation fitting was normalized to one.
Notice that this second normalization is performed for better presentation of the dose—response curves, and it does not affect the value of EC For single-channel studies Figs. Single-channel traces were inverted for a better visualization.
To ensure the accuracy of the number of functional channels in the patch, we applied GLPG in each experiment to determine the number of channels and analyzed data from patches yielding a maximum of four simultaneous opening steps for microscopic kinetics.
Both structures were downloaded from the PDB Berman et al. The recommended parameters were adopted and the two bound ATP molecules in 5W81 were included in the modeling work. As compared with the original version, which only provides the top 20 docking modes, our modified version offers a broader view of the docking landscape of our targets. The modeled CFTR protein structure was prepared as described in the last section.
Through the conversion process, partial charges were calculated by the AutoDock Vina with the Gasteiger method and assigned to the atoms of the protein, and the nonpolar hydrogen atoms were united with the heavy atoms to which they were bonded. In docking, the protein structure was treated as a rigid body, whereas the ligands were allowed to be flexible. The more negative the energy score, the better the rank of the binding mode. In this study, our goal was to identify their exact binding sites in the CFTR protein.
The output poses of GLPG were clustered visually, and the pose exhibiting most interactions with CFTR was selected as the representative conformation for each site.
We caution our readers that due to the limited accuracy of the existing docking scoring functions Grinter and Zou, ; Gaieb et al. Nonetheless, the docking methodology does provide a blunt yet valuable screening tool to preselect otherwise innumerable potential binding sites for functional studies. Among these residues, we then defined the ones that have a side chain pointing toward the bound ligand and thus can potentially interact with GLPG as the candidates for further examination with the patch-clamp functional assay.
The predicted binding sites were then tested by first making mutations at the above-identified positions and then measuring CFTR currents at different potentiator concentrations with the patch-clamp technique to quantitatively assess the effects of mutations in each binding site on the dose—response relationships of GLPG This straightforward assay, however, is impractical for VX, since we have previously determined that nM is already a saturating concentration for VX, and the actual EC 50 may be much lower Jih and Hwang, ; but compare Hadida et al.
As concentrations in the range of picomolars must be used to complete the dose—response curve for VX, it will take very long time to achieve a steady state. In addition, the stickiness of VX to the recording device also adds another level of technical difficulty in assuring the correct concentrations used in each experiment. Therefore, we used GLPG, which assumes a micromolar affinity and is readily removed from the system, in all the following dose—response experiments and took a different strategy to estimate the affinity for VX, as described latter in this section.
In this binding mode, the aspartate side chain appears to be hydrogen bonded with the two nitrogen atoms in the pyrazole ring of GLPG We reasoned that the binding affinity of GLPG may be changed by introducing mutation to disrupt the hydrogen bonding. As shown in Fig. We next replaced the aspartate with alanine DA in an attempt to observe a more drastic change in the affinity of GLPG Although a steady-state dose—response relationship and a corresponding EC 50 cannot be attained because we could not obtain macroscopic currents for DA-CFTR but a representative microscopic recording is shown in Fig.
While the dose—response experiment is a straightforward assay to obtain the apparent affinity, one can also measure the apparent dissociation rate or the off-rate of the drug by monitoring the time course of current decay upon sudden removal of the compound.
In Fig. The statistical comparison shown in Fig. It should be noted that the affinity of a drug is determined by both its on-rate and off-rate, and this relaxation analysis only assesses the off-rate. Despite this caveat, this measurement can be used as a complementary tool to evaluate any changes in the binding affinity of a molecule when performing dose—response experiment is not feasible, as in the case of VX see Figs.
The next amino acid in site I is N in transmembrane segment TM We thus speculated that substitutions of N with hydrophobic amino acids may actually improve the binding of GLPG First, due to its intrinsic low open probability P o ; Bompadre et al.
As seen in the docking mode shown in Fig. Compared with N, S is somewhat distant from the bound drug, suggesting a weaker interaction. Indeed, a leftward shift was observed, whereas the threonine substitution, which carries a hydroxyl group like serine, poses minimal effect Fig. To this point, we found that mutations on the three residues in site I either decrease D or increase N and S the affinity for GLPG, supporting the notion that these amino acids may contribute to the binding of GLPG We next tested the effect of alanine substitution on the amino acids in the three lower-scoring sites II, III, and IV predicted by docking.
Again, we reasoned that if any of the residues were involved in the binding of GLPG, substitution of the original side chain with alanine should disrupt the drug—protein interaction and hence decrease the affinity. Replacing the former three residues with alanine poses minimal effect on the affinity for GLPG Fig.
However, it should be noted that this slight change in apparent affinity could be attributed to a decreased P o of this mutant based on the idea of state-dependent binding Yeh et al. A simple calculation yields a maximum P o of 0. However, one caveat in the aforementioned study is that the docking simulation was based on a homology model, which could be different significantly from the actual structure of hCFTR.
We immediately performed molecular docking on this structure and compared the results with those shown in Fig. S1, E and F. This new site named site II N , because its score is between site I and II consists of four phenylalanine F, F, F, and F residues and one tyrosine Y residue, forming a hydrophobic cradle to accommodate the potentiators Fig. To test this idea, we determined the dose—response relationships for GLPG with each residue replaced with alanine one at a time.
According to the idea of state-dependent binding Yeh et al. This idea will be tested in Fig. We substituted Y with phenylalanine, threonine, and alanine. Although for technical reasons see Materials and methods we did not obtain a complete dose—response relationship of GLPG for YA- and YT-CFTR, the current relaxation analysis upon removal of GLPG, which reflects the apparent dissociation rate, did provide another line of supporting evidence for a significant decrease in the affinity Fig.
According to the idea of state-dependent binding, the positive correlation between apparent affinity and P o demands that the measured apparent affinity increases as the P o increases Yeh et al. In other words, the true binding affinity for GLPG is significantly decreased by the FA mutation, but the effect is masked by the opposing effect of its high P o. In this condition, the dose—response relationship is rightward shifted, and Hill equation fitting yields an EC 50 of 1.
As discussed earlier, it is impractical, at least in our experimental setting, to perform the same dose—response experiments for VX to confirm whether it binds to the same sites.
Here, we took the alternative strategy of relaxation analysis described in Fig. As the currents do not relax back to the initial level before the application of VX Fig. Nevertheless, some observed changes in the decay time constants are visually discernible e. Similar relaxation analysis was performed to test the changes in the affinity for VX by mutations in site II N.
Similarly, Fig. These data shown in Figs. In this study, we combined in silico docking and the patch-clamp functional assay to identify the molecular target sites for CFTR potentiators GLPG and VX, which were previously shown to share a common binding site Yeh et al. In this section, we will first discuss the intrinsic limitations of the techniques used in the current study, including homology model, molecular docking, and the functional assay using mutants as the main tool.
Next, we will delve into more detailed discussion on the complexity in interpreting affinity and efficacy in the context of the state-dependent binding and seek a possible yet imperfect solution to untangle the convoluted relationship between drug binding and channel gating.
We will then provide a more in-depth analysis of the pros and cons for each of the identified binding sites for GLGP and VX Finally, we will speculate on the implications of our results on structure-based drug design, and discuss translational significance based on our current understanding of drug effects on patients carrying pathogenic mutations with gating defects.
It is imperative to realize that the chemical interactions between the potentiators and their targets, which determine their affinity and efficacy, are dynamic as the channel itself usually undergoes multiple conformational changes Colquhoun, To hunt for the binding sites, we first need to ask which conformation of the channel is a better starting point. This issue is particularly critical, as the drug—protein interaction is highly sensitive to the local amino acid positioning in the binding sites.
The fact that our docking software failed to identify site II N in the homology model because the side chain of residue F in the modeled structure obstructs this binding pocket bespeaks this very point. Although the problem of homology modeling was solved by the timely atomic structure of hCFTR, docking simulation bears its own limitations. First, the computer program allows the drugs to assume different orientations but treats the protein as a rigid body with immobile amino acid side chains.
However, it is unrealistic to assume that the amino acids are positioned at the exact location without dynamic changes upon drug binding.
For example, according to the induced-fit model, binding of a ligand could cause considerable changes in the shape of its binding site Koshland, Thus, the docking software—calculated free energy of interaction between the ligand and its target may not represent the actual condition where the target could adjust to better interact with the ligand. Furthermore, the lipid bilayer is not considered in current docking.
The docking simulation assigned a better i. The affinity for site II N may in fact be greater than site I in a physiological condition due to a more favorable partition of hydrophobic compounds such as GLPG and VX into the lipid phase. Although what have been discussed here are of a more speculative nature, they highlight the potential limitations of the docking approach to identify the binding sites.
Despite these caveats, we recognized that the docking results can serve as a guide in our hunt for the drug binding sites. Once potential binding partners are identified through docking, we can then alter the amino acids involved in binding and measure the effects of the mutations on the sensitivity and efficacy of the drugs of interest.
One immediate problem in mutational study is that we cannot exclude the possibility that observed change in the apparent affinity is actually caused by allosteric effects of the mutation rather than by a direct alteration of the binding event. There are at least two scenarios where the word allosteric applies. First, by allosteric, we mean the amino acid being mutated alters the structure of the actual binding site through a long-distance effect.
Single channel recordings were obtained using an Axopatch B amplifier and recorded to digital tape. Traces were played back with Clampfit 9. Both macropatch and single channel data were analyzed with Clampfit Data were acquired using the Acquire and Analyze software and exported to Excel for analysis.
Unless otherwise noted, all reagents were obtained from Sigma Chemical Co. Louis, MO. PKA was used at, or diluted from, P2 was kindly provided by Cystic Fibrosis Foundation Therapeutics.
All chemicals used in this study were diluted to their final concentration in the relevant recording solution immediately prior to use Paired or unpaired t-tests were performed using Sigmaplot A representative single channel trace recorded from one patch and the associated all-points amplitude histogram are shown for WT-CFTR activated by 6.
Single channel amplitude was similar in both 6. In addition, the macroscopic currents of CFTR, which increased from low to high PKA here, may be affected not only by single channel amplitude no change and channel activity open probability, increase , but also influenced by an increase in the total number of active channels the total number of CFTR channel proteins per patch likely remained unchanged in this system.
All-points amplitude histograms are shown in the right panels, where solid lines are fit results to a Gaussian function. Based upon our prior data, we hypothesized that efficacy of VXmediated potentiation also would depend upon the level of phosphorylation 14 , Representative current traces are shown in Fig.
The degree of potentiation was related to the concentration of PKA 0. The results verified our working hypothesis that VX would potentiate WT-CFTR in a PKA concentration-dependent manner possibly by increase channel open probability and the number of active channels. TM6 is indicated in blue shades. Given previous evidence together with the above data, we expanded our hypothesis to include not only wildtype, but also disease-causing mutations in CFTR, to determine whether they, too, would be potentiated by VX in a phosphorylation-dependent manner We selected 6 variants based on the following criteria: 1 the most common mutations found in CF including Fdel We examined effects of 0.
Representative current traces and summary data are shown. These data provide strong evidence to support our hypothesis that VX potentiates wildtype as well as disease-associated mutants in a graded phosphorylation-dependent manner. Representative current traces and summary data are shown in Fig. We performed the following experiments to address these questions.
Although Fdel has been recognized to cause multiple defects including mistrafficking in mammalian cells, channel gating defects, and instability at the epithelial plasma membrane following rescue by low temperature or pharmacological correction, FC-CFTR is permissive for maturation with only a minor gating defect 11 , 30 , 31 , 32 , These data suggest that the apparent insensitivity of VXmediated potentiation of Fdel-CFTR under these conditions is not unique to this variant.
We tested the three variants according to the protocol shown in Fig. As summarized in Fig. We conclude that the unexpected behavior of these three mutants with respect to VXmediated potentiation may be attributable to a PKA-mediated effect, which is already saturated at 6. VX 0. Summary data for the ratio of three F mutants in high and low PKA conditions are shown in panel I and calculated based on the equation in Fig. P67 is located immediately adjacent to Lasso helix 2 Fig.
N66 is also situated in the Lasso domain and N66S has been associated with mild disease. P67L-CFTR exhibits trafficking and gating defects with normal single channel conductance, although the mechanism responsible for the gating defect remains unknown We defined activation duration as the time required to reach a plateau in the presence of high PKA.
These findings suggest that in our previous experiment Fig. The fractional increase by 0. Furthermore, both mutants exhibited potentiation seemingly insensitive to phosphorylation level when fully activated. Taken together, we conclude that the P67L gating defect is directly related to altered PKA sensitivity, a finding that supports previous reports that the R domain is possibly involved in CFTR gating regardless of its phosphorylation state and may interact with the Lasso motif 3 , 5.
TM6 is indicated in blue color. Summary data for activation durations are shown in panel J. GD is a representative Class III mutation with severe gating defects that include very low open probability and brief burst duration 3 , 34 , 35 , We hypothesized that the extreme sensitivity of GD-CFTR to potentiation may reflect an as yet undiscovered mechanism.
As shown in Fig. These results are consistent with an earlier report Summary data are shown in panel F. TM1—6 are indicated in blue shades and TM7—12 are indicated in orange shades.
Unfortunately, the complete mechanism behind this effect remains unclear. The same sort of abnormality could occur in GS. We functionally tested the GS mutation, but surprisingly, could not recover CFTR channels in oocytes even after multiple experiments.
We previously predicted based on molecular dynamics simulations using a CFTR homology model that D in G moved close to lysine at position during NBD dimerization, and might establish an aberrant electrostatic interaction Fig. We note that when channels were fully activated by PKA, the response to VX was greatly diminished. Summary data for effects of 0.
Cytoplasmic 0. Summary data are shown in C. F Summary data for mean activation durations. These observations led us to hypothesize that VX potentiates CFTR in multiple ways, and we tested this hypothesis as follows. A representative current trace is shown in Fig.
Summary data Fig. These data, combined with results in Fig. Summary data for deactivation are shown in F , G. Summary data for mean burst durations are shown in I. The experimental protocol was otherwise similar to that used to generate the macroscopic recordings shown in Fig. In summary, VX potentiated several CFTR disease-associated mutants expressed in oocytes, in a manner dependent upon phosphorylation level regardless of mutation location across the whole CFTR protein. Taken together, these data strongly suggest that VX possibly binds in more than one binding pocket in WT- as well as mutant CFTR in order to allosterically modulate the channel protein.
Our data thus far suggest that the potentiation of CFTR by VX is mediated by phosphorylation level when the channel is expressed heterologously in Xenopus oocytes. We next asked whether the activation level of CFTR affected potentiation efficacy in a more physiologically relevant system, FRT epithelial cells. This inverse relationship was observed over the entire 10 5 -fold range of FSK concentrations tested Fig. This suggests that with submaximal activation of P67L-CFTR, VX is efficacious enough to benefit patients bearing these mutations if channels are properly trafficked to the plasma membrane.
These data strongly suggest that the VXmediated potentiation of CFTR in primary bronchial epithelial cells is dependent upon phosphorylation level, similar to that observed in the heterologous expression systems.
It was commonly believed that the R domain simply moves out of the way after phosphorylation to allow the NBDs to form a dimer and the CFTR channel to open. One piece of evidence supporting this idea is that deleting the R domain from CFTR allows it to open in a manner independent of phosphorylation while still requiring MgATP However, the open probability and macroscopic current density of the R domain-deleted CFTR is only one third that of the WT channel, suggesting a fundamental difference between the two CFTR variants Another piece of evidence supporting the R domain contribution to channel gating of CFTR was proposed by Liu and colleagues recently, where they found that the R domain directly modulated CFTR channel gating from dephosphorylated D to phosphorylated P and maximally activated M states 5.
In addition, more and more evidence collected in the last decade supports the hypothesis that the phosphorylated R domain interacts directly with several intracellular parts of CFTR including the N-terminus, ICLs, NBDs, and the C-terminus, thus affecting channel activity 7 , 8 , 51 , 52 , 53 , The Lasso motif may be similar to extracellular loop 1 in that it requires a finely-tuned structure in order to precisely perform its function. Consequently, mutations like P67L and N66S could destabilize the loop structure and significantly shift interactions between the R domain and the Lasso motif, while a stable loop structure may be a prerequisite for R domain modulation of CFTR channel function As demonstrated previously, Fdel, the most common disease mutation in the American CF population, induces multiple defects including poor trafficking in mammalian cells, gating dysfunction, and fast endocytosis after the protein is trafficked to the plasma membrane In addition, the aromatic side chain of F was proposed to have a strong impact on CFTR function We found that similar to Fdel-CFTR, variants bearing FS and FC demonstrated altered PKA sensitivity suggesting that phenylalanine in position also might be necessary for maintaining local structure and interactions with the R domain.
Data presented here suggest that these graded interactions between the R domain and other parts of the channel, as regulated by the level of PKA-mediated phosphorylation, also determine the sensitivity to potentiation by VX and P2 for all variants we studied here. Hence, we conclude that phosphorylation-dependence of potentiation may be a fundamental characteristic of the allosteric regulation of CFTR function.
Our previous simulation results predicted that the D at position may form an electrostatic interaction with K at position that both perturbs ATP binding and slows down activation of GD-CFTR.
These results are also supported by previous observations 35 , Taken together, we propose that the unique and complicated behavior of GD-CFTR results from an electrostatic interaction between D at and K at This interaction between phosphorylation level and efficacy of VXmediated potentiation is conserved across cells expressing WT- or mutant CFTR variants endogenously or heterologously.
This finding has practical implications because phosphorylation levels that tune CFTR activity in bodily tissues likely are far below the maximum levels achieved with direct application of PKA, a notion supported by two observations. First, long-term PKA-mediated activation of CFTR by cholera toxin poisoning leads to levels of water loss that far exceed normal physiological levels Second, nasal potential difference NPDs in healthy individuals is increased by perfusion with isoproterenol due to an increment in CFTR activity from resting condition Hence, CFTR is not fully activated under normal physiological states.
Additionally, our data using P67L-FRT cells suggest that using a lower concentration of forskolin might enable the discovery of more potentiators by increasing the available dynamic range, thus avoiding the ceiling effect.
Since there are not enough patients with the P67L or other ultra-orphan mutation genotypes to run a full-scale clinical trial, in vitro data will be a large consideration in determining whether VX or other potentiators receive FDA approval Our results suggest that such drug screening should be performed on CFTR channels at a lower activation state.
The half-maximal activation concentration of FSK EC 50 was significantly reduced with VX chronic treatment while the maximal I sc remained unaffected. However, it is unknown whether VX would affect phosphorylation levels of CFTR channels when it is used chronically. In addition, while this manuscript was under revision there were two publications that reported a possible binding pocket in the TMs for VX 64 , There is no doubt that more studies are required to fully understand the mechanism and binding sites of VX in potentiation of CFTR to benefit the CF patient community.
Gadsby, D. The ABC protein turned chloride channel whose failure causes cystic fibrosis. Vx dissolved in 1-octanol shows strong absorption with a peak at nm. Absorption spectra were measured in a 1 ml quartz cuvette against 1-octanol saturated with water as a blank Figure 1b , blue spectra.
To extract Vx from the 1x saturated aqueous stock, two 15 ml conical tubes, each containing 14 ml of the 1x aqueous stock plus 0. To extract the remaining aqueous phase for a second time, the remnants of the 1-octanol phase were carefully discarded from both conical tubes, and The obtained calibration curve was identical to that shown in Figure 1b—c i.
The pH of the solution was therefore readjusted to 7. To measure Vx concentration in the 1x saturated stock solution, 0. Current recordings were obtained 1—3 days after injection.
Following patch excision the patch pipette was moved into a flow chamber, and recordings were done under continuous superfusion of the cytosolic patch surface. After each recording day perfusion tubings were extensively washed with distilled water. Although supersaturated solutions of Vx were not employed here, any perfusion tubing that had ever been in contact with a given concentration of Vx was subsequently used only for solutions that contained the same, or higher, concentrations of the drug.
After every 5—6 experimental days the entire perfusion system was replaced. For display purposes, currents are shown filtered at 10 Hz and sampled at 20 Hz, and expanded single-channel traces Figure 2b , Figure 4d are shown filtered at 50 Hz and sampled at Hz. Vx was applied after currents elicited by 2 mM ATP had stabilized. To allow for full development of its potentiator effect, Vx was superfused until the current reached steady state which, for low nanomolar Vx, required up to 5—6 min.
After full stimulation, Vx was washed off for at least 5 min to allow the current to decline to a post-drug steady state. Whereas this simple irreversible scheme is clearly unsuitable to explain reversibility of drug effects, and does not address the mechanism of the drug, it provided sufficient flexibility for a perfect fit of individual activation time courses.
Segments of current recording originating from 1 to 7 active channels were digitally filtered at 50 Hz, and idealized by half-amplitude threshold crossing. Steady-state open probabilities in 2 mM ATP, before or during application of Vx or after its removal Figure 2c , were calculated from the events lists as the time-average of the fraction of open channels.
The software is freely available upon request. The stability plot in Figure 4d was obtained by calculating open probability over sequential 46 s time windows. The dynamic equations that describe the kinetic models depicted in Figure 5 are as follows:.
For the ensemble fitting, all experimental current traces were normalized to their steady-state values observed prior to exposure to Vx Predicted normalized on- and off- current time courses for each model were calculated from the above dynamic equations by solving for the time-dependent evolution of the vector x , using a modified Euler's method Press, For the on- time courses the initial values for all x i were set to zero, for the off- time courses the final x vector of the preceding on- time course served as the initial vector while V c was set to zero.
The sets of predicted normalized on-off time courses were fitted to the sets of normalized experimental current traces by simple least squares, using a downhill simplex method for optimization Press, Under the conditions used here, that is upon prolonged exposure of the cytosolic face of the patch to a constant concentration of Vx, with no Vx present on the extracellular side, all four kinetic models shown in Figure 5 predict relaxation of the system to a steady state.
At steady state there is a constant outward directed transmembrane flux of Vx, while its concentration remains constant within the membrane or membrane leaflets , and close to zero in the extracellular pipette solution. Once the drug concentrations in the various compartments have stabilized, the channel pool approaches an equilibrium distribution between its available unliganded, ligandbound, and, for Schemes 3 and 4, diliganded forms. The concentration-dependence of steady-state current activation reflects the concentration-dependence of the fractional occupancy of the monoliganded state Y 1 for Schemes 1 and 2, but of the diliganded state Y 2 for Schemes 3 and 4.
The midpoints of these predicted concentration response curves K 0. The apparent drug affinities K 0. M from 3 to 13 independent measurements, as indicated in the figure legends.
All data generated or analyzed during this study are included in the manuscript or can be visualized in the figures. In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses.
A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.
Thank you for submitting your work entitled "Cystic fibrosis drug ivacaftor stimulates CFTR channels at picomolar concentrations" for consideration by eLife. Your article has been reviewed by three peer reviewers, including Leon D Islas as the Reviewing Editor and Reviewer 1, and the evaluation has been overseen by a Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Sidney Simon Reviewer 3.
Our decision has been reached after consultation between the reviewers. Based on these discussions and the individual reviews below, we regret to inform you that your work will not be considered further for publication in eLife. While the reviewers agree that the data presented in the manuscript is sound and represents an important technical development in understanding the action of ivacaftor on CFTR, they also think that the paper did not provide any insight into the molecular interaction of the drug with the channel and the determinants of solubility in biological tissues.
The reviewers point out that this diminishes the relevance and interest of the paper to a broader readership. Multiple mutations are associated with CFTR dysfunction and consequent induction of cystic fibrosis CF , which can be lethal.
One of the few treatments approved for CF is the drug Vx, which has been shown to be effective in potentiating CFTR mutants characterized by a low open probability, a gating defect. The experiments included in this manuscript show that Vx has an extremely low solubility in water.
As a consequence of this, the dose utilized in diverse laboratories is likely extremely high and mostly undetermined, accounting for the different values of EC 50 reported.
More interestingly, Vx effects are reportedly irreversible. This manuscript presents a determination of the solubility of Vx in several solvents, including water, and shows that when the correct activating doses below nM concentrations are applied, the drug effects are perfectly reversible. The experiments are of high quality and represent a solid foundation to start to understand the biophysics and molecular basis of the gating effects of this drug. In addition, this is an important contribution that might have clinical relevance, as it will help understand the pharmacokinetics of Vx I have a suggestion regarding the use of the multiple-step model employed to explain the delay in activation time course when Vx is applied.
The suggested model is an irreversible model, which may appear as a contradiction with the finding that the drug effects are indeed reversible. Also, it should be clear what state occupancy is being compared with the current time course.
And finally it should be clearly stated that this is not a scheme that explain gating, that is, states S1 to S4 are not to be interpreted as channel conformational states. In this study the authors determine the solubility profile and stimulatory potency of Vx ivacaftor , a CFTR potentiator drug in clinical use to treat people with cystic fibrosis, towards wild-type and mutant human CFTR channels using the patch-clamp technique with excised inside-out membrane patches.
They find evidence that several slow steps are involved in the activation pathway. The experiments are generally well done and of high quality.
The manuscript is well written. However, significance is reduced because no new insight is gained how Vx interacts with CFTR to activate channel activity. Usually a methodological progress is presented in the context of describing an important new insight that could be obtained because of the method. In addition, because solubility in biological fluids at physiological pH and body temperature is not studied, the results appear only relevant to a small group of readers who perform CFTR patch clamp electrophysiology with excised membrane patches.
The authors do not study solubility of Vx in biological fluids and tissues. For instance, protein present in a biological intra- or extracellular fluid may increase solubility substantially. Figure 2C: A one-tailed Student t-test appears not appropriate. First, since CFTR channel inactivation by Vx has been described among the effects of Vx in the literature see Introduction section final paragraph the statistical test should be two-tailed.
Second, repeated measurement ANOVA should be considered since technically there are two interventions with the same channel. In this paper the WT and two mutants of the CTFR channel are investigated in inside out patches in the absence and presence of continually perfused Vx, a clinically relevant drug used in CF patients.
In my opinion the most important finding of this work is that they found is that, in contrast to previous studies that found Vx to be irreversible, they found that by using lower concentrations that it is completely reversible. They measured its oil octanol etc. Thank you for submitting your article "Cystic fibrosis drug ivacaftor stimulates CFTR channels at picomolar concentrations" for consideration by eLife.
The editors have discussed the appeal with one another and are happy to consider a revised version of your manuscript.
This is an appeal of a previous decision and the author's proposed changes for improvement are reasonable and will improve the paper.
Our suggestion is that you make the changes that are proposed in your appeal letter, especially place more emphasis on the clinical and physiological relevance of the findings.
A discussion of the implication of the results for the pharmacokinetics of ivacaftor can also help to further the case for the clinical relevance of the present findings.
I kindly ask for the reevaluation of the reasons that have led to the rejection of our manuscript, and for a potential reconsideration of the editorial decision. The Reviewers all agreed that the results support the conclusions. The rejection was based on two issues: 1 a lack of insight into the molecular interaction of the drug with the channel, and 2 a lack of information on its solubility in biological tissues.
I have serious objections against both of these major criticisms, as discussed in detail below. As to the question of significance, I had sent a presubmission inquiry to Senior Editor Richard Aldrich, describing him the main findings of this study, and explicitly pointing out that no biological mechanisms are addressed. In his response, Dr.
Aldrich judged the findings of sufficient interest, and encouraged submission to eLife. It comes therefore as a surprise that the findings are judged insignificant at the present stage. I do agree that in general determination of solubility and potency of a drug is in itself not a major advance. But there are not many drugs that make it to the clinics, and ivacaftor is one of them. Therefore, I do think that in the present case the simple establishment of the drug's true solubility profile, true potency, and concentration dependence of its apparent on- and off-rates, is a major breakthrough which will put all published studies into a new perspective, and allow all future studies to be planned adequately.
As to the second point, reviewers 2 and 3 seem to have misunderstood the relevance of a saturated vs. However, cell membranes are at equilibrium with FREE dissolved Vx, not with the protein-bound drug. The composition of the aqueous saline in which Vx solubility was determined resembled that of an intracellular fluid with respect to ionic strength, osmolarity, as well as pH 7.
We did not test solubility at 37 o C, but this could be very easily added. Of note, although solubility at 37 o C is clearly of academic interest, we believe that solubility at 25 o C is practically more relevant: solubility has to be known under the conditions that are used in experiments aimed at understanding the mechanisms of the drug. The majority of such experiments have been conducted at room temperature.
This is a misunderstanding.
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