Steven Molinski

Functional Rescue of F508del-CFTR Using Small Molecule Correctors

High-throughput screens for small molecules that are effective in “correcting” the functional expression of F508del-CFTR have yielded several promising hits. Two such compounds are currently in clinical trial. Despite this success, it is clear that further advances will be required in order to restore 50% or greater of wild-type CFTR function to the airways of patients harboring the F508del-CFTR protein. Progress will be enhanced by our better understanding of the molecular and cellular defects caused by the F508del mutation, present in 90% of CF patients. The goal of this chapter is to review the current understanding of defects caused by F508del in the CFTR protein and in CFTR-mediated interactions important for its biosynthesis, trafficking, channel function, and stability at the cell surface. Finally, we will discuss the gaps in our knowledge regarding the mechanism of action of existing correctors, the unmet need to discover compounds which restore proper CFTR structure and function in CF affected tissues and new strategies for therapy development.

The major cystic fibrosis causing mutation exhibits defective propensity for phosphorylation

The major cystic fibrosis causing mutation, F508del‐CFTR (where CFTR is cystic fibrosis transmembrane conductance regulator), impairs biosynthetic maturation of the CFTR protein, limiting its expression as a phosphorylation‐dependent channel on the cell surface. The maturation defect can be partially rescued by low‐temperature (27°C) cell culture conditions or small‐molecule corrector compounds. Following its partial rescue, the open probability of F508del‐CFTR is enhanced by the potentiator compound, VX‐770. However, the channel activity of rescued F508del‐CFTR remains less than that of the Wt‐CFTR protein in the presence of VX‐770. In this study, we asked if there are allosteric effects of F508del on the phosphorylation‐regulated R domain. To identify defects in the R domain, we compared the phosphorylation status at protein kinase A sites in the R domain of Wt and F508del‐CFTR. Here we show that phosphorylation of Ser‐660, quantified by SRM‐MS, is reduced in F508del‐CFTR. Although the generation of a phosphomimic at this site (substituting aspartic acid for serine) did not modify the maturation defect, it did enhance F508del‐CFTR channel function after pharmacological rescue with corrector VX‐809, and treatment with the potentiator, VX‐770. These findings support the concept that defective phosphorylation of F508del‐CFTR partially accounts for its altered channel activity at the cell surface.

Facilitating Structure-Function Studies of CFTR Modulator Sites with Efficiencies in Mutagenesis and Functional Screening

There are nearly 2000 mutations in the CFTR gene associated with cystic fibrosis disease, and to date, the only approved drug, Kalydeco, has been effective in rescuing the functional expression of a small subset of these mutant proteins with defects in channel activation. However, there is currently an urgent need to assess other mutations for possible rescue by Kalydeco, and further, definition of the binding site of such modulators on CFTR would enhance our understanding of the mechanism of action of such therapeutics. Here, we describe a simple and rapid one-step PCR-based site-directed mutagenesis method to generate mutations in the CFTR gene. This method was used to generate CFTR mutants bearing deletions (p.Gln2_Trp846del, p.Ser700_Asp835del, p.Ile1234_Arg1239del) and truncation with polyhistidine tag insertion (p.Glu1172-3Gly-6-His*), which either recapitulate a disease phenotype or render tools for modulator binding site identification, with subsequent evaluation of drug responses using a high-throughput (384-well) membrane potential–sensitive fluorescence assay of CFTR channel activity within a 1 wk time frame. This proof-of-concept study shows that these methods enable rapid and quantitative comparison of multiple CFTR mutants to emerging drugs, facilitating future large-scale efforts to stratify mutants according to their “theratype” or most promising targeted therapy.

Genetic, cell biological, and clinical interrogation of the CFTR mutation c.3700 A>G (p.Ile1234Val) informs strategies for future medical intervention

Purpose:The purpose of this study was to determine the molecular consequences of the variant c.3700 A>G in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, a variant that has been predicted to cause a missense mutation in the CFTR protein (p.Ile1234Val).Methods:Clinical assays of CFTR function were performed, and genomic DNA from patients homozygous for c.3700 A>G and their family members was sequenced. Total RNA was extracted from epithelial cells of the patients, transcribed into complementary DNA, and sequenced. CFTR complementary DNA clones containing the missense mutation p.Ile1234Val or a truncated exon 19 (p.Ile1234_Arg1239del) were constructed and heterologously expressed to test CFTR protein synthesis and processing.Results:In vivo functional measurements revealed that the individuals homozygous for the variant c.3700 A>G exhibited defective CFTR function. We show that this mutation in exon 19 activates a cryptic donor splice site 18 bp upstream of the original donor splice site, resulting in deletion of six amino acids (r.3700_3717del; p.Ile1234_Arg1239del). This deletion, similar to p.Phe508del, causes a primary defect in folding and processing. Importantly, Lumacaftor (VX-809), currently in clinical trial for cystic fibrosis patients with the major cystic fibrosis–causing mutation, p.Phe508del, partially ameliorated the processing defect caused by p.Ile1234_Arg1239del.Conclusion:These studies highlight the need to verify molecular and clinical consequences of CFTR variants to define possible therapeutic strategies.Genet Med 16 8, 625–632.Genetics in Medicine (2014); 16 8, 625–632. doi:10.1038/gim.2014.4

Conformational defects underlie proteasomal degradation of Dent's disease-causing mutants of ClC-5.

Mutations in the CLCN5 (chloride channel, voltage-sensitive 5) gene cause Dents disease because they reduce the functional expression of the ClC-5 chloride/proton transporter in the recycling endosomes of proximal tubule epithelial cells. The majority (60%) of these disease-causing mutations in ClC-5 are misprocessed and retained in the ER (endoplasmic reticulum). Importantly, the structural basis for misprocessing and the cellular destiny of such ClC-5 mutants have yet to be defined. A ClC-5 monomer comprises a short N-terminal region, an extensive membrane domain and a large C-terminal domain. The recent crystal structure of a eukaryotic ClC (chloride channel) transporter revealed the intimate interaction between the membrane domain and the C-terminal region. Therefore we hypothesized that intramolecular interactions may be perturbed in certain mutants. In the present study we examined two misprocessed mutants: C221R located in the membrane domain and R718X, which truncates the C-terminal domain. Both mutants exhibited enhanced protease susceptibility relative to the normal protein in limited proteolysis studies, providing direct evidence that they are misfolded. Interestingly, the membrane-localized mutation C221R led to enhanced protease susceptibility of the cytosolic N-terminal region, and the C-terminal truncation mutation R718X led to enhanced protease susceptibility of both the cytosolic C-terminal and the membrane domain. Together, these studies support the idea that certain misprocessing mutations alter intramolecular interactions within the full-length ClC-5 protein. Further, we found that these misfolded mutants are polyubiquitinated and targeted for proteasomal degradation in the OK (opossum kidney) renal epithelial cells, thereby ensuring that they do not elicit the unfolded protein response.

Phenotypic profiling of CFTR modulators in patient-derived respiratory epithelia

Pulmonary disease is the major cause of morbidity and mortality in patients with cystic fibrosis, a disease caused by mutations in the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) gene. Heterogeneity in CFTR genotype–phenotype relationships in affected individuals plus the escalation of drug discovery targeting specific mutations highlights the need to develop robust in vitro platforms with which to stratify therapeutic options using relevant tissue. Toward this goal, we adapted a fluorescence plate reader assay of apical CFTR-mediated chloride conductance to enable profiling of a panel of modulators on primary nasal epithelial cultures derived from patients bearing different CFTR mutations. This platform faithfully recapitulated patient-specific responses previously observed in the “gold-standard” but relatively low-throughput Ussing chamber. Moreover, using this approach, we identified a novel strategy with which to augment the response to an approved drug in specific patients. In proof of concept studies, we also validated the use of this platform in measuring drug responses in lung cultures differentiated from cystic fibrosis iPS cells. Taken together, we show that this medium throughput assay of CFTR activity has the potential to stratify cystic fibrosis patient-specific responses to approved drugs and investigational compounds in vitro in primary and iPS cell-derived airway cultures.Cystic fibrosis: toward personalized therapiesA new method for evaluating drug responses in patient-derived respiratory tissue promises to help determine the best treatment for each patient with cystic fibrosis (CF). CF patients are highly susceptible to lung infections due to the build-up of thick mucus in the airways. Over 2000 mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene have been identified in patients with CF, which partly explains their varied response to treatment. Saumel Ahmadi, Christine E. Bear, and colleagues at the Hospital for Sick Children in Toronto developed a fluorescence-based method for measuring improvements in mutant CFTR function in patient-derived nasal and induced pluripotent stem cell-derived lung tissue. This method enables comparison of approved and investigational drugs on airway cells from each individual patient and in the longer term will accelerate the development of personalized therapeutic strategies.

Orkambi® and amplifier co‐therapy improves function from a rare CFTR mutation in gene‐edited cells and patient tissue

The combination therapy of lumacaftor and ivacaftor (Orkambi®) is approved for patients bearing the major cystic fibrosis (CF) mutation: ΔF508. It has been predicted that Orkambi® could treat patients with rarer mutations of similar “theratype”; however, a standardized approach confirming efficacy in these cohorts has not been reported. Here, we demonstrate that patients bearing the rare mutation: c.3700 A>G, causing protein misprocessing and altered channel function—similar to ΔF508‐CFTR, are unlikely to yield a robust Orkambi® response. While in silico and biochemical studies confirmed that this mutation could be corrected and potentiated by lumacaftor and ivacaftor, respectively, this combination led to a minor in vitro response in patient‐derived tissue. A CRISPR/Cas9‐edited bronchial epithelial cell line bearing this mutation enabled studies showing that an “amplifier” compound, effective in increasing the levels of immature CFTR protein, augmented the Orkambi® response. Importantly, this “amplifier” effect was recapitulated in patient‐derived nasal cultures—providing the first evidence for its efficacy in augmenting Orkambi® in tissues harboring a rare CF‐causing mutation. We propose that this multi‐disciplinary approach, including creation of CRISPR/Cas9‐edited cells to profile modulators together with validation using primary tissue, will facilitate therapy development for patients with rare CF mutations.

Sphingosine-1-Phosphate Is a Novel Regulator of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Activity

The cystic fibrosis transmembrane conductance regulator (CFTR) attenuates sphingosine-1-phosphate (S1P) signaling in resistance arteries and has emerged as a prominent regulator of myogenic vasoconstriction. This investigation demonstrates that S1P inhibits CFTR activity via adenosine monophosphate-activated kinase (AMPK), establishing a potential feedback link. In Baby Hamster Kidney (BHK) cells expressing wild-type human CFTR, S1P (1μmol/L) attenuates forskolin-stimulated, CFTR-dependent iodide efflux. S1P’s inhibitory effect is rapid (within 30 seconds), transient and correlates with CFTR serine residue 737 (S737) phosphorylation. Both S1P receptor antagonism (4μmol/L VPC 23019) and AMPK inhibition (80μmol/L Compound C or AMPK siRNA) attenuate S1P-stimluated (i) AMPK phosphorylation, (ii) CFTR S737 phosphorylation and (iii) CFTR activity inhibition. In BHK cells expressing the ΔF508 CFTR mutant (CFTRΔF508), the most common mutation causing cystic fibrosis, both S1P receptor antagonism and AMPK inhibition enhance CFTR activity, without instigating discernable correction. In summary, we demonstrate that S1P/AMPK signaling transiently attenuates CFTR activity. Since our previous work positions CFTR as a negative S1P signaling regulator, this signaling link may positively reinforce S1P signals. This discovery has clinical ramifications for the treatment of disease states associated with enhanced S1P signaling and/or deficient CFTR activity (e.g. cystic fibrosis, heart failure). S1P receptor/AMPK inhibition could synergistically enhance the efficacy of therapeutic strategies aiming to correct aberrant CFTR trafficking.

Identification and Validation of Hits from High Throughput Screens for CFTR Modulators

These are exciting times with the appearance of small molecule compounds in clinical trials which target the basic defects caused by mutation in the CFTR gene. This progress was enabled by years of basic research probing the molecular and cellular consequences caused by mutation and the development of methods by which to study the primary anion transport defect in a high-throughput manner by robotics. Future progress with the development of new, more effective corrector compounds is needed. Such discovery will require further progress in defining the molecular targets for effective intervention using a multidisciplinary approach, merging computational, molecular, proteomic and cell biological methods. There is also an urgent need to develop means to link the right therapeutic compound to the right patients given the heterogeneity of the CF patient population. We envision a time when mid to high-throughput methods will be married with stem cell biology to enable testing a compendium of compounds on cells derived from each individual patient. Given the rate of progress in this field- this scenario may exist in the not too distant future.

Correctors of the major Cystic Fibrosis mutant interact through membrane spanning domains

The most common cystic fibrosis causing mutation is deletion of phenylalanine at position 508 (F508del), a mutation that leads to protein misassembly with defective processing. Small molecule corrector compounds: VX-809 or Corr-4a (C4) partially restores processing of the major mutant. These two prototypical corrector compounds cause an additive effect on F508del/cystic fibrosis transmembrane conductance regulator (CFTR) processing, and hence were proposed to act through distinct mechanisms: VX-809 stabilizing the first membrane-spanning domain (MSD) 1, and C4 acting on the second half of the molecule [consisting of MSD2 and/or nucleotide binding domain (NBD) 2]. We confirmed the effect of VX-809 in enhancing the stability of MSD1 and showed that it also allosterically modulates MSD2 when coexpressed with MSD1. We showed for the first time that C4 stabilizes the second half of the CFTR protein through its action on MSD2. Given the allosteric effect of VX-809 on MSD2, we were prompted to test the hypothesis that the two correctors interact in the full-length mutant protein. We did see evidence supporting their interaction in the full-length F508del-CFTR protein bearing secondary mutations targeting domain:domain interfaces. Disruption of the MSD1:F508del-NBD1 interaction (R170G) prevented correction by both compounds, pointing to the importance of this interface in processing. On the other hand, stabilization of the MSD2:F508del-NBD1 interface (by introducing R1070W) led to a synergistic effect of the compound combination on the total abundance of both the immature and mature forms of the protein. Together, these findings suggest that the two correctors interact in stabilizing the complex of MSDs in F508del-CFTR.