Written by Dr Sophie Rose and Professor David Sheppard
Transformative therapies targeting the root cause of disease are now available for around 90% of individuals living with Cystic Fibrosis (CF) following the recent FDA and EMA approval of the triple drug combination of Elexacaftor, Tezacaftor and Ivacaftor. Professor David Sheppard (DS) from the University of Bristol presented work undertaken in collaboration with both the University of Manchester and Pfizer investigating the dysfunction of the cystic fibrosis transmembrane conductance regulator (CFTR) channel in CF and its rescue with small molecules. The presentation focused initially on targeted therapies for the most common cause of CF, the F508del variant in the CFTR gene. This was followed by a summary of work undertaken with Pfizer on a novel CFTR potentiator, which enhances CFTR activity and its use in conjunction with Ivacaftor to restore greater function to faulty channels in CF.
Identification of the defective gene responsible for CF
DS began by highlighting the long road to developing therapies for CF that target the root cause of disease. Work by an army of researchers led by Collins, Riordan and Tsui led to the identification and cloning of the defective gene responsible for CF in 1989. With the CFTR gene identified, researchers raced to identify the function of the CFTR protein and understand how disease-causing CFTR variants lead to a loss of function of this protein.
The structure of CFTR
When the CFTR gene was identified, it was recognised that its protein product was a membrane protein composed of five domains: two transmembrane domains, each with six a-helices which span the lipid bilayer; two nucleotide-binding domains, containing amino acid sequences known to interact with ATP and a fifth regulatory domain, a unique feature of CFTR distinguished by multiple consensus phosphorylation sites. This structure of CFTR placed it in a large family of transport proteins called ATP-binding cassette transporters, found in bacteria, plants and animals including humans. Advances in structural biology, led to the publication in 2016 of the first atomic resolution structure of CFTR, showing a dephosphorylated, ATP-free configuration of CFTR. In this configuration, the transmembrane domains form an inverted V-shape closed towards the outside of the channel, and the nucleotide-binding domains are separated by the regulatory domain. The structure of CFTR in a phosphorylated, ATP-bound configuration reveals that upon phosphorylation, the regulatory domain moves out of the way allowing the nucleotide-binding domains to dimerise and the transmembrane domains to align vertically.
Understanding how the protein functions
In contrast to most ATP-binding cassette transporters, CFTR is an anion channel with complex regulation. DS reviewed the ATP-driven nucleotide-binding domain model of CFTR channel gating developed by Vergani and Gadsby to explain how ATP controls CFTR activity before atomic resolution structures of CFTR were solved. Two ATP-binding sites are formed at the interface of the nucleotide-binding domain dimer. These ATP-binding sites have distinct properties. The first is a site of stable ATP binding, whereas the second is a site of rapid ATP hydrolysis. Once the regulatory domain has been phosphorylated, cycles of ATP binding and hydrolysis at the nucleotide-binding domains control channel gating. ATP binding at both ATP-binding sites is required for channel opening, whereas ATP hydrolysis at the second ATP-binding site leads to dimer separation and prompt channel closure. DS highlighted how transitions between the closed and open channel observed in single-channel recordings represent conformational changes in the CFTR protein driven by cycles of ATP binding and hydrolysis at the nucleotide-binding domains.
How does F508del cause loss of CFTR function?
The CFTR variant F508del (in frame deletion of the phenylalanine residue at position 508 of the CFTR amino acid sequence) primarily causes CFTR dysfunction because it is missing from its correct cellular location, the apical membrane of epithelia lining ducts and tubes in the body. However, if F508del-CFTR reaches the plasma membrane two further defects are observed: defective channel gating and reduced plasma membrane stability.
DS discussed electrophysiology data his group had captured with colleagues at the University of Manchester to demonstrate the impact of the F508del-CFTR variant on channel gating and plasma membrane stability. They used the excised inside-out configuration of the patch-clamp technique and baby hamster kidney (BHK) cells stably expressing wild-type and F508del-CFTR. To deliver F508del-CFTR to the plasma membrane, they incubated BHK cells expressing the variant at 27 °C for 24 hours prior to study. To magnify the small size of CFTR channel openings, a large chloride concentration gradient was used and voltage was clamped at -50 mV. ATP and protein kinase A (PKA) were continuously present in the intracellular solution to activate and sustain CFTR channel activity. By studying CFTR channels at 37 °C, the impact of the F508del variant on channel rundown, a measure of the plasma membrane stability of CFTR was assessed.
F508del slows CFTR channel opening
Low temperature-rescued F508del-CFTR has a severe gating defect which greatly slows channel opening. As a result, the open probability (a measure of channel activity) of F508del-CFTR is greatly reduced compared to that of wild-type CFTR. Using prolonged channel recordings, DS demonstrated that at 37 °C F508del-CFTR is unstable and runs down within 10 minutes even in the continuous presence of PKA and ATP in the intracellular solution. He explained that the rundown of F508del-CFTR reflects not only changes in channel gating, but also current flow through the channel evident by openings to a sub-conductance state during rundown. DS emphasized that channel rundown makes studying the function of F508del-CFTR and its rescue by small molecules particularly challenging.
DS summarised that F508del-CFTR has multiple mechanisms of CFTR dysfunction including defective delivery of protein to the plasma membrane, perturbed channel gating and reduced protein stability at the plasma membrane. He emphasized that most CFTR variants that had been studied to date disrupt CFTR function in multiple ways. Very few variants, including the CFTR gating variant G551D, cause CFTR dysfunction by only a single defect.
Combinations of correctors and potentiators repair F508del-CFTR
To rescue F508del and other disease-causing CFTR variants, requires two types of CFTR-targeted therapies, correctors and potentiators. Correctors, such as Tezacaftor and Elexacaftor, allow misfolded CFTR variants to escape from the endoplasmic reticulum and traffic to the Golgi apparatus for maturation before delivery to the plasma membrane. By contrast, potentiators, such as Ivacaftor, enhance CFTR channel gating once the protein is phosphorylated by PKA. The combination therapy Elexacaftor-Tezacaftor-Ivacaftor is a CFTR-targeted therapy for most people with CF.
A novel CFTR potentiator – CP-628006
DS then spoke about the characterisation of a new efficacious CFTR potentiator developed by Pfizer, CP-628006 (referred to as CP). CP was identified by Pfizer following a high-throughput screen of a 150k compound library. It has a distinct chemical structure to known CFTR potentiators and efficaciously potentiated F508del- and G551D-CFTR in Fischer Rat Thyroid (FRT) epithelia heterologously expressing CFTR and human bronchial epithelia from individuals with CF and the genotypes F508del/F508del and F508del/G551D.
Using single-channel recording, CP was shown to have reduced potency, but similar efficacy to Ivacaftor, enhancing channel activity by increasing the frequency and duration of channel openings. Interestingly, CP restored wild-type CFTR levels of channel activity to F508del-CFTR, but not G551D-CFTR.
To learn about how CP enhances CFTR activity, the group at Pfizer/ University of Bristol examined the ATP-dependence of channel gating for WT-CFTR, F508del-CFTR and G551D-CFTR in the absence and presence of either CP or Ivacaftor. For F508del-CFTR, channel activity was weakly ATP-dependent. Both potentiators restored some ATP-dependent channel gating to F508del-CFTR. In the case of G551D-CFTR, channel gating was ATP-independent. Ivacaftor potentiated G551D-CFTR activity similarly at all ATP concentrations tested, demonstrating that it enhances ATP-independent channel gating of G551D-CFTR. By contrast, potentiation of G551D-CFTR by CP was ATP-dependent. This result indicates that CP restores some ATP-dependence to G551D-CFTR.
The distinct effects of CP compared to Ivacaftor suggest a different mechanism of action. This encouraged the group to test combinations of the two potentiators. They found that CP and Ivacaftor together enhanced the channel activity of G551D-CFTR but not that of F508del-CFTR. DS explained that studies by other investigators have also demonstrated that some CFTR variants are receptive to combinations of two potentiators, whereas others are not. DS speculated that greater clinical benefit might be achieved by combinations of CFTR potentiators.
CFTR-targeted therapies have transformed the treatment of CF. Around 90% of people with CF will likely benefit from Elexacaftor-Tezacaftor-Ivacaftor combination therapy. However, DS emphasized that there is still much research to be done. An urgent priority is to develop drug therapies for the last 10% of individuals with CF who have CFTR variants unresponsive to current CFTR-targeted therapies. Ultimately, the aim is to cure CF.