Recommended Tool Compounds for Modifying the Cystic Fibrosis Transmembrane Conductance Regulator Channel Variants

Abstract

Cystic fibrosis (CF) is a genetic disorder arising from variations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, leading to multiple organ system defects. CFTR tool compounds are molecules that can modify the activity of the CFTR channel. Especially, patients that are currently not able to benefit from approved CFTR modulators, such as patients with rare CFTR variants, benefit from further research in discovering novel tools to modulate CFTR. This Review explores the development and classification of CFTR tool compounds, including CFTR blockers (CFTRinh-172, GlyH-101), potentiators (VRT-532, Genistein), correctors (VRT-325, Corr-4a), and other approved and unapproved modulators, with detailed descriptions and discussions for each compound. The challenges and future directions in targeting rare variants and optimizing drug delivery, and the potential synergistic effects in combination therapies are outlined. CFTR modulation holds promise not only for CF treatment but also for generating CF models that contribute to CF research and potentially treating other diseases such as secretory diarrhea. Therefore, continued research on CFTR tool compounds is critical.

Cystic Fibrosis

Cystic fibrosis (CF) is an autosomal recessive genetic disorder caused by variants in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, resulting in a deficient or dysfunctional CFTR protein and ultimately leading to multisystem malfunction.¹ Current estimates indicate that CF impacts over 100,000 people globally.^2,3 The CFTR protein is an anion channel located on the epithelial cell membrane, and its primary function includes the transportation of chloride ions, which plays a critical role in regulating cellular chloride concentration.¹ Additionally, CFTR also exerts inhibitory effects on sodium channels, reducing the absorption of sodium ions and further influencing overall ion balance within the cells.^1,4,5 These functions highlight the central role of CFTR in ensuring proper overall homeostasis of ions and fluid across multiple organ systems.

In people with CF (pwCF), the functionality of the CFTR protein is compromised in different ways depending on the mutation of the CFTR gene, there are more than 2000 variants that have been identified.^3,6,7 The defective or absent CFTR chloride channel results in the accumulation of chloride ions within the cell, and hyperabsorption of sodium. The altered cellular osmotic pressure increases the passive movement of water into the cell, leading to thicker and dehydrated mucus in the airway, resulting in impaired mucus clearance and decreased airflow.^8,9 The gastrointestinal tract (GIT), reproductive tract, sweat and salivary glands, and lungs of people with CF (pwCF) are affected and are usually characterized by chronic bacterial infection of the airways due to the CF patients commonly have dysregulated immunity and impaired mucus clearance.^1,4,10−13 The CFTR variants are traditionally categorized into 6 classes, and each class has a unique CFTR defect (Table 1). Class I variants cause defective CFTR production, ultimately leading to the absence of the CFTR protein on the cell surface.¹⁴ Moreover, it is further proposed that Class I be differentiated into Class IA and Class IB by Marson et al., where Class IA variants lead to no mRNA transcription (e.g., Dele 2,3 (21kb)), and Class IB mutations lead to no protein production (e.g., G542X), and both classes ultimately cause CFTR protein production deficiency.^14,15 Class II variants cause intracellular production of misfolded proteins, including the most common mutation, due to a deletion of phenylalanine at position 508 in the CFTR gene, the Delta F508-CFTR variant. It has been estimated that 85% of all CF related alleles in the United States are the Delta F508 variant.^14,16,17 In this mutation, the protein is rapidly recognized as misfolded and degraded shortly after synthesis, therefore failing to reach the cell membrane.¹⁸ Class III variants such as G551D cause gating impairment; the CFTR proteins do not open properly for chloride transportation.¹⁴ In Class IV variants, there are sufficient CFTR proteins reaching the cell membrane but with reduced chloride conductance (e.g., A455E).¹⁶ Class V variants do not alter the protein function but reduce the production of CFTR (e.g., 3272-26A>G).^14,19,20 Lastly, the class VI variants lead to destabilization of the protein at the cell membrane by reducing its conformational stability or generating additional internalization signals, which results in accelerated CFTR turnover (e.g., Q1412X).²¹⁻²³

Table 1. Classification of CFTR Gene Mutations That Cause CF¹⁰⁷.

classification	Class I	Class II	Class III	Class IV	Class V	Class VI
CFTR presence	no	no	yes	yes	yes	yes
phenotype	no mRNA/No protein	no traffic	gating defects	conductance impairment	less protein	less stable
CFTR function	no	yes (decreased)	no	yes (decreased)	yes	yes
example	G542X	F508Del	G551D	A455E	3272-26A>G	Q1412X
severity	more-severe disease			less-severe disease

CFTR and the Role of CFTR in Different Organs

The CFTR protein is classified into the superfamily of ATP-binding cassette transporter ATPases based on its amino acid sequence. Unlike other common ATP-binding cassette transporter proteins, it functions as an ion channel rather than as a typical ATPase.^24,25 CFTR channels still exhibit the characteristic structure of the ATP-binding cassette protein superfamily.²⁶ It is composed of five functional domains overall, two membrane spanning domains (MSDs) that form an ion transport pathway, two cytosolic nucleotide-binding domains (NBDs) which ATP binds to, and a cytosolic regulatory domain (R-domain) located between two MSDs, and needs to be phosphorylated by Protein Kinase A for CFTR to function effectively (Figure 1).^24,27

Figure 1.

Schematic of cystic fibrosis transmembrane conductance regulator (CFTR) protein, which is composed of five functional domains including two membrane spanning domains (MSDs), each composed of six transmembrane domain (TMDs) and forming an ion transport pathway; two cytosolic nucleotide-binding domains (NBD) which ATP binds to, and a cystolic regulatory domain (R-domain) that needs to be phosphorylated by protein kinase A for CFTR normal function. Reproduced from ref (41). Copyright 2023 American Chemical Society.

One of the primary functions of CFTR is working as an anion channel; it is selectively permeable to various halide ions with the highest conductance to chloride, as well as allows various polyatomic anions to permeate such as nitrate and bicarbonate.^26,28−30 Nevertheless, it acts not only as an ion channel, but also as a regulator of other channels, such as exerting inhibitory activity on the epithelial sodium channel (ENaC).^5,31,32

The CFTR protein is located predominantly on the epithelial cells of organs such as the pancreas, liver, salivary glands, gastrointestinal tract, lungs, and the male and female reproductive systems.³³ Not restricted to these organs, CFTR is also detected to have regulatory functions in immunity; for instance, CFTR normal expression was found to impact various immune cell cytokine production and their normal functions.^26,34−36 Hence, CF patients not only exhibit abnormalities in organ functions but also experience immune dysregulation, such as chronic airway inflammation.^33,37 Besides, defective CFTR is often associated with a wide spectrum of comorbidities since it affects the viscosity and pH of mucus on the surface of multiple organs; the lung, pancreas, and GIT are the most frequently affected among CF patients.^11,38−41

CFTR in GIT

The GIT serves as the site for food digestion, nutrient absorption, and elimination of waste products.⁴² The CFTR protein is responsible for regulating intestinal homeostasis, and it affects the processes of food digestion, nutrient absorption, and waste excretion in the GIT. Apart from malnutrition, CFTR dysfunction also contributes to various manifestations of the GIT in CF disease, such as constipation, meconium ileus (MI), distal intestinal obstruction syndrome (DIOS), intestinal dysbiosis, and inflammation.⁴³⁻⁴⁵ Furthermore, MI is often the earliest manifestation that appears in CF infants, it affects approximately 20% pwCF.⁴⁶ The loss-of-function CFTR protein fails to mediate bicarbonate secretion, resulting in a relatively dehydrated and acidic luminal environment, promoting mucus thickening.⁴⁷ The thick, unclearable mucus mixes with the partially digested food, obstructing the terminal ileum and leading to MI.^46,47

Moreover, studies indicate CF patients have an abnormal gut microbiome composition.⁴⁸⁻⁵⁰ Among CF children, there was an elevation of the pathogenic bacteria such as Escherichia coli and a reduction in beneficial bacteria such as bifidobacteria, which contribute to CF-related intestinal dysbiosis and inflammation.^41,48,50,51

CFTR in Lung

CFTR on the epithelial cell membrane in the airway plays an essential role in maintaining proper lung function. The presence of bacterial infections, such as Pseudomonas aeruginosa (P. aeruginosa) has become a hallmark of CF pathogenesis and disease progression, which directly contributes to the pulmonary function decline, and ultimately leads to respiratory failure.⁵²⁻⁵⁴

In healthy individuals, CFTR helps produce thin and nonviscous mucus on the airway surface, aiding in the trapping and clearance of foreign particles and bacteria.^55,56 In comparison, mutations in the CFTR gene among CF patients lead to defective or absent CFTR protein in airway epithelial cells, causing an imbalance in chloride concentration and water movement.^3,37 This results in the production of thick, sticky mucus, impairing the mucus clearance process. Consequently, the accumulated mucus forms a “mucous protective screen” for foreign pathogens, such as bacteria. Moreover, the lung environment is further conducive to bacterial infections, since they are neither physically removed by the mucus clearance mechanism nor directly killed by immune cells, as pwCF are usually accompanied by immune dysregulation.^41,54 The bacteria colonization lasts longer, and eventually leads to chronic infection and inflammation.⁵⁴

CFTR in Pancreas

The pancreas creates enzymes to assist in breaking down fats, proteins, and carbohydrates in foods, and helps the absorption of nutrients in the human body.⁵⁷ In the pancreas, CFTR also plays an essential role in the secretion of digestive enzymes into the small intestine to facilitate food digestion and nutrient absorption.⁵⁸ Nevertheless, among those suffering from CF, faulty CFTR results in the thickening of mucus secretions in the pancreas, leading to obstructions of the pancreatic ducts.^58,59 Consequently, digestive enzymes accumulate within the pancreas, instead of flowing into the digestive system, triggering pancreatic inflammation (pancreatitis).⁵⁹ In addition, the obstruction of the pancreatic duct will cause insufficient enzyme release to the digestive system.^11,59,60 Thereby individuals with CF frequently suffer from exocrine pancreatic insufficiency, which impairs food digestion and nutrient absorption processs and eventually leads to malnutrition.^11,59−62 This connection between CF and pancreatic abnormalities underscores the systemic nature of the disease and the broad impact of CFTR mutations on multiple organ systems. Overall, these CF manifestations highlight the vital role of CFTR in multiple organs and underscore the urgent need for more compounds targeting CFTR protein, since it directly targets the underlying cause of the disease rather than being a temporary symptomatic reliever, providing more therapeutic options for patients.

Tool Compounds for CFTR

The development of small-molecule compounds that directly interact with CFTR to modulate its expression or function has proven to be highly effective in the therapeutic treatment and CF disease research, as they target the cause of the disease.^63,64 In this Review, 4 classes of CFTR tool compounds with specific examples will be discussed based on their function, potential clinical applications, and relevant research studies (Table 2).

Table 2. Classification of CFTR Tool Compounds.

CFTR Blockers

CFTR blockers are a category of CFTR compounds that are specifically designed to inhibit the function of the CFTR channel, which can be utilized to create CFTR knock-out models to mimic CF conditions, and contribute to CF research and drug development.⁶⁵ 4-[4-Oxo-2-thioxo-3-(3-trifluoromethyl-phenyl)-thiazolidin-5-ylidenemethyl]-benzoic acid (CFTRinh-172) and (naphthalen-2-ylamino)-acetic acid (3,5-dibromo-2,4,-dihydroxy-benzylidene)-hydrazide (GlyH-101) are blockers that target but are not solely selective for CFTR protein as some of them also inhibit other transepithelial ion channels.^66,67 These compounds play crucial roles in the research of CFTR and CF model development.^63,68

CFTRinh-172

CFTRinh-172 acts as a potent, voltage-independent CFTR inhibitor, reversibly inhibiting CFTR short-circuit current with potency ≈ 300 nM.⁶⁵ It was found that CFTRinh-172 increases the mean closed time and decreases the mean open time of the CFTR channel. This induces CFTR dysfunction similar to what is observed in CF, therefore could be utilized in CF research and study designs. Although the exact mechanism of action is not fully clear, it has been illustrated that CFTRinh-172 may interact with one of the NBDs of CFTR to exert its inhibitory effects.^65,69 Consequently, there is less chloride ion secretion, thereby reducing water secretion on the cell surface. The compound was investigated to reduce the intestinal fluid loss in cholera and other forms of secretory diarrhea, and it effectively decreased 90% of the fluid secretion in the small intestine over 6 h in a mouse cholera model.^63,70

GlyH-101

Likewise, GlyH-101 has a similar reversible inhibitory action on CFTR; however, it has more rapidity of action, greater water solubility, and higher inhibitory potency of approximately 5 μM.⁶⁸ GlyH-101 blocks CFTR by occlusion of the CFTR pore near its external surface, and it was initially demonstrated to be effective in exerting its function in vivo in cholera rodent models.⁶⁸ Moreover, both compounds were used as a tool to develop CF animal models in the past and were shown to effectively block CFTR and mimic the CF conditions in mice, and the airways of these mouse models exhibit CF-like features, such as impaired mucociliary clearance in the trachea.^67,71 Nevertheless, despite the compounds not being feasible for clinical research, it is still worth mentioning that both compounds were shown to exhibit off-target effects. For example, GlyH-101 exerts its action on voltage-gated calcium and potassium channels in cardiomyocytes.^67,72−74

CFTR Potentiators

CFTR potentiators enhance the function of the existing CFTR proteins on the cell membrane by increasing the ion channel activity.^75,76 In in vitro and preclinical studies, CFTR potentiators have exhibited their ability to increase chloride ion transport, ultimately improving fluid balance, reducing mucus viscosity, and leading to mucus clearance improvement.^76,77 Notable examples within this category, such as ivacaftor, the only approved CFTR potentiator available for the treatment of CF patients, have demonstrated remarkable success in modulating CFTR function and, in some cases, leading to enhanced lung function (such as forced expiratory volume in 1 s (FEV₁) improvement) and even reduced bacterial infection in individuals with CF.^13,78,79 Further research on mechanism of action studies may prove instrumental in advancing CF pharmacotherapy and potentially discovery of a novel class of medications.

VRT-532

4-Methyl-2-(5-phenyl-1H-pyrazol-3-yl)-phenol (VRT-532) was found by a using high-throughput screening technique in 2006.⁸⁰ It is a CFTR potentiator that primarily targets the G551D CFTR variant, and it is a major type of CFTR variants that has a problem with ATP-dependent gating, resulting in a defect in CFTR activation. This compound effectively enhances the chloride ion conductance activity of the channel, bringing it to a level similar to that of the wild-type CFTR.⁸¹⁻⁸³

Notably, VRT-532 has also has been shown to rescue the most common deletion mutation in the CFTR, Delta F508. This variant leads to improper protein folding and trafficking, resulting in reduced CFTR expression at the cell surface, which indicates that it has both the CFTR corrector potentiator functions, although with only one-fifth of the affinity compared to its action on the G551D CFTR variant.^80,84 The mechanism of action of VRT-532 involves boosting CFTR ATPase activity, with an EC₅₀ of approximately 3 μM, thereby enhancing the channel open state by accelerating the rate of channel opening and slowing the rate of channel closure, leading to subsequent chloride ion conductance improvement.^84,85 Critically, VRT-532 has demonstrated a synergistic effect when administered in combination with another CFTR corrector, VRT-325.^80,85 This combined treatment, when used in conjunction with cAMP agonists, resulted in CFTR-mediated transepithelial chloride ion secretion levels of Delta F508-CFTR expressing human bronchial epithelial cells exceeding 20% of those observed in healthy human bronchial epithelial cells, showing promising outcomes.^80,85

UCCF-853

1-(3-Chlorophenyl)-5-trifluoromethyl-3-hydrobenzimidazol-2-one (UCCF-853) is a benzimidazolone found in early 2000s through high-throughput screening.⁸⁶ It was then identified as the best benzimidazolone to activate CFTR Cl^– conductance with a potency of 5–20 μM.⁸⁷ In combination with the best 7,8-benzoflovone UCCF-029, they elicit additive effects on CFTR activation, 50–80% greater than that of either compound alone.⁸⁷

The detailed mechanism of action for activating CFTR remains unknown. However, other benzimidazolones such as NS004 have been demonstrated as CFTR potentiators to increase the opening time and also decrease the closed time of the Delta F508-CFTR channel.⁸⁸ It has been proposed that benzimidazolones can stabilize the open state by inhibiting ATP hydrolysis at the second nucleotide binding fold. Since UCCF-853 has a structure similar to that of NS004, its mechanism of action could resemble that of NS004 as described above. Apart from CFTR Cl^– potentiation, a study on human airway epithelial cells also suggests that UCCF-853 may activate basolateral K⁺ channels since it is still effective after the maximal stimulation of CFTR.⁸⁷

In conclusion, UCCF-853 could be a promising CFTR potentiator to treat CF, whereas basic study is significantly absent to understand its mechanism of action and pharmacokinetic properties.

Genistein

5,7-Dihydroxy-3-(4-hydroxy-phenyl)-chroman-4-one (genistein) is a naturally occurring isoflavone found in various plants, primarily in soybeans.⁸⁹⁻⁹¹ Its impact on CFTR is multifaceted, and it has garnered significant attention in the CF therapy development.

Genistein has been recognized and investigated as one of the CFTR potentiators for more than 2 decades.⁹²⁻⁹⁴ It was found to increase the CFTR channel’s opening rate and decrease the closing rate to enhance the chloride ion transportation with an EC₅₀ of around 16 μM.^95,96 Previous studies have suggested that genistein exerts its function by interacting with NBDs. Nonetheless, it is also suggested to alter lipid bilayer mechanics, and the exact detailed mechanism of action on CFTR remains unclear.^95,97,98 A bioinformatics study has predicted the possible genistein binding sites on the CFTR protein, which provides crucial information that can not only help to discover its mechanism of action but also assist chemists in designing similar action treatment compounds, which provides more therapeutic options to pwCF.⁹⁹

The CFTR potentiation action has been proved and elucidated by several preclinical studies; Previously, Zegarra-Moran et al. reported that genistein is capable of rescuing the gating defect of G551D-CFTR mutation, achieving up to 20% of the activity observed in the wild-type CFTR.¹⁰⁰ Nevertheless, the concentrations needed for this effect is too high for a safe blood drug concentration (produce drug effects with minimum side effects) and might cause adverse effects if used invivo. Notably, the combination administration of low-concentration genistein and curcumin showed promising outcomes: it restored the defective G551D-CFTR up to approximately 50% of the wild-type CFTR level, which seems safer and more effective compared to monotherapy.^101,102

Moreover, in a recent study of Delta F508-CF mice, genistein supplementation has shown a dramatic reduction effect in the need for laxatives which were used to prevent constipation and DIOS caused by CF in a rodent model. This may be evidence to support its effectiveness in treating CF, since symptoms like constipation and DIOS frequently occur in CF.¹⁰³ In addition, an early study proved that genistein activates CFTR-mediated chloride ion secretion in the murine trachea and distal colon, which may explain the improvement of intestinal relative symptoms in the previous study.^103,104 A clinical trial was initiated in 2003 to evaluate the effectiveness of a combination of genistein and phenylbutyrate in treating CF patients with the Delta F508 mutation. However, the trial halted in 2008 since the results were unconclusive and unsatisfactory.^105,106 Overall, even though genistein has been investigated extensively over the last few decades, most of the research on genistein is preclinical, and further studies on this compound are needed if it is to be used clinically in the future.

PG-01

2-[(2-1H-Indol-3-yl-acetyl)-methylamino]-N-(4-isopropylphenyl)-2-phenylacetamide (PG-01) was discovered during the screening of small molecules to identify novel compounds capable of correcting defective gating in CFTR variants. It emerged as the most potent member of the phenylglycine compound class, demonstrating the ability to reversibly restore Delta F508del CFTR function in human airway epithelial cells when added with forskolin (a cyclic adenosine monophosphate activator that can improve the CFTR opening) with an affinity of approximately 70 nM.¹⁰⁷ Its mechanism of action was subsequently analyzed through patch-clamp measurements where Pedemonte et al. revealed that PG-01 at a concentration of 100 nm was able to increase the channel opening probability to a level comparable to that of the wild-type CFTR channels by reducing the channel closing time. The ability to modulate gating kinetics prompted further research on whether PG-01 could correct defective gating within other CFTR mutations of G551D and G1349D (Class III mutations that caused severe gating defects). Notably, PG-01 demonstrated a synergistic effect when combined with forskolin, correcting the CFTR mutations of G551D and G1349D with K_a values of 1 μM and 40 nM, respectively, whereas the addition of forskolin alone had little effect on the chloride currents. Building upon its success in correcting NBD-affecting mutations of G551D and G1329D, Caputo et al. found that PG-01 was also strongly effective in correcting mutations of E193K and G970R that affected the intracellular loops of CFTR protein with K_d values of 0.22 μM and 0.45 μM respectively.¹⁰⁸ Despite some promising findings, the investigations of PG-01 did not progress to the clinical stages.

SF-03

6-(Ethyl-phenyl-sulfonyl)-4-oxo-1,4-dihydro-quinoline-3-carboxylic acid 2- methoxy-benzylamide (SF-03) is a compound from the novel sulfonamide class that arose in the same high throughput screening as PG-01.¹⁰⁷ Its potentiator activity was verified by its ability to cause a concentration-dependent I- influx when acting on the Delta F508-CFTR expressing Fisher rat thyroid cells with an EC50 of approximately 0.1 μM.¹⁰⁹In vitro studies exploring the hepatic clearance of SF-03 through incubation with liver microsomes found a 35% degradation after 1 h. This metabolism process is significantly slower in comparison to the 90% degradation of PG-01 under the same incubation conditions, thus highlighting SF-03 as a more metabolically stable compound.¹⁰⁷ Unlike the wider applicable range of CFTR variants PG-01 has shown efficacy against, SF-03 has only been found to correct defective gating within Delta F508-CFTR variants and ultimately did not progress into any clinical phases.

CFTR Correctors

CFTR correctors primarily target Class II CFTR variants, which result in misfolding, premature degradation by the endoplasmic reticulum, and impaired protein biogenesis. This extensively reduces the amount of CFTR protein that reaches the cell membrane.^76,110−112 Notably, the Delta F508-CFTR variant belongs to Class II CFTR variations, and nearly 90% of people with CF carry at least one copy of the Delta F508 variant.^113,114 CFTR correctors are specifically designed to enhance and correct the protein folding, maturation, and trafficking of CFTR to the cell membrane. They are often used in combination with CFTR potentiators to further boost the channels’ functionality. For example, the triple combination consisting of elexacaftor–tezacaftor–ivacaftor received approval from the U.S. FDA for the treatment of CF patients aged ≥2 years who carry at least one Delta F508 variant in the CFTR gene.^115,116 However, many previously invented CFTR corrector compounds need further investigation to expand the potential therapeutic options.

VRT-640

2-{1-[4-(4-Chloro-benzensulfonyl)-piperazin-1-yl]-ethyl}-4-piperidin-1-yl-quinazolin (VRT-640) is a CFTR corrector discovered through high-throughput screens, it works by increasing the trafficking of Delta F508-CFTR to the cell surface.^117,118 Its potency and mechanism of actions are yet to be elucidated. An in vitro study reported that VRT-640 increased the conductance of Delta F508-CFTR on the Fisher Rat Thyroid (FRT) cell surface but elicited no effects in human CF bronchial epithelial cells.¹¹⁹ The contradictory results may be attributed to differences in the Delta F508-CFTR structure as well as cAMP responsiveness in different cell lines. Generally, the effects of correctors are hugely affected by cell type variations, and the lack of responsiveness in human CF bronchial cell lines may hinder the development of VRT-640 to treat CF in the future. Future in vivo studies are required to investigate its effectiveness in human patients.

VRT-325

4-Cyclohexyloxy-2-{1-[4-(4-methoxy-benzensulfonyl)-piperazin-1-yl]-ethyl}-quinazoline (VRT-325) is one of the earliest CFTR correctors that was discovered in 2005.¹¹⁸ It was shown to promote CFTR folding of the Delta F508 variant with EC₅₀ ≈ 2 μM, and it also rescues the misprocessed P-glycoprotein mutants, which is a close relative of CFTR protein.^80,118

Although there no clinical studies have been performed, VRT-325 still showed promising effects in in vitro studies by increasing cell surface expression of mature Delta F508-CFTR and increasing chloride ion conductance activity by at least 10% compared to non-CF human bronchial epithelial cultures.⁸⁰ Notably, VRT-325 also exhibited additive properties with other drugs that also target CFTR mutation.¹²⁰ For instance, it increased the efficiency of Delta F508-CFTR maturation in a baby hamster kidney (BHK) stable cell model by 4-fold greater when used in combination with Corr-2b than when used with VRT-325 alone.^120,121 This evidence may showcase that the use of multiple correctors may be a useful technique for improving CFTR forming, trafficking, and activity.

The mechanism of action has been investigated; previous results indicated that VRT-325 acts at the endoplasmic reticulum to promote CFTR folding.⁸⁰ Chiaw et al. suggested that VRT-325 directly binds Delta F508-CFTR and exerts its action, possibly enabling the protein to exit and transport from the endoplasmic reticulum to the cell membrane, and argued that specific binding sites need to be found.¹²² Furthermore, it was then illustrated that the binding site of VRT-325 on Delta F508-CFTR is NBD1.^123,124 However, another study proposed that VRT-325 may act on multiple proteins or on a more general mechanism involved in protein processing.¹²⁵ It may not be further investigated to treat CF as it is nonspecific to CFTR; however further research on its mechanism of action is needed, and exact binding site discovery may be a critical step to fully understand how it works, which will help to develop other drugs for CF.

Cmpd4a (Corr-4a)

Bisaminomethylbithiazole (N-[2-(5-chloro-2-methoxy-phenylamino)-4′-methyl-[4,5′]bithiazolyl-2′-yl]-benzamide), Corr-4a, is another CFTR corrector compound which was discovered in a screening of 150,000 chemical compounds in 2005.¹²¹ Similar to VRT-325, it has also been documented to rescue Delta F508-CFTR folding defects through direct interaction, facilitating correct trafficking and glycosylation, and enhancing surface expression in both transfected cells and primary airway epithelial cells from Delta F508 CF patients, and achieving levels of CFTR function around 8% of that of healthy airway epithelia.^121,126

Corr-4a enhances the CFTR folding efficiency of Delta F508 and wild-type CFTR and delays the endoplasmic reticulum-associated degradation of the core-glycosylated Delta F508-CFTR.¹²¹ In addition, it not only augments the CFTR expression but also appears to stabilize the Delta F508-CFTR on the plasma membrane, and extends its timespan which may also contribute to its functional rescue of CFTR mutant.^121,127 Furthermore, it exhibits a specific action on CFTR, distinct from the P-glycoprotein maturation promotion effect as seen with VRT-325, indicating a lower drug side-effect potential.¹²⁸ Even though inconclusive, Okyoneda et al. suggested that the Corr-4a binding site may be located in NBD2 of CFTR.¹²⁹

Intriguingly, this compound also exerts synergetic effects when combined with other drugs. For example, when in the presence of VRT-325 and Corr-4a, Delta F508-CFTR maturation efficiency was doubled compared with expression in the presence of a single compound.¹²⁰ In addition, combination of RMA1 or CHIP (both are E3 ligases) inactivation and Corr-4a enhanced Delta F508-CFTR folding to 3–7-fold greater than that of Corr-4a alone in the HEK293 cell model.¹³⁰

Nonsense-Mediated mRNA Decay (NMD) Inhibitor

SMG1i

SMG1i (2-chloro-N,N-diethyl-5-((4-(2-(4-(3-methylureido)phenyl)pyridin-4-yl)pyrimidin-2-yl) amino)benzenesulfonamide) is a small molecule and selective inhibitor of the NMD effector SMG1 which was found in 2012.¹³¹ SMG1 plays a crucial role in the RNA surveillance pathway named nonsense-mediated RNA decay (NMD), which influences the integrity of translated proteins by eliminating mRNAs bearing a premature termination codon (PTC), thereby it is essentially involved in the RNA processing, storage, transport and translation.^132−134

Class I CFTR variants such as those that have PTCs produce incomplete protein, hence classic CFTR modulators such as CFTR potentiators and correctors are unable to treat CF patients with Class I variants.¹³⁵ NMD inhibitors such as SMG1i that can potentially treat Class I CFTR variants have attracted attention, as they directly interfere with the NMD pathway.¹³⁶ It is noteworthy that SMG1i has been demonstrated to enhance the efficacy of readthrough agents and the expression of W1282X and G542X CFTR mRNA, which are both nonsense mutations, indicating a potential therapeutic option for Class I CFTR variants.¹³⁷ Furthermore, others have shown that SMG1i has synergistic effects when used in combination with G418, a readthrough-inducing agent among G542X nonsense mutation mouse model.¹³⁸

Approved CFTR Modulators

Ivacaftor Monotherapy

Ivacaftor is the first clinically approved CFTR potentiator. It functions by binding to the CFTR protein, increasing its opening time and the transport of chloride ions.⁷⁵ It has been demonstrated to improve lung function significantly and improve mucociliary clearance.⁷⁹ Ivacaftor monotherapy is approved for class 3 mutations, including gating mutations such as G551D mutations (∼2–3% of patients) and rare gating mutations such as G1244E, S1251N, S1255P, G1349D, G178R, G551S, S549N, S1252N, S549R, and R117H.¹¹² These mutations result in sufficient CFTR protein on the cell surface but impair function.⁷⁵ In clinical trials, ivacaftor monotherapy improved lung function in patients with this mutation by 6.7% on average.⁷⁹ It was shown that ivacaftor acts by directly binding on the CFTR protein, as its capability of potentiating the purified CFTR.^139,140 Interestingly, another study indicated that it binds to multiple sites in the CFTR.¹⁴¹

Ivacaftor Combination Therapies

Ivacaftor also forms part of all other CFTR modulator regimens with either lumacaftor, tezacaftor, or elexacaftor–tezacaftor. The ivacaftor–lumacaftor combination has been shown to improve lung function by 3%, as well as increase body mass index. Despite modest improvements in lung function, ivacaftor–lumacaftor therapy has reduced hospitalisations of patients carrying the homozygous F508del-CFTR mutation.¹⁴² Moreover, ivacaftor combination therapies (with lumacaftor or tezacaftor) have been shown to decrease the pro-inflammatory cytokine IL-1β in serum from patients with the F508del CFTR mutation, as well as increase the anti-inflammatory IL-10.¹⁴³

Tezacaftor–ivacaftor (Symdeko) is a new combination therapy for CF treatment, which is approved for CF patients with a homozygous F508del CFTR mutation. Like lumacaftor, tezacaftor is a CFTR corrector and increases the CFTR protein that transports to the cell membrane.¹⁴⁴ While tezacaftor corrects CFTR protein folding and presentation on the cell surface, ivacaftor additionally improves the CFTR opening duration, therefore reconstructing the ion transportation, and balancing the concentration. Consequently, less water moves into the cell, improving mucus clearance.¹⁴⁵ In an in vitro study, tezacaftor–ivacaftor significantly increased the F508del CFTR protein processing and trafficking to the cell surface, with an improvement in chloride transport as well.¹⁴⁴ Tezacaftor–ivacaftor combination therapy increased the FEV₁ of CF patients who have a homozygous F508del CFTR mutation by 4% after 24 weeks of administration compared to the placebo group. It has also demonstrated a 6.8% improvement in patients with a heterozygous F508del CFTR mutation compared to the placebo group after 4 and 8 weeks of administration.¹⁴⁶

Elexacaftor–tezacaftor–ivacaftor is a recently approved triple-combination therapy for treating CF patients with one or two F508del CFTR mutations. It contains two correctors and one potentiator.¹⁴⁷ Elexacaftor–tezacaftor–ivacaftor resulted in a 14.3% improvement in FEV₁ and a 63% lower pulmonary exacerbation rate than in the placebo group after 24 weeks of administration.¹⁴⁸ Triple-combination therapy also showed greater performance in reducing sweat chloride concentration, which means better performance in fixing the CFTR ion transportation function in comparison with tezacaftor–ivacaftor therapy among CF patients with an F508del CFTR mutation.¹⁴⁷

CFTR Modulators by AbbVie/Galapagos (Clinical Trials)

4-[(2R,4R)-4-({[1-(2,2-Difluoro-1,3-benzodioxol-5-yl)cyclopropyl]carbonyl}amino)-7-(difluoromethoxy)-3,4-dihydro-2H-chromen-2-yl]benzoic acid (GLPG2222), a potent CFTR corrector discovered in 2018 that targets NBD1, has been demonstrated to rescue Delta F508 and various rare CFTR variants.¹⁴⁹ It has been tested in HEK293 cells that expressing E92K CFTR, P67L CFTR, V232D CFTR, or Delta F508-CFTR on the surface, and the effects of GLPG2222 on those were observed with potencies of 503, 206, 14, and 14 nM respectively.¹⁵⁰ Unlike other modulators discussed in this Review, GLPG2222 has been assessed in multiple Phase II clinical trials, and the results indicated that GLPG2222 was well tolerated with CFTR activity enhancement in subjects with the Delta F508-CFTR variant.¹⁵¹

GLPG2737 is another novel CFTR corrector in development for treatment of CF, which has been found to rescue the defective folding and trafficking process of Delta F508-CFTR variant.¹⁵⁰ GLPG2737 was evaluated on top of the corrector GLPG2222, and it was examined and proved to correct various CFTR mutations in HEK293 cells, including but not limited to V232D CFTR and Delta F508-CFTR; Intriguingly, the EC₅₀ of GLPG2737 on V232D CFTR is around 10 times that of Delta F508-CFTR (161 and 2151 nM, respectively).¹⁵⁰ It is worth mentioning that the potency of GLPG2737 is strongly dependent on the presence of the co-corrector, which can be seen from the GLPG2737 and GLPG2222 combination. The combination is 25 times more potent than GLPG2737 alone, indicating GLPG2222 induces the CFTR variant a conformational change that enables GLPG2737 to better exert its effect.¹⁵⁰ Moreover, the addition of GLPG2737 to the corrector/potentiator combination GLPG2222/GLPG3067 greatly increased the expression of Delta F508-CFTR on the cell membrane.¹⁵⁰ Surprisingly, the compound was further demonstrated to inhibit wild-type CFTR in a dose dependent manner in both cellular assay and non-CF human bronchial epithelial cells.¹⁵⁰ Although the exact mechanism of action and potential binding site is not fully validated, it still represents a valuable novel class of CFTR corrector both on its own and in combination with other modulators, this was corroborated by the fact that GLPG2737 has been evaluated in clinical trials for safety, tolerability and pharmacokinetics.^152,153

Less Common CFTR Tool Compounds

Diarylsulfonylurea (DASU-01), a low-affinity CFTR blocker introduced in 2004, has been used to perform noise analysis on CFTR studies.¹⁵⁴ The CFTR inhibitor, PPQ-102 (7,9-dimethyl-11-phenyl-6-(5-methylfuran-2-yl)-5,6-dihydro- pyrimido-[4′,5′-3,4]pyrrolo[1,2-a]quinoxaline-8,10-(7H,9H)-dione) reversibly inhibits the CFTR chloride currents by stabilizing the channel closing state with IC₅₀ ≈ 90 nM.¹⁵⁵ It was initially discovered in 2009 by Tradtrantip et al. Unlike other inhibitors, it is uncharged and thus membrane potential insensitive, with demonstrated effects of ameliorating kidney cysts in a neonatal kidney organ culture model of polycystic kidney disease.¹⁵⁵ Tetrazolo-172 (5-[[4-(2h-tetrazol-5-yl)phenyl]methylene]-2-thioxo-3-[3-(trifluoromethyl)phenyl]-4-thiazolidinoneand) and Oxo-172 (4-[[3-[3-(trifluoromethyl)phenyl]-2,4- dioxo-5-thiazolidinylidene]methyl]benzoic acid) are CFinh-172 analogues that were found in 2008. They both inhibit CFTR with a potency of ∼1 μM; however, they are 10-fold more water-soluble than CFTRinh-172, which means they have a higher dissolution rate and subsequent bioavailability as they are able to be absorbed through the GIT epithelium, thereby making them potentially useful in treating secretory diarrhea and polycystic kidney disease.¹⁵⁶

DeltaF508_act-02 (2-(2-chloro-benzoylamino)-4,5,6,7-tetrahydro-benzo[b]thiophene-3-carboxylic acid amide) is a tetrahydrobenzothiophene found through a cell-based functional high-throughput screen in 2003.^157,158 It is a CFTR potentiator with high potency (EC₅₀ ≈ 600 nM) and efficacy on the Delta F508-CFTR variant instead of the G551D variant.¹⁵⁸ NS004 (1-(5-chloro-2-hydroxy-phenyl)-5-trifluoromethyl-1,3-dihydro-indol-2-one) is an early CFTR potentiator discovered in 1994 by Gribkoff et al.¹⁵⁹ It was demonstrated to reversibly augment wild-type and Delta F508-CFTR channel activity; furthermore, it has been shown to restore the function of G551D CFTR variants when in the presence of forskolin, but neither can stimulate the mutant channel alone.^159,160 UCCF-029 (4-(4-oxo-4H-benzo[H]chromen-2-yl)-pyridinium bisulfate) is another CFTR potentiator the improves wild-type and G551D CFTR activation with EC₅₀ ≈ 3 μM.¹⁶¹ Ferrera et al. initially stated that low concentrations of UCCF-029 work by increasing the CFTR channel opening probability; intriguingly, another study performed by AI-Nakkash illustrated that UCCF-029 did not affect the CFTR gating time within the NIH3T3 cells that were transfected with K1250A CFTR. Hence, they hypothesized that the compound stabilizes the open state by inhibiting ATP hydrolysis at NBD.¹⁶² In addition, CFTR potentiators UCCF-180 (3-but-3-ynyl-5-methoxy-1-phenyl-1H-pyrazole-4-carbaldehyde) and UCCF-152 (3-(2-benzyloxy-phenyl)-5- chloromethyl-isoxazole) were discovered from the same assay of UCCF-029, with EC₅₀ values of approximately 10 and 50 μM (least potent), respectively.^161,163,164 Unlike other compounds that activate both wild-type and G551D CFTR, UCCF-180 was initially advised to preferentially activate wild-type CFTR compared to other mutants.¹⁶¹ Comparatively, UCCF-152 was suggested to not only activate wild-type and G551D CFTR but also rescue Delta F508-CFTR.¹⁶⁴ Moreover, it was also suggested that it exerts its effects by inducing R-Domain phosphorylation.¹⁶⁴

Corr-3a (6-(1H-benzoimidazol-2-ylsulfanylmethyl-2-(6-methoxy-4-methyl-quinazolin-2-ylamino)-pyrimidin-4-ol), Corr-5a (4,5,7-trimethyl-N-phenylquinolin-2-amine), Corr-5c (N-(4-bromophenyl)-4- methylquinolin-2-amine), Corr-2i (N-(4-fluorophenyl)-4-p- tolylthiazol-2-amine), Corr-4c (N-(2-(3-acetylphenylamino)-4′-methyl-4,5′-bithiazol-2′-yl)benzamide), Corr-4d (N-(2′-(2-methoxypheny-lamino)-4-methyl-5,5′-bithiazol-2-yl)benzamide), Corr-2b (N-phenyl-4-(4-vinylphenyl)thiazol-2-amine), and Corr-3d (2-(6-methoxy-4-methylquinazolin-2-ylamino)-5,6-dimethylpyrimidin-4(1H)-one) were all identified by high-throughput screening of 150,000 compounds in 2005 by Pedemonte et al.¹²¹ All the small molecules were initially elucidated to improve Delta F508-CFTR trafficking and expression on the plasma surface.¹²¹ The exact mechanism of action is unclear, but Pedemonte et al. suggest that it may involve improved CFTR folding at the endoplasmic reticulum and stability at the cell surface.¹²¹ CFTR correctors KM11060 (7-chloro-4-(4-(4-chlorophenylsulfonylpiperazin-1-yl)quinoline) and KM11057 (7-chloro-4-(4-(phenylsulfonyl)piperazin-1-yl)quinoline) were found by high-throughput screening using baby hamster kidney cells in 2008.¹⁶⁵ Intriguingly, although KM11057 differs from KM11060 by only a single chlorine atom, it shows much weaker effects on promoting protein trafficking than KM11060 of the Delta F508-CFTR mutation, with potencies of approximately 100 and 1 μM, respectively.¹⁶⁵ Moreover, KM11060 has been further demonstrated to elevate plasma lipoxin A4 level in Delta F508 and wild-type mice, indicating a potential anti-inflammatory property, which is beneficial to CF treatment as inflammation is frequently seen in CF patients, especially those variants with severe symptoms such as Class II variants.¹⁶⁶ Another interesting compound is Dynasore ((Z)-N′-(3,4- dihydroxybenzylidene)-3-hydroxy-2-naphthohydrazide). It is a potent inhibitor of dynamin found by Macia et al. through high-throughput screening.¹⁶⁷ Dynamin is a protein required for vesicle formation during synaptic vesicle endocytosis. Dynasore inhibits it by interfering with its GTPase activity, thereby blocking both the wild-type and mutant CFTR endocytosis process and increasing the surface expression of CFTR.¹⁶⁷ Furthermore, Young et al. indicated that coadministration of Corr-4 and dynasore leads to a critical improvement on the level of cell surface CFTR expression than Corr-4 alone, which further emphasizes the significance of combination therapy in CFTR modulation.¹⁶⁸ Compound 15jf (N-(2-(5-chloro-2-methoxyphenylamino)-4′-methyl-4,5′-bithiazol-2′-yl)pivalamide) is a CFTR corrector defined in 2008 with EC₅₀ ≈ 1 μM.¹⁶⁹ In addition, it was stated to correct the defective Delta F508-CFTR processing; although the exact mechanism of the correction process is not clearly understood, 15jf was found to enhance glycosylation of CFTR TMDs, which may involve in its mechanism of action.¹⁷⁰ Lastly, VRT-534, also known as CF-106951 or C18, is a CFTR corrector, an analogue of VX-809 (lumacaftor) which can partially rescue Delta F508-CFTR misfolding and improve protein maturation and trafficking to the cell surface with an EC₅₀ of around 0.6 μM.^171,172 In addition, previous findings suggested that C18 binds directly to the CFTR protein and exerts its action.¹⁷²

Challenges and Future Directions

CFTR modulation therapy has been recognized as a promising therapeutic target in treating CF, as it directly addresses the underlying cause of disease rather than acts solely as a symptomatic reliver. Patients who currently do not benefit from approved CFTR modulators could benefit from further research aimed at discovering novel tools to modulate CFTR. In CFTR tool compound development, several challenges and exciting future directions are shaping the landscape of CF research.

One of the primary challenges in the development of CFTR tool compounds is targeting rare CFTR variants. Many promising compounds have been shown to effectively treat specific CFTR variants, e.g., VX-770 (Ivacaftor) for G551D CFTR variants.⁹⁶ In addition, VX-770 was proven effective against other gating mutations, including Class III mutations like G551S, G1244E, S1251N, S1255P, and G1349D, and Class IV mutation R117H.^173−175 Furthermore, CFTR corrector VX-809 (Lumacaftor) is capable of treating Delta F508-CFTR variants, when used with VX-770.¹⁷⁶ Currently, there are four different CFTR modulator/combinations available: elexacaftor–tezacaftor–ivacaftor, tezacaftor–ivacaftor, lumacaftor–ivacaftor, and ivacaftor monotherapy.^177−181 However, up to 20% of individuals in the CF population carry rare CFTR variants that are not eligible for CFTR modulator therapy.¹⁸² This issue underpins the need for molecules that act via completely different mechanisms compared to current modulators, and a clearer understanding of the molecular defects associated with these genetic variants. Therefore, developing models that can help researchers study the characteristics of these rare CFTR mutations is crucial.

Conclusion

In conclusion, the discovery and research of CFTR tool compounds play a pivotal role in investigating and developing therapies for CF, especially for patients with rare variants. Promising CFTR tool compounds such as CFTRinh-172, CFinh-172, GlyH-101, VRT-532, genistein, VRT-325, and Corr-4a exemplify diverse approaches to modulating CFTR function and addressing the underlying genetic variations that cause CF. While promising CFTR modulators are already available, targeting rare or less common CFTR variants remains a primary concern. However, due to the complexity of CFTR function, developing novel CFTR modulators requires a broad understanding of the molecular defects associated with these genetic variations. Although numerous modulators discussed in this Review have not undergone clinical trials due to suboptimal preclinical effects or intense drug toxicity, the imperative for further development of CFTR modulators is still necessary. Additionally, there remains an unmet need for comprehensive investigations into the applicability of novel CFTR modulators for patients with rare variants to benefit from novel CFTR modulators. Such endeavors are essential to facilitate the development of personalized therapeutic strategies for individuals with distinct CFTR genetic profiles.

Glossary

Abbreviations

BHK

Baby hamster kidney

CF

Cystic fibrosis

CFTR

Cystic fibrosis transmembrane conductance regulator

DIOS

Distal intestinal obstruction syndrome

ENaC

Epithelial sodium channel

FEV₁

Forced expiratory volume in 1 s

GIT

Gastrointestinal tract

MI

Meconium ileus

MSD

Membrane spanning domain

NBDs

Nucleotide-binding domains

P. aeruginosa

Pseudomonas aeruginosa

pwCF

People with CF

R-domain

Regulatory domain

TMD

Transmembrane domain

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request. Some data may not be made available because of privacy or ethical restrictions.

Author Contributions

Conceptualization (X.X.H., D.L., Y.Z., E.K.S.-F.); Funding acquisition (E.K.S.-F.); Supervision (E.K.S.-F.); Writing - review and editing (X.X.H., D.I., Y.Z., E.K.S.-F.).

E.K.S.-F. is supported by the National Health and Medical Research Council (Grant ID: APP1157287), Cystic Fibrosis Australia (Innovation Award 2021), and The University of Melbourne.

The authors declare no competing financial interest.