Innovative Strategy toward Mutant CFTR Rescue in Cystic Fibrosis: Design and Synthesis of Thiadiazole Inhibitors of the E3 Ligase RNF5.

In cystic fibrosis (CF), deletion of phenylalanine 508 (F508del) in the CF transmembrane conductance regulator (CFTR) is associated to misfolding and defective gating of the mutant channel. One of the most promising CF drug targets is the ubiquitin ligase RNF5, which promotes F508del-CFTR degradation. Recently, the first ever reported inhibitor of RNF5 was discovered, i.e., the 1,2,4-thiadiazol-5-ylidene inh-2. Here, we designed and synthesized a series of new analogues to explore the structure-activity relationships (SAR) of this class of compounds. SAR efforts ultimately led to compound 16, which showed a greater F508del-CFTR corrector activity than inh-2, good tolerability, and no toxic side effects. Analogue 16 increased the basal level of autophagy similar to what has been described with RNF5 silencing. Furthermore, co-treatment with 16 significantly improved the F508del-CFTR rescue induced by the triple combination elexacaftor/tezacaftor/ivacaftor in CFBE41o- cells. These findings validate the 1,2,4-thiadiazolylidene scaffold for the discovery of novel RNF5 inhibitors and provide evidence to pursue this unprecedented strategy for the treatment of CF.

Rescue by elexacaftor-tezacaftor-ivacaftor of the G1244E cystic fibrosis mutation's stability and gating defects are dependent on cell background.

BACKGROUND: Cystic fibrosis is caused by mutations impairing expression, trafficking, stability and/or activity of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel. The G1244E mutation causes a severe gating defect that it is not completely rescued by ivacaftor but requires the use of a second compound (a co-potentiator). Recently, it has been proposed that the corrector elexacaftor may act also as a co-potentiator. METHODS: By using molecular, biochemical and functional analyses we performed an in-depth characterization of the G1244E-CFTR mutant in heterologous and native cell models. RESULTS: Our studies demonstrate that processing and function of the mutant protein, as well as its pharmacological sensitivity, are markedly dependent on cell background. In heterologous expression systems, elexacaftor mainly acted on G1244E-CFTR as a co-potentiator, thus ameliorating the gating defect. On the contrary, in the native nasal epithelial cell model, elexacaftor did not act as a co-potentiator, but it increased mature CFTR expression possibly by improving mutant's defective stability at the plasma membrane. CONCLUSIONS: Our study highlights the importance of the cell background in the evaluation of CFTR modulator effects. Further, our results draw attention to the need for the development of novel potentiators having different mechanisms with respect to ivacaftor to improve channel activity for mutants with severe gating defect.

Proteostasis Regulators in Cystic Fibrosis: Current Development and Future Perspectives.

In cystic fibrosis (CF), the deletion of phenylalanine 508 (F508del) in the CF transmembrane conductance regulator (CFTR) leads to misfolding and premature degradation of the mutant protein. These defects can be targeted with pharmacological agents named potentiators and correctors. During the past years, several efforts have been devoted to develop and approve new effective molecules. However, their clinical use remains limited, as they fail to fully restore F508del-CFTR biological function. Indeed, the search for CFTR correctors with different and additive mechanisms has recently increased. Among them, drugs that modulate the CFTR proteostasis environment are particularly attractive to enhance therapy effectiveness further. This Perspective focuses on reviewing the recent progress in discovering CFTR proteostasis regulators, mainly describing the design, chemical structure, and structure-activity relationships. The opportunities, challenges, and future directions in this emerging and promising field of research are discussed, as well.

Targeting of Ubiquitin E3 Ligase RNF5 as a Novel Therapeutic Strategy in Neuroectodermal Tumors.

RNF5, an endoplasmic reticulum (ER) E3 ubiquitin ligase, participates to the ER-associated protein degradation guaranteeing the protein homeostasis. Depending on tumor model tested, RNF5 exerts pro- or anti-tumor activity. The aim of this study was to elucidate the controversial role of RNF5 in neuroblastoma and melanoma, two neuroectodermal tumors of infancy and adulthood, respectively. RNF5 gene levels are evaluated in publicly available datasets reporting the gene expression profile of melanoma and neuroblastoma primary tumors at diagnosis. The therapeutic effect of Analog-1, an RNF5 pharmacological activator, was investigated on in vitro and in vivo neuroblastoma and melanoma models. In both neuroblastoma and melanoma patients the high expression of RNF5 correlated with a better prognostic outcome. Treatment of neuroblastoma and melanoma cell lines with Analog-1 reduced cell viability by impairing the glutamine availability and energy metabolism through inhibition of F1Fo ATP-synthase activity. This latter event led to a marked increase in oxidative stress, which, in turn, caused cell death. Similarly, neuroblastoma- and melanoma-bearing mice treated with Analog-1 showed a significant delay of tumor growth in comparison to those treated with vehicle only. These findings validate RNF5 as an innovative drug target and support the development of Analog-1 in early phase clinical trials for neuroblastoma and melanoma patients.

Targeting the E1 ubiquitin-activating enzyme (UBA1) improves elexacaftor/tezacaftor/ivacaftor efficacy towards F508del and rare misfolded CFTR mutants.

The advent of Trikafta (Kaftrio in Europe) (a triple-combination therapy based on two correctors-elexacaftor/tezacaftor-and the potentiator ivacaftor) has represented a revolution for the treatment of patients with cystic fibrosis (CF) carrying the most common misfolding mutation, F508del-CFTR. This therapy has proved to be of great efficacy in people homozygous for F508del-CFTR and is also useful in individuals with a single F508del allele. Nevertheless, the efficacy of this therapy needs to be improved, especially in light of the extent of its use in patients with rare class II CFTR mutations. Using CFBE41o- cells expressing F508del-CFTR, we provide mechanistic evidence that targeting the E1 ubiquitin-activating enzyme (UBA1) by TAK-243, a small molecule in clinical trials for other diseases, boosts the rescue of F508del-CFTR induced by CFTR correctors. Moreover, TAK-243 significantly increases the F508del-CFTR short-circuit current induced by elexacaftor/tezacaftor/ivacaftor in differentiated human primary airway epithelial cells, a gold standard for the pre-clinical evaluation of patients' responsiveness to pharmacological treatments. This new combinatory approach also leads to an improvement in CFTR conductance on cells expressing other rare CF-causing mutations, including N1303K, for which Trikafta is not approved. These findings show that Trikafta therapy can be improved by the addition of a drug targeting the misfolding detection machinery at the beginning of the ubiquitination cascade and may pave the way for an extension of Trikafta to low/non-responding rare misfolded CFTR mutants.

The L467F-F508del Complex Allele Hampers Pharmacological Rescue of Mutant CFTR by Elexacaftor/Tezacaftor/Ivacaftor in Cystic Fibrosis Patients: The Value of the Ex Vivo Nasal Epithelial Model to Address Non-Responders to CFTR-Modulating Drugs.

Loss-of-function mutations of the CFTR gene cause cystic fibrosis (CF) through a variety of molecular mechanisms involving altered expression, trafficking, and/or activity of the CFTR chloride channel. The most frequent mutation among CF patients, F508del, causes multiple defects that can be, however, overcome by a combination of three pharmacological agents that improve CFTR channel trafficking and gating, namely, elexacaftor, tezacaftor, and ivacaftor. This study was prompted by the evidence of two CF patients, compound heterozygous for F508del and a minimal function variant, who failed to obtain any beneficial effects following treatment with the triple drug combination. Functional studies on nasal epithelia generated in vitro from these patients confirmed the lack of response to pharmacological treatment. Molecular characterization highlighted the presence of an additional amino acid substitution, L467F, in cis with the F508del variant, demonstrating that both patients were carriers of a complex allele. Functional and biochemical assays in heterologous expression systems demonstrated that the double mutant L467F-F508del has a severely reduced activity, with negligible rescue by CFTR modulators. While further studies are needed to investigate the actual prevalence of the L467F-F508del allele, our results suggest that this complex allele should be taken into consideration as plausible cause in CF patients not responding to CFTR modulators.

Partial Rescue of F508del-CFTR Stability and Trafficking Defects by Double Corrector Treatment.

Deletion of phenylalanine at position 508 (F508del) in the CFTR chloride channel is the most frequent mutation in cystic fibrosis (CF) patients. F508del impairs the stability and folding of the CFTR protein, thus resulting in mistrafficking and premature degradation. F508del-CFTR defects can be overcome with small molecules termed correctors. We investigated the efficacy and properties of VX-445, a newly developed corrector, which is one of the three active principles present in a drug (Trikafta®/Kaftrio®) recently approved for the treatment of CF patients with F508del mutation. We found that VX-445, particularly in combination with type I (VX-809, VX-661) and type II (corr-4a) correctors, elicits a large rescue of F508del-CFTR function. In particular, in primary bronchial epithelial cells of CF patients, the maximal rescue obtained with corrector combinations including VX-445 was close to 60-70% of CFTR function in non-CF cells. Despite this high efficacy, analysis of ubiquitylation, resistance to thermoaggregation, protein half-life, and subcellular localization revealed that corrector combinations did not fully normalize F508del-CFTR behavior. Our study indicates that it is still possible to further improve mutant CFTR rescue with the development of corrector combinations having maximal effects on mutant CFTR structural and functional properties.

A high-content drug screening strategy to identify protein level modulators for genetic diseases: A proof-of-principle in autosomal dominant leukodystrophy.

In genetic diseases, the most prevalent mechanism of pathogenicity is an altered expression of dosage-sensitive genes. Drugs that restore physiological levels of these genes should be effective in treating the associated conditions. We developed a screening strategy, based on a bicistronic dual-reporter vector, for identifying compounds that modulate protein levels, and used it in a pharmacological screening approach. To provide a proof-of-principle, we chose autosomal dominant leukodystrophy (ADLD), an ultra-rare adult-onset neurodegenerative disorder caused by lamin B1 (LMNB1) overexpression. We used a stable Chinese hamster ovary (CHO) cell line that simultaneously expresses an AcGFP reporter fused to LMNB1 and a Ds-Red normalizer. Using high-content imaging analysis, we screened a library of 717 biologically active compounds and approved drugs, and identified alvespimycin, an HSP90 inhibitor, as a positive hit. We confirmed that alvespimycin can reduce LMNB1 levels by 30%-80% in five different cell lines (fibroblasts, NIH3T3, CHO, COS-7, and rat primary glial cells). In ADLD fibroblasts, alvespimycin reduced cytoplasmic LMNB1 by about 50%. We propose this approach for effectively identifying potential drugs for treating genetic diseases associated with deletions/duplications and paving the way toward Phase II clinical trials.

Identification, Structure-Activity Relationship, and Biological Characterization of 2,3,4,5-Tetrahydro-1H-pyrido[4,3-b]indoles as a Novel Class of CFTR Potentiators.

Cystic fibrosis (CF) is a life-threatening autosomal recessive disease, caused by mutations in the CF transmembrane conductance regulator (CFTR) chloride channel. CFTR modulators have been reported to address the basic defects associated with CF-causing mutations, partially restoring the CFTR function in terms of protein processing and/or channel gating. Small-molecule compounds, called potentiators, are known to ameliorate the gating defect. In this study, we describe the identification of the 2,3,4,5-tetrahydro-1H-pyrido[4,3-b]indole core as a novel chemotype of potentiators. In-depth structure-activity relationship studies led to the discovery of enantiomerically pure 39 endowed with a good efficacy in rescuing the gating defect of F508del- and G551D-CFTR and a promising in vitro druglike profile. The in vivo characterization of γ-carboline 39 showed considerable exposure levels and good oral bioavailability, with detectable distribution to the lungs after oral administration to rats. Overall, these findings may represent an encouraging starting point to further expand this chemical class, adding a new chemotype to the existing classes of CFTR potentiators.

Characterization of the mechanism of action of RDR01752, a novel corrector of F508del-CFTR.

Despite progress in developing pharmacotherapies to rescue F508del-CFTR, the most prevalent Cystic Fibrosis (CF)-causing mutation, individuals homozygous for this mutation still face several disease-related symptoms. Thus, more potent compound combinations are still needed. Here, we investigated the mechanism of action (MoA) of RDR01752, a novel F508del-CFTR trafficking corrector. F508del-CFTR correction by RDR01752 was assessed by biochemical, immunofluorescence microscopy and functional assays in cell lines and in intestinal organoids. To determine the MoA of RDR01752, we assessed its additive effects to those of genetic revertants of F508del-CFTR, the FDA-approved corrector drugs VX-809 and VX-661, and low temperature. Our data demonstrated that RDR01752 rescues F508del-CFTR processing and plasma membrane (PM) expression to similar levels of VX-809 in cell lines, although RDR01752 produced lower functional rescue. However, in functional assays using intestinal organoids (F508del/F508del), RDR01752, VX-809 and VX-661 had similar efficacy. RDR01752 demonstrated additivity to revertants 4RK and G550E, but not to R1070W, as previously shown for VX-809. RDR01752 was also additive to low temperature. Co-treatment of RDR01752 and VX-809 did not increase F508del-CFTR PM expression and function compared to each corrector alone. The lack of additivity of RDR01752 with the genetic revertant R1070W suggests that this compound has the same effect as the insertion of tryptophan at 1070, i.e., filling the pocket at the NBD1:ICL4 interface in F508del-CFTR, similarly to VX-809. Combination of RDR01752 with correctors mimicking the rescue by revertants G550E or 4RK could thus maximize rescue of F508del-CFTR.

Discovery of a picomolar potency pharmacological corrector of the mutant CFTR chloride channel.

F508del, the most frequent mutation causing cystic fibrosis (CF), results in mistrafficking and premature degradation of the CFTR chloride channel. Small molecules named correctors may rescue F508del-CFTR and therefore represent promising drugs to target the basic defect in CF. We screened a carefully designed chemical library to find F508del-CFTR correctors. The initial active compound resulting from the primary screening underwent extensive chemical optimization. The final compound, ARN23765, showed an extremely high potency in bronchial epithelial cells from F508del homozygous patients, with an EC50 of 38 picomolar, which is more than 5000-fold lower compared to presently available corrector drugs. ARN23765 also showed high efficacy, synergy with other types of correctors, and compatibility with chronic VX-770 potentiator. Besides being a promising drug, particularly suited for drug combinations, ARN23765 represents a high-affinity probe for CFTR structure-function studies.

High-Content Screening Identifies Vanilloids as a Novel Class of Inhibitors of NET Formation.

Neutrophils migrate to sites of infection where they phagocytose, degranulate, and/or, in the presence of appropriate stimuli, release decondensed chromatin strands (called neutrophil extracellular traps, NETs) for trapping and possibly killing microorganisms. NET formation is characterized by marked morphological cell changes, in particular within the nucleus. Lytic NET formation can be observed in neutrophils undergoing cell death, which is referred to as NETosis. Dysregulation of NET production and/or degradation can exert pathogenic effects, contributing to the pathogenesis of various diseases, including cystic fibrosis, autoimmune diseases and inflammatory conditions. By employing a phenotypic assay based on high-content imaging and analysis, we screened a library of biologically active compounds and identified vanilloids as a novel class of chemical compounds able to hinder NETosis induction and NET release. Vanilloids also markedly decrease cytosolic ROS production. The identification of novel vanilloid NET inhibitors, able to stop excessive or aberrant NET production might offer new therapeutic options for those disorders displaying NET overproduction.

Chemically modified hCFTR mRNAs recuperate lung function in a mouse model of cystic fibrosis.

Gene therapy has always been a promising therapeutic approach for Cystic Fibrosis (CF). However, numerous trials using DNA or viral vectors encoding the correct protein resulted in a general low efficacy. In the last years, chemically modified messenger RNA (cmRNA) has been proven to be a highly potent, pulmonary drug. Consequently, we first explored the expression, function and immunogenicity of human (h)CFTR encoded by cmRNAhCFTR in vitro and ex vivo, quantified the expression by flow cytometry, determined its function using a YFP based assay and checked the immune response in human whole blood. Similarly, we examined the function of cmRNAhCFTR in vivo after intratracheal (i.t.) or intravenous (i.v.) injection of the assembled cmRNAhCFTR together with Chitosan-coated PLGA (poly-D, L-lactide-co-glycolide 75:25 (Resomer RG 752 H)) nanoparticles (NPs) by FlexiVent. The amount of expression of human hCFTR encoded by cmRNAhCFTR was quantified by hCFTR ELISA, and cmRNAhCFTR values were assessed by RT-qPCR. Thereby, we observed a significant improvement of lung function, especially in regards to FEV0.1, suggesting NP-cmRNAhCFTR as promising therapeutic option for CF patients independent of their CFTR genotype.

Pharmacological Inhibition of the Ubiquitin Ligase RNF5 Rescues F508del-CFTR in Cystic Fibrosis Airway Epithelia.

In cystic fibrosis (CF), deletion of phenylalanine 508 (F508del) in the CFTR channel is associated with misfolding and premature degradation of the mutant protein. Among the known proteins associated with F508del-CFTR processing, the ubiquitin ligase RNF5/RMA1 is particularly interesting. We previously demonstrated that genetic suppression of RNF5 in vivo leads to an attenuation of intestinal pathological phenotypes in CF mice, validating the relevance of RNF5 as a drug target for CF. Here, we used a computational approach, based on ligand docking and virtual screening, to discover inh-02, a drug-like small molecule that inhibits RNF5. In in vitro experiments, treatment with inh-02 modulated ATG4B and paxillin, both known RNF5 targets. In immortalized and primary bronchial epithelial cells derived from CF patients homozygous for the F508del mutation, long-term incubation with inh-02 caused significant F508del-CFTR rescue. This work validates RNF5 as a drug target for CF, providing evidence to support its druggability.

Thymosin α-1 does not correct F508del-CFTR in cystic fibrosis airway epithelia.

In cystic fibrosis (CF), deletion of phenylalanine 508 (F508del) in the cystic fibrosis transmembrane conductance regulator (CFTR) anion channel causes misfolding and premature degradation. Considering the numerous effects of the F508del mutation on the assembly and processing of CFTR protein, combination therapy with several pharmacological correctors is likely to be required to treat CF patients. Recently, it has been reported that thymosin α-1 (Tα-1) has multiple beneficial effects that could lead to a single-molecule-based therapy for CF patients with F508del. Such effects include suppression of inflammation, improvement in F508del-CFTR maturation and gating, and stimulation of chloride secretion through the calcium-activated chloride channel (CaCC). Given the importance of such a drug, we aimed to characterize the underlying molecular mechanisms of action of Tα-1. In-depth analysis of Tα-1 effects was performed using well-established microfluorimetric, biochemical, and electrophysiological techniques on epithelial cell lines and primary bronchial epithelial cells from CF patients. The studies, which were conducted in 2 independent laboratories with identical outcome, demonstrated that Tα-1 is devoid of activity on mutant CFTR as well as on CaCC. Although Tα-1 may still be useful as an antiinflammatory agent, its ability to target defective anion transport in CF remains to be further investigated.

The Autophagy Inhibitor Spautin-1 Antagonizes Rescue of Mutant CFTR Through an Autophagy-Independent and USP13-Mediated Mechanism.

The mutation F508del, responsible for a majority of cystic fibrosis cases, provokes the instability and misfolding of the CFTR chloride channel. Pharmacological recovery of F508del-CFTR may be obtained with small molecules called correctors. However, treatment with a single corrector in vivo and in vitro only leads to a partial rescue, a consequence of cell quality control systems that still detect F508del-CFTR as a defective protein causing its degradation. We tested the effect of spautin-1 on F508del-CFTR since it is an inhibitor of USP10 deubiquitinase and of autophagy, a target and a biological process that have been associated with cystic fibrosis and mutant CFTR. We found that short-term treatment of cells with spautin-1 downregulates the function and expression of F508del-CFTR despite the presence of corrector VX-809, a finding obtained in multiple cell models and assays. In contrast, spautin-1 was ineffective on wild type CFTR. Silencing and upregulation of USP13 (another target of spautin-1) but not of USP10, had opposite effects on F508del-CFTR expression/function. In contrast, modulation of autophagy with known activators or inhibitors did not affect F508del-CFTR. Our results identify spautin-1 as a novel chemical probe to investigate the molecular mechanisms that prevent full rescue of mutant CFTR.

High-throughput screening identifies FAU protein as a regulator of mutant cystic fibrosis transmembrane conductance regulator channel.

In cystic fibrosis, deletion of phenylalanine 508 (F508del) in the cystic fibrosis transmembrane conductance regulator (CFTR) anion channel causes misfolding and premature degradation. One possible approach to reducing the detrimental health effects of cystic fibrosis could be the identification of proteins whose suppression rescues F508del-CFTR function in bronchial epithelial cells. However, searches for these potential targets have not yet been conducted, particularly in a relevant airway background using a functional readout. To identify proteins associated with F508del-CFTR processing, we used a high-throughput functional assay to screen an siRNA library targeting 6,650 different cellular proteins. We identified 37 proteins whose silencing significantly rescued F508del-CFTR activity, as indicated by enhanced anion transport through the plasma membrane. These proteins included FAU, UBE2I, UBA52, MLLT6, UBA2, CHD4, PLXNA1, and TRIM24, among others. We focused our attention on FAU, a poorly characterized protein with unknown function. FAU knockdown increased the plasma membrane targeting and function of F508del-CFTR, but not of wild-type CFTR. Investigation into the mechanism of action revealed a preferential physical interaction of FAU with mutant CFTR, leading to its degradation. FAU and other proteins identified in our screening may offer a therapeutically relevant panel of drug targets to correct basic defects in F508del-CFTR processing.

RNF5, DAB2 and Friends: Novel Drug Targets for Cystic Fibrosis.

BACKGROUND: Deletion of phenylalanine 508 is the most frequent mutation causing cystic fibrosis. It causes multiple defects: 1) misfolding of the protein causing retention at the ER (processing defect); 2) reduced channel activity (gating defect); 3) reduced plasma membrane residency time due to increased internalization rate and defective recycling. METHODS: Druggability of F508del-CFTR was demonstrated by several studies. Correctors are molecules able to improve maturation and trafficking to the membrane of F508del- CFTR. Correctors could act as pharmacological chaperones or as proteostasis regulators. Pharmacological chaperones act directly on mutant CFTR, while proteostasis regulators modify the proteostasis environment leading to beneficial effects on CFTR maturation. RESULTS: The use of a single compound is not sufficient to promote a therapeutically relevant F508del-CFTR rescue. Drug therapy for CF will require combinations of correctors exploiting different mechanisms of action, i.e. pharmacological chaperones combined together or with a proteostasis regulator. CONCLUSION: Development of more effective CF drugs could therefore rely on a better understanding of the molecular events underlying CFTR processing/degradation. This review will focus on most promising pathways and related targets for the development of novel CF pharmacotherapies.

Genetic Inhibition Of The Ubiquitin Ligase Rnf5 Attenuates Phenotypes Associated To F508del Cystic Fibrosis Mutation.

Cystic fibrosis (CF) is caused by mutations in the CFTR chloride channel. Deletion of phenylalanine 508 (F508del), the most frequent CF mutation, impairs CFTR trafficking and gating. F508del-CFTR mistrafficking may be corrected by acting directly on mutant CFTR itself or by modulating expression/activity of CFTR-interacting proteins, that may thus represent potential drug targets. To evaluate possible candidates for F508del-CFTR rescue, we screened a siRNA library targeting known CFTR interactors. Our analysis identified RNF5 as a protein whose inhibition promoted significant F508del-CFTR rescue and displayed an additive effect with the investigational drug VX-809. Significantly, RNF5 loss in F508del-CFTR transgenic animals ameliorated intestinal malabsorption and concomitantly led to an increase in CFTR activity in intestinal epithelial cells. In addition, we found that RNF5 is differentially expressed in human bronchial epithelia from CF vs. control patients. Our results identify RNF5 as a target for therapeutic modalities to antagonize mutant CFTR proteins.