Discovery of GLPG2737, a Potent Type 2 Corrector of CFTR for the Treatment of Cystic Fibrosis in Combination with a Potentiator and a Type 1 Co-corrector.

Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR) protein. This epithelial anion channel regulates the active transport of chloride and bicarbonate ions across membranes. Mutations result in reduced surface expression of CFTR channels with impaired functionality. Correctors are small molecules that support the trafficking of CFTR to increase its membrane expression. Such correctors can have different mechanisms of action. Combinations may result in a further improved therapeutic benefit. We describe the identification and optimization of a new pyrazolol3,4-bl pyridine-6-carboxylic acid series with high potency and efficacy in rescuing CFTR from the cell surface. Investigations showed that carboxylic acid group replacement with acylsulfonamides and acylsulfonylureas improved ADMET and PK properties, leading to the discovery of the structurally novel co-corrector GLPG2737. The addition of GLPG2737 to the combination of the potentiator GLPG1837 and C1 corrector 4 led to an 8-fold increase in the F508del CFTR activity.

Investigating wound healing potential of sesamol loaded solid lipid nanoparticles: Ex-vivo, in vitro and in-vivo proof of concept.

Sesamol, a lignan, obtained from sesame seeds (Sesamum indicum Linn., Pedaliaciae) has a promising antioxidant, and anti-inflammatory profile. When applied topically, free sesamol rapidly crosses skin layers and gets absorbed in systemic circulation. Its encapsulation into solid lipid nanoparticles not only improved its localised delivery to skin but also resulted in better skin retention, as found in ex-vivo skin retention studies. Free and encapsulated sesamol was compared for antimicrobial and antibiofilm activity against some common skin pathogens and it was found that encapsulation improved the antimicrobial profile by 200%. In vivo evaluation in diabetic open excision wound model suggested that encapsulation of sesamol in SLNs substantially enhanced its wound healing potential when investigated for biophysical, biochemical and histological parameters. It was envisaged that this was achieved via inhibiting bacterial growth and clearing the bacterial biofilm at the wound site, and by regulating oxidative stress in skin tissue.

Comprehensive Assessment of CFTR Modulators' Therapeutic Efficiency for N1303K Variant.

p.Asn1303Lys (N1303K) is a common missense variant of the CFTR gene, causing cystic fibrosis (CF). In this study, we initially evaluated the influence of CFTR modulators on the restoration of N1303K-CFTR function using intestinal organoids derived from four CF patients expressing the N1303K variant. The forskolin-induced swelling assay in organoids offered valuable insights about the beneficial effects of VX-770 + VX-661 + VX-445 (Elexacaftor + Tezacaftor + Ivacaftor, ETI) on N1303K-CFTR function restoration and about discouraging the prescription of VX-770 + VX-809 (Ivacaftor + Lumacaftor) or VX-770 + VX-661 (Ivacaftor + Tezacaftor) therapy for N1303K/class I patients. Then, a comprehensive assessment was conducted on an example of one patient with the N1303K/class I genotype to examine the ETI effect on the restoration of N1303K-CFTR function using in vitro the patient's intestinal organoids, ex vivo the intestinal current measurements (ICM) method and assessment of the clinical status before and after targeted therapy. All obtained results are consistent with each other and have proven the effectiveness of ETI for the N1303K variant. ETI produced a significant positive effect on forskolin-induced swelling in N1303K/class I organoids indicating functional improvement of the CFTR protein; ICM demonstrated that ETI therapy restored CFTR function in the intestinal epithelium after three months of treatment, and the patient improved his clinical status and lung function, increased his body mass index (BMI) and reduced the lung pathogenic flora diversity, surprisingly without improving the sweat test results.

Organic Synthesis and Current Understanding of the Mechanisms of CFTR Modulator Drugs Ivacaftor, Tezacaftor, and Elexacaftor.

The monogenic rare disease Cystic Fibrosis (CF) is caused by mutations in the gene encoding the CF transmembrane conductance (CFTR) protein, an anion channel expressed at the apical plasma membrane of epithelial cells. The discovery and subsequent development of CFTR modulators-small molecules acting on the basic molecular defect in CF-have revolutionized the standard of care for people with CF (PwCF), thus drastically improving their clinical features, prognosis, and quality of life. Currently, four of these drugs are approved for clinical use: potentiator ivacaftor (VX-770) alone or in combination with correctors lumacaftor, (VX-809), tezacaftor (VX-661), and elexacaftor (VX-445). Noteworthily, the triple combinatorial therapy composed of ivacaftor, tezacaftor, and elexacaftor constitutes the most effective modulator therapy nowadays for the majority of PwCF. In this review, we exploit the organic synthesis of ivacaftor, tezacaftor, and elexacaftor by providing a retrosynthetic drug analysis for these CFTR modulators. Furthermore, we describe the current understanding of the mechanisms of action (MoA's) of these compounds by discussing several studies that report the key findings on the molecular mechanisms underlying their action on the CFTR protein.

Tissue-specific variations of piperine in ten populations of Piper longum L.: bioactivities and toxicological profile.

P. longum L., one of the most significant species of the genus Piperaceae, is most frequently employed in Indian-Ayurvedic and other traditional medicinal-systems for treating a variety of illnesses. The alkaloid piperine, is the key phytoconstituent of the plant, primarily responsible for its' pharmacological-impacts. The aim of the study is to analyse the intra-specific variation in piperine content among different chemotypes (PL1 to PL 30) and identify high piperine yielding chemotype (elite-chemotype) collected from 10 different geographical regions of West Bengal by validated HPTLC chromatography method. The study also focused on the pharmacological-screening to better understand the antioxidant activity of the methanol extracts of P. longum by DPPH and ABTS radical-scavenging activity and genotoxic activity by Allium cepa root tip assay. It was found that the P. longum fruit chemotypes contain high amount piperine (highest 16.362 mg/g in chemotype PL9) than the stem and leaf chemotypes. Both DPPH and ABTS antioxidant assays revealed that P. longum showed moderate radical-scavenging activity and the highest activity was found in PL9 (fruit) chemotype with IC50 values of 124.2 ± 0.97 and 104 ± 0.78 µg/ml respectively. The A. cepa root tip assay showed no such significant genotoxic-effect and change in mitotic-index. The quick, reproducible, and validated HPTLC approach offers a useful tool for determining quantitative variations of piperine among P. longum chemotypes from different geographical-regions and also according to the different tissues and choose elite genotypes with high piperine production for continued propagation and commercialization for the pharmaceutical sector. Additionally, the plant's in-vitro antioxidant property and lack of genotoxicity directly supports its' widespread and long history of use as a medicinal and culinary plant.

PTI-801 (posenacaftor) shares a common mechanism with VX-445 (elexacaftor) to rescue p.Phe508del-CFTR.

The deletion of a phenylalanine at position 508 (p.Phe508del) in the CFTR anion channel is the most prevalent variant in people with Cystic Fibrosis (CF). This variant impairs folding and stability of the CF transmembrane conductance regulator (CFTR) protein, resulting in its defective trafficking and premature degradation. Over the last years, therapeutic accomplishments have been attained in developing small molecules that partially correct p.Phe508del-CFTR defects; however, the mechanism of action (MoA) of these compounds has only started to be uncovered. In this study, we employed biochemical, fluorescence microscopy, and functional assays to examine the efficacy and properties of PTI-801, a newly developed p.Phe508del-CFTR corrector. To exploit its MoA, we assessed PTI-801 effects in combination with low temperature, genetic revertants of p.Phe508del-CFTR (the in cis p.Val510Asp, p.Gly550Glu, p.Arg1070Trp, and 4RK) and other correctors. Our results demonstrated that PTI-801 rescues p.Phe508del-CFTR processing, PM trafficking, and channel function (upon agonist stimulation) with greater correction effects in combination with ABBV-2222, FDL-169, VX-661, or VX-809, but not with VX-445. Although PTI-801 exhibited no potentiator activity on low temperature- and corrector-rescued p.Phe508del-CFTR, this compound displayed similar behavior to that of VX-445 on genetic revertants. Such evidence associated with the lack of additivity when PTI-801 and VX-445 were combined indicates that they share a common binding site to correct p.Phe508del-CFTR defects. Despite the high efficacy of PTI-801 in combination with ABBV-2222, FDL-169, VX-661, or VX-809, these dual corrector combinations only partially restored p.Phe508del-CFTR conformational stability, as shown by the lower half-life of the mutant protein compared to that of WT-CFTR. In summary, PTI-801 likely shares a common MoA with VX-445 in rescuing p.Phe508del-CFTR, thus being a feasible alternative for the development of novel corrector combinations with greater capacity to rescue mutant CFTR folding and stability.

Piperine, a Plant Alkaloid, Exhibits Efficient Disintegration of the Pre-existing Biofilm of Staphylococcus aureus: a Step Towards Effective Management of Biofilm Threats.

Staphylococcus aureus causes a range of chronic infections in humans by exploiting its biofilm machinery and drug-tolerance property. Although several strategies have been proposed to eradicate biofilm-linked issues, here, we have explored whether piperine, a bioactive plant alkaloid, can disintegrate an already existing Staphylococcal biofilm. Towards this direction, the cells of S. aureus were allowed to develop biofilm first followed by treatment with the test concentrations (8 and 16 µg/mL) of piperine. In this connection, several assays such as total protein recovery assay, crystal violet assay, extracellular polymeric substances (EPS) measurement assay, fluorescein diacetate hydrolysis assay, and fluorescence microscopic image analysis confirmed the biofilm-disintegrating property of piperine against S. aureus. Piperine reduced the cellular auto-aggregation by decreasing the cell surface hydrophobicity. On further investigation, we observed that piperine could down regulate the dltA gene expression that might reduce the cell surface hydrophobicity of S. aureus. It was also observed that the piperine-induced accumulation of reactive oxygen species (ROS) could enhance biofilm disintegration by decreasing the cell surface hydrophobicity of the test organism. Together, all the observations suggested that piperine could be used as a potential molecule for the effective management of the pre-existing biofilm of S. aureus.

Small-molecule correctors divert CFTR-F508del from ERAD by stabilizing sequential folding states.

Over 80% of people with cystic fibrosis (CF) carry the F508del mutation in the cystic fibrosis transmembrane conductance regulator (CFTR), a chloride ion channel at the apical plasma membrane (PM) of epithelial cells. F508del impairs CFTR folding causing it to be destroyed by endoplasmic reticulum associated degradation (ERAD). Small-molecule correctors, which act as pharmacological chaperones to divert CFTR-F508del from ERAD, are the primary strategy for treating CF, yet corrector development continues with only a rudimentary understanding of how ERAD targets CFTR-F508del. We conducted genome-wide CRISPR/Cas9 knockout screens to systematically identify the molecular machinery that underlies CFTR-F508del ERAD. Although the ER-resident ubiquitin ligase, RNF5 was the top E3 hit, knocking out RNF5 only modestly reduced CFTR-F508del degradation. Sublibrary screens in an RNF5 knockout background identified RNF185 as a redundant ligase and demonstrated that CFTR-F508del ERAD is robust. Gene-drug interaction experiments illustrated that correctors tezacaftor (VX-661) and elexacaftor (VX-445) stabilize sequential, RNF5-resistant folding states. We propose that binding of correctors to nascent CFTR-F508del alters its folding landscape by stabilizing folding states that are not substrates for RNF5-mediated ubiquitylation.

Cyclic diacyl thioureas enhance activity of corrector Lumacaftor on F508del-CFTR.

Cystic fibrosis is a genetic disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) protein. In the search of novel series of CFTR modulators, a library of mono and diacyl thioureas were prepared by sequential synthesis. When tested alone, the obtained compounds 5 and 6 poorly affected F508del-CFTR conductance but, in combination with Lumacaftor, selected derivatives showed the ability to increase the activity of the approved modulator. Analogue 6 i displayed the most marked enhancing effect and acylthioureas 6 d and 6 f were also able to improve efficacy of Lumacaftor. All compounds proved to be non-cytotoxic against different cancer cell lines. Good pharmacokinetic properties were predicted for derivatives 5 and 6, thus supporting the value of these compounds for the development of novel modulators potentially useful for cystic fibrosis.

The detection and modulation of piperine in the human oral cavity.

Piperine is an alkaloid that is responsible for the pungency of black pepper and long pepper. This hydrophobic compound causes a spicy sensation when it comes in contact with trigeminal neurons of the oral cavity. Piperine has low solubility in water, which presents difficulties in examining the psychophysical properties of this stimulus by standard aqueous chemosensory tests. This report describes approaches that utilize novel edible film formulations for delivering precise amounts of piperine to the human oral cavity. These films were then used to identify detection thresholds for piperine, and to identify the chemosensory properties of this compound at suprathreshold amounts. When incorporated into edible films, mean detection thresholds for piperine were approximately 35 nanomoles. For suprathreshold studies, edible films that contained 4000 nanomole amounts of piperine yielded variable intensity responses in subjects, with mean intensities in the moderate range. This amount of piperine caused significant self-desensitization, which was partially reversed after 60-90 min. In contrast, edible films that contained lower amounts of piperine yielded mean intensity ratings in the weak range and showed essentially no self-desensitization. The application of piperine to the circumvallate region of the tongue caused moderate intensity responses that were identified as primarily spicy, and rarely bitter. In addition, oral rinses with aqueous sucrose solutions decreased mean intensities for piperine by approximately twenty-five percent over sixty seconds. Blockage of nasal airflow significantly decreased piperine intensities in the oral cavity. These two findings indicate that oral sucrose or blockage of nasal airflow can modulate piperine perception in the human oral cavity. Finally, these results indicate that a variety of excipients can be included in edible film formulations for presenting piperine to the oral cavity at stimulus amounts that cause quantifiable chemosensory responses.

Structure-Activity Relationship of FL118 Platform Position 7 Versus Position 9-Derived Compounds and Their Mechanism of Action and Antitumor Activity.

Structurally, FL118 is a camptothecin analogue and possesses exceptional antitumor efficacy against human cancer through a novel mechanism of action (MOA). In this report, we have synthesized and characterized 24 FL118 Position 7-substituted and 24 FL118 Position 9-substituted derivatives. The top compounds were further characterized for their MOA in colorectal cancer (CRC) models using CRC patient-derived xenograft (PDX) models and pancreatic cancer PDX models to evaluate their antitumor activities. Four FL118 Position 7-substituted derivatives showed significantly better antitumor efficacy than the FL118 Position 9-substituted derivatives. The four identified compounds also appeared to have better antitumor activity than their parental platform FL118. Interestingly, RNA-Seq analyses indicated that three of the four compounds exerted antitumor effects via an MOA similar to FL118, which provided an intriguing opportunity for follow-up studies. Extended in vivo studies revealed that FL77-6 (7-(4-ethylphenyl)-FL118), FL77-9 (7-(4-methoxylphenyl)-FL118), and FL77-24 (7-(3, 5-dimethoxyphenyl)-FL118) exhibit potential for further development toward clinical trials.

Piperine: Chemistry and Biology.

Piperine is a plant-derived promising piperamide candidate isolated from the black pepper (Piper nigrum L.). In the last few years, this natural botanical product and its derivatives have aroused much attention for their comprehensive biological activities, including not only medical but also agricultural bioactivities. In order to achieve sustainable development and improve survival conditions, looking for environmentally friendly pesticides with low toxicity and residue is an extremely urgent challenge. Fortunately, plant-derived pesticides are rising like a shining star, guiding us in the direction of development in pesticidal research. In the present review, the recent progress in the biological activities, mechanisms of action, and structural modifications of piperine and its derivatives from 2020 to 2023 are summarized. The structure-activity relationships were analyzed in order to pave the way for future development and utilization of piperine and its derivatives as potent drugs and pesticides for improving the local economic development.

Sesamol: A lignan in sesame seeds with potent anti-inflammatory and immunomodulatory properties.

Inflammation is associated with the development and progression of a plethora of diseases including joint, metabolic, neurological, hepatic, and renal disorders. Sesamol, derived from the seeds of Sesamum indicum L., has received considerable attention due to its well-documented multipotent phytotherapeutic effects, including its anti-inflammatory and immunomodulatory properties. However, to date, no comprehensive review has been established to highlight or summarize the anti-inflammatory and immunomodulatory properties of sesamol. Herein, we aim to address this gap in the literature by presenting a thorough review encapsulating evidence surrounding the range of inflammatory mediators and cytokines shown to be targeted by sesamol in modulating its anti-inflammatory actions against a range of inflammatory disorders. Additionally, evidence highlighting the role that sesamol has in modulating components of adaptive immunity including cellular immune responses and Th1/Th2 balance is underscored. Moreover, the molecular mechanisms and the signaling pathways underlying such effects are also highlighted. Findings indicate that this seemingly potent lignan mediates its anti-inflammatory actions, at least in part, via suppression of various pro-inflammatory cytokines like IL-1ß and TNFα, and downregulation of a multitude of signaling pathways including NF-κB and MAPK. In conclusion, we anticipate that sesamol may be employed in future therapeutic regimens to aid in more effective drug development to alleviate immune-related and inflammatory conditions.

CFTR Function Restoration upon Elexacaftor/Tezacaftor/Ivacaftor Treatment in Patient-Derived Intestinal Organoids with Rare CFTR Genotypes.

Cystic fibrosis (CF) is caused by mutations in the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) gene. The combination of the CFTR modulators elexacaftor, tezacaftor, and ivacaftor (ETI) enables the effective rescue of CFTR function in people with the most prevalent F508del mutation. However, the functional restoration of rare CFTR variants remains unclear. Here, we use patient-derived intestinal organoids (PDIOs) to identify rare CFTR variants and potentially individuals with CF that might benefit from ETI. First, steady-state lumen area (SLA) measurements were taken to assess CFTR function and compare it to the level observed in healthy controls. Secondly, the forskolin-induced swelling (FIS) assay was performed to measure CFTR rescue within a lower function range, and to further compare it to ETI-mediated CFTR rescue in CFTR genotypes that have received market approval. ETI responses in 30 PDIOs harboring the F508del mutation served as reference for ETI responses of 22 PDIOs with genotypes that are not currently eligible for CFTR modulator treatment, following European Medicine Agency (EMA) and/or U.S. Food and Drug Administration (FDA) regulations. Our data expand previous datasets showing a correlation between in vitro CFTR rescue in organoids and corresponding in vivo ppFEV1 improvement upon a CFTR modulator treatment in published clinical trials, and suggests that the majority of individuals with rare CFTR variants could benefit from ETI. CFTR restoration was further confirmed on protein levels using Western blot. Our data support that CFTR function measurements in PDIOs with rare CFTR genotypes can help to select potential responders to ETI, and suggest that regulatory authorities need to consider providing access to treatment based on the principle of equality for people with CF who do not have access to treatment.

Antibacterial and immunological properties of piperine evidenced by preclinical studies: a systematic review.

Aim: To review in vitro, in vivo, and in silico studies examining the antibacterial and immunomodulatory properties of piperine (PPN). Methods: This systematic review followed PRISMA guidelines, and five databases were searched. Results: A total of 40 articles were included in this study. Six aspects of PPN activity were identified, including antibacterial spectrum, association with antibiotics, efflux pump inhibition, biofilm effects, protein target binding, and modulation of immune functions/virulence factors. Most studies focused on Mycobacterium spp. and Staphylococcus aureus. Cell lineages and in vivo models were employed to study PPN antibacterial effects. Conclusion: We highlight PPN as a potential adjuvant in the treatment of bacterial infections. PPN possesses several antibacterial properties that need further exploration to determine the mechanisms behind its pharmacological activity.

Significant Advancement in Various Synthetic Strategies and Pharmacotherapy of Piperine Derivatives: A Review.

BACKGROUND: Piperine is a natural compound found in black pepper that has been traditionally used for various therapeutic purposes. In the ayurvedic system of medication there is a lot of evidence which shows that the piperine is widely used for different therapeutic purpose. In recent years, there has been an increasing interest in the pharmacological and therapeutic potential of piperine and its derivatives in modern medicine. In order to increase the bioavailability and therapeutic effectiveness of piperine and its analogs, researchers have been looking at various extraction methods and synthesis approaches. Many studies have been conducted in this area because of the promise of piperine as a natural substitute for synthetic medications. OBJECTIVES: The objective of this review article is to provide an up-to-date analysis of the literature on the synthesis of piperine analogs, including their extraction techniques and various biological activities such as antihypertensive, antidiabetic, insecticidal, antimicrobial, and antibiotic effects. Additionally, the review aims to discuss the potential of piperine in modern medicine, given its traditional use in various medicinal systems such as Ayurveda, Siddha, and Unani. The article also provides a comprehensive analysis of the plant from which piperine is derived. CONCLUSION: This review article provides a thorough examination of piperine and the source plant. The best extraction technique for the extraction of piperine and the synthesis of its analogs with various biological activities, including antihypertensive, antidiabetic, insecticidal, antibacterial, and antibiotic properties, are covered in the article. This review aims to provide an updated analysis of the literature on the synthesis of piperine analogs.

Elexacaftor/VX-445-mediated CFTR interactome remodeling reveals differential correction driven by mutation-specific translational dynamics.

Cystic fibrosis (CF) is one of the most prevalent lethal genetic diseases with over 2000 identified mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Pharmacological chaperones such as lumacaftor (VX-809), tezacaftor (VX-661), and elexacaftor (VX-445) treat mutation-induced defects by stabilizing CFTR and are called correctors. These correctors improve proper folding and thus facilitate processing and trafficking to increase the amount of functional CFTR on the cell surface. Yet, CFTR variants display differential responses to each corrector. Here, we report that variants P67L and L206W respond similarly to VX-809 but divergently to VX-445 with P67L exhibiting little rescue when treated with VX-445. We investigate the underlying cellular mechanisms of how CFTR biogenesis is altered by correctors in these variants. Affinity purification-mass spectrometry multiplexed with isobaric tandem mass tags was used to quantify CFTR protein-protein interaction changes between variants P67L and L206W. VX-445 facilitates unique proteostasis factor interactions especially in translation, folding, and degradation pathways in a CFTR variant-dependent manner. A number of these interacting proteins knocked down by siRNA, such as ribosomal subunit proteins, moderately rescued fully glycosylated P67L. Importantly, these knockdowns sensitize P67L to VX-445 and further enhance the trafficking correction of this variant. Partial inhibition of protein translation also mildly sensitizes P67L CFTR to VX-445 correction, supporting a role for translational dynamics in the rescue mechanism of VX-445. Our results provide a better understanding of VX-445 biological mechanism of action and reveal cellular targets that may sensitize nonresponsive CFTR variants to known and available correctors.

Personalized CFTR Modulator Therapy for G85E and N1303K Homozygous Patients with Cystic Fibrosis.

CFTR modulator therapy with elexacaftor/tezacaftor/ivacaftor (ETI) has been approved for people with CF and at least one F508del allele in Europe. In the US, the ETI label has been expanded to 177 rare CFTR mutations responsive in Fischer rat thyroid cells, including G85E, but not N1303K. However, knowledge on the effect of ETI on G85E or N1303K CFTR function remains limited. In vitro effects of ETI were measured in primary human nasal epithelial cultures (pHNECs) of a G85E homozygous patient and an N1303K homozygous patient. Effects of ETI therapy in vivo in these patients were assessed using clinical outcomes, including multiple breath washout and lung MRI, and the CFTR biomarkers sweat chloride concentration (SCC), nasal potential difference (NPD) and intestinal current measurement (ICM), before and after initiation of ETI. ETI increased CFTR-mediated chloride transport in G85E/G85E and N1303K/N1303K pHNECs. In the G85E/G85E and the N1303K/N1303K patient, we observed an improvement in lung function, SCC, and CFTR function in the respiratory and rectal epithelium after initiation of ETI. The approach of combining preclinical in vitro testing with subsequent in vivo verification can facilitate access to CFTR modulator therapy and enhance precision medicine for patients carrying rare CFTR mutations.

A mechanistic updated overview on Cepharanthine as potential anticancer agent.

The antitumor effects of traditional drugs have received increasing attention and active antitumor components extracted from traditional drugs have shown good efficacy with minimal adverse events. Cepharanthine(CEP for short) is an active component derived from the Stephania plants of Menispermaceae, which can regulate multiple signaling pathways alone or in combination with other therapeutic drugs to inhibit tumor cell proliferation, induce apoptosis, regulate autophagy, and inhibit angiogenesis, thereby inhibiting tumor progression. Therefore, we retrieved studies concerning CEP's antitumor effects in recent years and summarized the antitumor mechanism and targets, in order to gain new insights and establish a theoretical basis for further development and application of CEP.

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.