A Class of High-affinity Bicyclooctane G551D-CFTR Activators Identified by High Throughput Screening

The glycine-to-aspartic acid missense mutation at the codon 551(G551D) of the cystic fibrosis transmem-brane conductance regulator (CFTR) is one of the five most frequent cystic fibrosis (CF) mutations associated with a severe CF phenotype. To explore the feasibility of pharmacological correction of disrupted activation of CFTR chloride channel caused by G551D mutation, we developed a halide-sensitive fluorescence miniassay for G551D-CFTR in Fisher rat thyroid(FRT) epithelial cells for the discovery of novel activators of G551DCFTR. A class of bicyclooctane small molecule compounds that efficiently stimulate G551D-CFTR chloride channel activity was identified by high throughput screening via the FRT cell-based assay. This class of compounds selectively activates G551D-CFTR with a high affinity, whereas little effect of the compounds on wildtype CFTR can be seen. The discovery of a class of bicyclooctane G551D-CFTR activators will permit the analysis of structure-activity relationship of the compounds to identify ideal leads for in vivo therapeutic studies.     

Activation of G551D-CFTR by Bicyclooctane Compounds Is cAMP-dependent and Exhibits Low Sensitivity to Thiazolidinone CFTR Inhibitor CFTRinh-172

The G551D-CFTR mutation causing cystic fibrosis(CF) results from a missense mutation at codon 551 (G551D) in the gene encoding of the cystic fibrosis transmembrane conductance regulator (CFTR). The G551D mutation in CFTR results in a reduced functional channel but G551D-CFTR is appropriately inserted in the apical membrane. In previous studies we discovered a class of high-affinity bicyclooctane (BCO) G551D-CFTR activators(G551DBCOS) with Kd down to 1μmol/L. In this study, we analyzed the pharmacological activation of G551D-CFTR by the G551DBCOS by means of short circuit current analysis and cell-based fluorescence quenching assay. The G551DBCOS-induced G551D-CFTR activation is cAMP-dependent and is less sensitive to thiazolidinone CFTR inhibitor CFTRinh-172. These data suggest that (1) the phosphorylation of G551D-CFTR by protein kinase A is required for the activation by G551DBCOS; (2) G551DBCOS and CFTRinh-172 may act at the same site on the G551D-CFTR molecule.     

Natural Compound Curcumin—— a Channel Potentiator Rather Than a Corrector of the Defective Intracellular Processing of △F508 Mutant Cystic Fibrosis Transmembrane Conductance Regulator

Cystic fibrosis（CF） is a severe genetic disease caused by the gene mutation of the cystic fibrosis transmembrane conductance regulator（CFTR） chloride channel. The most common point mutation AF508, which leads to impaired intracellular processing and channel gating of CFTR, appears in about 90% CF patients. The natural compound curcumin was reported to correct the processing defect of AF508-CFTR and proposed as a potential therapeutic drug to cure CF. In the present study, we analyzed the effect of curcumin on AF508-CFTR and demonstrated that curcumin can restore the impaired chloride conductance of AF508 mutant CFTR. The activity is rapid, reversible and cAMP-dependent. However, we couldn＇t reproduce the previously reported correction of the defective membrane trafficking of AF508-CFTR by curcumin. Therefore, curcumin may not be a superior lead compound for developing anti-CF drugs.     

Natural Compound Curcumin-a Channel Potentiator Rather Than a Corrector of the Defective Intracellular Processing of △F508 Mutant Cystic Fibrosis Transmembrane Conductance Regulator

Cystic fibrosis(CF)is a severe genetic disease caused by the gene mutation of the cystic fibrosis transmembrane conductance regulator(CFTR)chloride channel.The most common point mutation △F508,which leads to impaired intracellular processing and channel gating of CFTR, appears in about 90? patients.The natural compound curcumin was reported to correct the processing defect of △F508-CFTR and proposed as a potential therapeutic drug to cure CF.In the present study.we analyzed the efrect of curcumin on △F508-CFTR and demonstrated that curcumin can restore the impaired chloride conductance of △F508 mutant CFTR.The activity is rapid,reversible and cAMP-dependent.However,we couldn't reproduce the previously reported correction of the defective membrane trafficking of △F508-CFTR by curcumin.Therefore,curcumin may not be a superior lead compound for developing anti-CF drugs.     

A High-affinity Activator of G551D-CFTR Chloride Channel Identified By High Throughput Screening

A stably transfected CHO cell line coexpressing G551D-CFTR and iodide-sensitive yellow fluorescent protein mutant EYFP-H148Q-I152L was successfully established and used as assay model to identify smallmolecule activators of G551D-CFTR chloride channel from 100000 diverse combinatorial compounds by high throughput screening on a customized Beckman robotic system. A bicyclooctane compound was identified to activate G551D-CFTR chloride channel with high-affinity(Kd=1.8 μmol/L). The activity of the bicyclooctane compound is G551D-CFTR-specific, reversible and non-toxic. The G551D-CFTR activator may be useful as a tool to study the mutant G551D-CFTR chloride channel structure and transport properties and as a candidate drug to cure cystic fibrosis caused by G551D-CFTR mutation,     

20(S)-原人参二醇促进CFTR氯离子通道开放

利用氯离子通道细胞荧光测定模型对386种中药单体化合物进行筛选, 发现20(S)-原人参二醇对依赖于cAMP的CFTR氯离子通道具有激活作用. 20(S)-原人参二醇能够以剂量依赖的方式激活野生型CFTR氯离子通道, 其激活作用通过更为可靠的氯离子通道短路电流测定系统得到证实. 20(S)-原人参二醇对CFTR氯离子通道的激活效应具有作用迅速且可逆的特点. 其在发挥激活作用时依赖于腺苷环化酶激动剂Forskolin的存在, 单独与细胞孵育不提高细胞内cAMP的水平, 表明对CFTR氯离子通道的激活作用是通过与CFTR直接结合实现的. 该化合物对ΔF508-CFTR突变氯离子通道的开放也具有特征相似的激活作用.     

Curcumin Inhibits In Vitro SARS-CoV-2 Infection In Vero E6 Cells through Multiple Antiviral Mechanisms

Due to the scarcity of therapeutic approaches for COVID-19, we investigated the antiviral and anti-inflammatory properties of curcumin against SARS-CoV-2 using in vitro models. The cytotoxicity of curcumin was evaluated using MTT assay in Vero E6 cells. The antiviral activity of this compound against SARS-CoV-2 was evaluated using four treatment strategies (i. pre–post infection treatment, ii. co-treatment, iii. pre-infection, and iv. post-infection). The D614G strain and Delta variant of SARS-CoV-2 were used, and the viral titer was quantified by plaque assay. The anti-inflammatory effect was evaluated in peripheral blood mononuclear cells (PBMCs) using qPCR and ELISA. By pre–post infection treatment, Curcumin (10 µg/mL) exhibited antiviral effect of 99% and 99.8% against DG614 strain and Delta variant, respectively. Curcumin also inhibited D614G strain by pre-infection and post-infection treatment. In addition, curcumin showed a virucidal effect against D614G strain and Delta variant. Finally, the pro-inflammatory cytokines (IL-1β, IL-6, and IL-8) released by PBMCs triggered by SARS-CoV-2 were decreased after treatment with curcumin. Our results suggest that curcumin affects the SARS-CoV-2 replicative cycle and exhibits virucidal effect with a variant/strain independent antiviral effect and immune-modulatory properties. This is the first study that showed a combined (antiviral/anti-inflammatory) effect of curcumin during SARS-CoV-2 infection. However, additional studies are required to define its use as a treatment for the COVID-19.     

Evaluation of 1,2,3-Triazoles as Amide Bioisosteres In Cystic Fibrosis Transmembrane Conductance Regulator Modulators VX-770 and VX-809

The 1,2,3-triazole has been successfully utilized as an amide bioisostere in multiple therapeutic contexts. Based on this precedent, triazole analogues derived from VX-809 and VX-770, prominent amide-containing modulators of the cystic fibrosis transmembrane conductance regulator (CFTR), were synthesized and evaluated for CFTR modulation. Triazole 11 , derived from VX-809, displayed markedly reduced efficacy in F508del-CFTR correction in cellular TECC assays in comparison to VX-809. Surprisingly, triazole analogues derived from potentiator VX-770 displayed no potentiation of F508del, G551D, or WT-CFTR in cellular Ussing chamber assays. However, patch clamp analysis revealed that triazole 60 potentiates WT-CFTR similarly to VX-770. The efficacy of 60 in the cell-free patch clamp experiment suggests that the loss of activity in the cellular assay could be due to the inability of VX-770 triazole derivatives to reach the CFTR binding site. Moreover, in addition to the negative impact on biological activity, triazoles in both structural classes displayed decreased metabolic stability in human microsomes relative to the analogous amides. In contrast to the many studies that demonstrate the advantages of using the 1,2,3-triazole, these findings highlight the negative impacts that can arise from replacement of the amide with the triazole and suggest that caution is warranted when considering use of the 1,2,3-triazole as an amide bioisostere.     

Rheological properties of lyotropic liquid crystals encapsulating curcumin

This study constructed new curcumin-loaded lyotropic liquid crystals containing pharmaceutically accepted oil, and ethyl oleate (EtOL). Three liquid crystalline phases including lamellar, hexagonal, and cubic phases were identified by means of the polarized optical microscopy and rheology method. By analyzing the shear viscosity (η_0.1), the viscosity of curcumin-liquid crystals is smaller than those without curcumin. Dynamic rheological results show that: Dissolved curcumin in EtOL can make the elastic modulus of hexagonal and cubic phase increase compared with that without curcumin, while the elastic modulus of lamellar phase decreases. Dissolved curcumin in Brij 97 can lead to the decreasing of the elastic modulus for cubic and lamellar phases, whereas it has little influence on hexagonal phase. When the curcumin is solubilized in both EtOL and Brij 97, the elastic modus of hexagonal phase increase, the elastic modus of lamellar and cubic phases decrease compared with that without curcumin. Furthermore, three temperature turning points were identified by the change in the slope of tanδ (G″/G′) for curcumin-hexagonal liquid crystal. These studies might be a help to study the storage of drug carrier and in vitro release properties of lyotropic liquid crystals containing curcumin.     

Activation of CFTR-mediated Cl- Transport by Magnolin

Magnolin is a herbal compound from Magnolia biondii Pamp. It possesses numerous biological activities. Cystic fibrosis transmembrane conductance regulator（CFTR） is an epithelial chloride channel that plays a key role in the fluid secretion of various exocrine organs. In the present study, the activation of CFTR-mediated chloride transport by magnolin is indentified and characterized. In CFTR stably transfected FRT cells, magnolin increases CFTR CI- currents in a concentration-dependent manner. The activation of magnolin on CFTR is rapid, reversible, and cAMP-dependent. Magnolin does not elevate cellular cAMP level, indicating that it activates CFTR by direct binding and interaction with CFTR protein. Magnolin selectively activates wildtype CFTR rather than mutant CFTR. Magnolin may present a novel class of therapeutic lead compound tbr the treatment of diseases associated with reduced CFTR function such as keratoconjunctivitis sicca, idiopathic chronic pancreatiti, and chronic constipation.     

Epithelial-to-Mesenchymal Transition Is Not a Major Modulating Factor in the Cytotoxic Response to Natural Products in Cancer Cell Lines

Numerous natural products exhibit antiproliferative activity against cancer cells by modulating various biological pathways. In this study, we investigated the potential use of eight natural compounds (apigenin, curcumin, epigallocatechin gallate, fisetin, forskolin, procyanidin B2, resveratrol, urolithin A) and two repurposed agents (fulvestrant and metformin) as chemotherapy enhancers and mesenchymal-to-epithelial (MET) inducers of cancer cells. Screening of these compounds in various colon, breast, and pancreatic cancer cell lines revealed anti-cancer activity for all compounds, with curcumin being the most effective among these in all cell lines. Although some of the natural products were able to induce MET in some cancer cell lines, the MET induction was not related to increased synergy with either 5-FU, irinotecan, gemcitabine, or gefitinib. When synergy was observed, for example with curcumin and irinotecan, this was unrelated to MET induction, as assessed by changes in E-cadherin and vimentin expression. Our results show that MET induction is compound and cell line specific, and that MET is not necessarily related to enhanced chemosensitivity.     

Evaluation of Fused Pyrrolothiazole Systems as Correctors of Mutant CFTR Protein

Cystic fibrosis (CF) is a genetic disease caused by mutations that impair the function of the CFTR chloride channel. The most frequent mutation, F508del, causes misfolding and premature degradation of CFTR protein. This defect can be overcome with pharmacological agents named “correctors”. So far, at least three different classes of correctors have been identified based on the additive/synergistic effects that are obtained when compounds of different classes are combined together. The development of class 2 correctors has lagged behind that of compounds belonging to the other classes. It was shown that the efficacy of the prototypical class 2 corrector, the bithiazole corr-4a, could be improved by generating conformationally-locked bithiazoles. In the present study, we investigated the effect of tricyclic pyrrolothiazoles as analogues of constrained bithiazoles. Thirty-five compounds were tested using the functional assay based on the halide-sensitive yellow fluorescent protein (HS-YFP) that measured CFTR activity. One compound, having a six atom carbocyle central ring in the tricyclic pyrrolothiazole system and bearing a pivalamide group at the thiazole moiety and a 5-chloro-2-methoxyphenyl carboxamide at the pyrrole ring, significantly increased F508del-CFTR activity. This compound could lead to the synthesis of a novel class of CFTR correctors.     

Ultrasound mediates the release of curcumin from microemulsions

Ultrasound is a powerful noninvasive modality for biomedical imaging, and holds much promise for noninvasive drug delivery enhancement and targeting. However, the optimal design of sound sensitive carriers is still poorly understood. In this study, curcumin, an important natural antioxidant and anticancer compound, was stably entrapped into microemulsion droplets with average size 20-35 nm. To release curcumin, low frequency (40 kHz) ultrasound at an intensity of 3.8 or 9.8 W/cm2 was applied to the microemulsions, using a probe sonicator. On insonation, much of the curcumin was released from the microemulsions and formed insoluble aggregates, as evidenced by decreased UV-vis absorption at 420 nm. The initial release rate (assayed by the rate of change of absorption) was as high as 0.11 microg/s (1.87%/sec) in phosphate buffered saline solution at neutral pH, but decreased at acidic pH. Interestingly, lower curcumin loading led to a more rapid release under insonation. Measurements of emulsion droplet size implicate droplet reorganization (fusion or fission) as an important contributing mechanism for the ultrasonic release of this compound. Although cargo in microemulsions is partitioned, rather than encapsulated (as in, for example, liposomes), these new results demonstrate that microemulsion carriers are feasible for some ultrasonic drug delivery applications.     

Curcumin reduces cold storage-induced damage in human cardiac myoblasts

Curcumin is a polyphenolic compound possessing interesting anti-inflammatory and antioxidant properties and has the ability to induce the defensive protein heme oxygenase-1 (HO-1). The objective of this study was to investigate whether curcumin protects against cold storage-mediated damage of human adult atrial myoblast cells (Girardi cells) and to assess the potential involvement of HO-1 in this process. Girardi cells were exposed to either normothermic or hypothermic conditions in Celsior preservation solution in the presence or absence of curcumin. HO-1 protein expression and heme oxygenase activity as well as cellular damage were assessed after cold storage or cold storage followed by re-warming. In additional experiments, an inhibitor of heme oxygenase activity (tin protoporphyrin IX, 10 microM) or siRNA for HO-1 were used to investigate the participation of HO-1 as a mediator of curcumin-induced effects. Treatment with curcumin produced a marked induction of cardiac HO-1 in normothermic condition but cells were less responsive to the polyphenolic compound at low temperature. Cold storage-induced damage was markedly reduced in the presence of curcumin and HO-1 contributed to some extent to this effect. Thus, curcumin added to Celsior preservation solution effectively prevents the damage caused by cold-storage; this effect involves the protective enzyme HO-1 but also other not yet identified mechanisms.     

Dictamine Stimulates Cystic Fibrosis Transmembrane Conductance Regulator CL Transport

Dictamine is a furoquinoline alkaloid isolated from Dictamus dasycarpus Turcz.In the present study,we found that dictamine is able to stimulate the chloride transport activity of wild-type and ΔF508 mutant CFTR.The activity is cAMP-dependent and can be completely reversed by specific CFTR inhibitor CFTRinh-172.In addition,dictamine can further increase the chloride transport activity when CFTR is maximally activated by the combination of cAMP stimulators forskolin(FSK)and IBMX,suggesting direct interaction of dictamine with CFTR.Dictamine may be useful for probing CFTR channel gating mechanisms and used as a lead compound to develop the pharmacological therapy of CFTR-related diseases such as idiopathic chronic pancreatitis and keratoconjunctivitis sicca and cystic fibrosis.     

Effects of Curcumin Analogues DMC and EF24 in Combination with the Cytokine TRAIL against Kidney Cancer

The natural compound curcumin has been shown to have therapeutic potential against a wide range of diseases such as cancer. Curcumin reduces cell viability of renal cell carcinoma (RCC) cells when combined with TNF-related apoptosis-inducing ligand (TRAIL), a cytokine that specifically targets cancer cells, by helping overcome TRAIL resistance. However, the therapeutic effects of curcumin are limited by its low bioavailability. Similar compounds to curcumin with higher bioavailability, such as demethoxycurcumin (DMC) and 3,5-bis(2-fluorobenzylidene)-4-piperidone (EF24), can potentially have similar anticancer effects and show a similar synergy with TRAIL, thus reducing RCC viability. This study aims to show the effects of DMC and EF24 in combination with TRAIL at reducing ACHN cell viability and ACHN cell migration. It also shows the changes in death receptor 4 (DR4) expression after treatment with these compounds individually and in combination with TRAIL, which can play a role in their mechanism of action.     

Activation Effect of Cathartic Natural Compound Rhein to CFTR Chloride Channel

Introduction Aloe,cascaraandsennaarewidelyusedasall purposelaxativemedicine.Itisgenerallyregardedthat thecatharticingredientsofaloe,cascaraandsennaare anthraquinoneandtheirderivatives[1].Ithasbeenwell definedthattheincreasedleakingofplasmaintointesti nall…     

DFT Study of Physisorption Effect of the Curcumin on CNT(8,0-6) Nanotube for Biological Applications

In the given work the adsorption properties of molecule curcumin((1 E,6 E)-1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione) on CNT(8,0-6) nanotube were investigated by the density functional theory(DFT) in the solvent water for the first time. The non-bonded interaction effects of compounds curcumin and CNT(8,0-6) nanotube on the electronic properties, UV/Vis spectra, chemical shift tensors and natural charges were determined and discussed. The electronic spectra of the compound curcumin and the complex CNT(8,0-6)/curcumin in the solvent water were calculated by time dependent density functional theory(TD-DFT) for investigation of the maximum wavelength value of molecule Curcumin before and after the non-bonded interaction with the CNT(8,0-6) nanotube and molecular orbitals involved in the formation of absorption spectrum of the complex CNT(8,0-6)/curcumin at maximum wavelength.     

Azobioisosteres of Curcumin with Pronounced Activity against Amyloid Aggregation,Intracellular Oxidative Stress,and Neuroinflammation

Many (poly-)phenolic natural products, for example, curcumin and taxifolin, have been studied for their activity against specific hallmarks of neurodegeneration, such as amyloid-β 42 (Aβ42) aggregation and neuroinflammation. Due to their drawbacks, arising from poor pharmacokinetics, rapid metabolism, and even instability in aqueous medium, the biological activity of azobenzene compounds carrying a pharmacophoric catechol group, which have been designed as bioisoteres of curcumin has been examined. Molecular simulations reveal the ability of these compounds to form a hydrophobic cluster with Aβ42, which adopts different folds, affecting the propensity to populate fibril-like conformations. Furthermore, the curcumin bioisosteres exceeded the parent compound in activity against Aβ42 aggregation inhibition, glutamate-induced intracellular oxidative stress in HT22 cells, and neuroinflammation in microglial BV-2 cells. The most active compound prevented apoptosis of HT22 cells at a concentration of 2.5 μm (83 % cell survival), whereas curcumin only showed very low protection at 10 μm (21 % cell survival).     

Circular dichroism spectroscopic studies reveal pH dependent binding of curcumin in the minor groove of natural and synthetic nucleic acids

For the first time, an interaction between the non-toxic, cancer chemopreventive agent curcumin and both natural and synthetic DNA duplexes has been demonstrated by using circular dichroism (CD) and absorption spectroscopy techniques. Upon addition of curcumin to calf thymus DNA, poly(dG-dC).poly(dG-dC) and poly(dA-dT).poly(dA-dT) solutions, an intense positive induced CD band centered around 460-470 nm was observed depending on the actual pH and Na+ ion concentration of the medium; no CD signal was obtained, however, with single stranded poly(dC). Interaction of curcumin with calf thymus DNA was observed already at pH 6.5 in contrast with poly(dG-dC).poly(dG-dC) which induces no extrinsic Cotton effect above a pH value of 5. The protonated, Hoogsteen base-paired structure of poly(dG-dC).poly(dG-dC) is necessary for curcumin binding while the alternating AT-rich polymer formed complexes with curcumin only at certain Na+ concentrations. Evaluation of the spectral data and molecular modeling calculations suggested that curcumin, this dietary polyphenolic compound binds in the minor groove of the double helix. The mechanism of the induced CD activity, the effects of the pH and Na+ ions on the ligand binding and conformation of the double helix are discussed in detail. As well as being an essentially new phenolic minor groove binder agent curcumin is also a promising molecular probe to study biologically important, pH and cation induced conformational polymorphisms of nucleic acids.